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The Virtual Child

Gilbertson RJ, Behjati S, Böttcher AL, Bronner ME, Burridge M, Clausing H, Clifford H, Danaher T, Donovan LK, Drost J, Eggermont AMM, Emerson C, Flores MG, Hamerlik P, Jabado N, Jones A, Kaessmann H, Kleinman CL, Kool M, Kutscher LM, Lindberg G, Linnane E, Marioni JC, Maris JM, Monje M, Macaskill A, Niederer S, Northcott PA, Peeters E, Plieger-van Solkema W, Preußner L, Rios AC, Rippe K, Sandford P, Sgourakis NG, Shlien A, Smith P, Straathof K, Sullivan PJ, Suvà ML, Taylor MD, Thompson E, Vento-Tormo R, Wainwright BJ, Wechsler-Reya RJ, Westermann F, Winslade S, Al-Lazikani B, Pfister SM. The Virtual Child. Cancer Discov. 2024 Apr 4;14(4):663-668. doi: 10.1158/2159-8290.CD-23-1500. PMID: 38571421.

Abstract

We are building the world’s first Virtual Child-a computer model of normal and cancerous human development at the level of each individual cell. The Virtual Child will “develop cancer” that we will subject to unlimited virtual clinical trials that pinpoint, predict, and prioritize potential new treatments, bringing forward the day when no child dies of cancer, giving each one the opportunity to lead a full and healthy life.

CryoEM and CryoET NMR SgourakisLab

Synthetic Efforts toward Lannotinidine G Based on an Aziridinium-Mediated Ring Contraction and Dienyne Metathesis

Peitsinis Z, Trauner D. Synthetic Efforts toward Lannotinidine G Based on an Aziridinium-Mediated Ring Contraction and Dienyne Metathesis. Org Lett. 2024 Apr 2. doi: 10.1021/acs.orglett.4c00791. Epub ahead of print. PMID: 38564423.

Abstract

Lannotinidine G is a unique Lycopodium alkaloid that features a tricyclic [6/6/6] core with 3 contiguous stereocenters and a 1,3-diene moiety in addition to a 7-membered lactone. Herein, we disclose our efforts toward the synthesis of this natural product, which achieved the construction of the aza-tricyclic core with the correct configuration at its three stereocenters. Key features of our strategy include a highly diastereoselective Fráter-Seebach alkylation and Corey-Chaykovsky type epoxide formation, an unusual aziridinium-mediated ring contraction for the formation of the piperidine moiety, and a regioselective dienyne metathesis.

Chemical Biology CryoEM and CryoET Trauner Group

Iterative oxidation by TET1 is required for reprogramming of imprinting control regions and patterning of mouse sperm hypomethylated regions

Prasasya RD, Caldwell BA, Liu Z, Wu S, Leu NA, Fowler JM, Cincotta SA, Laird DJ, Kohli RM, Bartolomei MS. Iterative oxidation by TET1 is required for reprogramming of imprinting control regions and patterning of mouse sperm hypomethylated regions. Dev Cell. 2024 Apr 1:S1534-5807(24)00144-8. doi: 10.1016/j.devcel.2024.02.012. Epub ahead of print. PMID: 38569549.

Abstract

Ten-eleven translocation (TET) enzymes iteratively oxidize 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxylcytosine to facilitate active genome demethylation. Whether these bases are required to promote replication-coupled dilution or activate base excision repair during mammalian germline reprogramming remains unresolved due to the inability to decouple TET activities. Here, we generated two mouse lines expressing catalytically inactive TET1 (Tet1-HxD) and TET1 that stalls oxidation at 5hmC (Tet1-V). Tet1 knockout and catalytic mutant primordial germ cells (PGCs) fail to erase methylation at select imprinting control regions and promoters of meiosis-associated genes, validating the requirement for the iterative oxidation of 5mC for complete germline reprogramming. TET1V and TET1HxD rescue most hypermethylation of Tet1-/- sperm, suggesting the role of TET1 beyond its oxidative capability. We additionally identify a broader class of hypermethylated regions in Tet1 mutant mouse sperm that depend on TET oxidation for reprogramming. Our study demonstrates the link between TET1-mediated germline reprogramming and sperm methylome patterning.

Chemical Biology CryoEM and CryoET Kohli Lab

Engineering Substrate Channeling in Assembly-Line Terpene Biosynthesis

Wenger ES, Schultz K, Marmorstein R, Christianson DW. Engineering Substrate Channeling in Assembly-Line Terpene Biosynthesis. bioRxiv [Preprint]. 2024 Mar 28:2024.03.25.586617. doi: 10.1101/2024.03.25.586617. PMID: 38586022; PMCID: PMC10996616.

Abstract

Fusicoccadiene synthase from P. amygdala (PaFS) is a bifunctional assembly-line terpene synthase containing a prenyltransferase domain that generates geranylgeranyl diphosphate (GGPP) from dimethylallyl diphosphate (DMAPP) and three equivalents of isopentenyl diphosphate (IPP), and a cyclase domain that converts GGPP into fusicoccadiene, a precursor of the diterpene glycoside Fusicoccin A. The two catalytic domains are linked by a flexible 69-residue polypeptide segment. The prenyltransferase domain mediates oligomerization to form predominantly octamers, and cyclase domains are randomly splayed out around the prenyltransferase core. Previous studies suggest that substrate channeling is operative in catalysis, since most of the GGPP formed by the prenyltransferase remains on the protein for the cyclization reaction. Here, we demonstrate that the flexible linker is not required for substrate channeling, nor must the prenyltransferase and cyclase domains be covalently linked to sustain substrate channeling. Moreover, substrate competition experiments with other diterpene cyclases indicate that the PaFS prenyltransferase and cyclase domains are preferential partners regardless of whether they are covalently linked or not. The cryo-EM structure of engineered “linkerless” construct PaFSLL, in which the 69-residue linker is spliced out and replaced with the tripeptide PTQ, reveals that cyclase pairs associate with all four sides of the prenyltransferase octamer. Taken together, these results suggest that optimal substrate channeling is achieved when a cyclase domain associates with the side of the prenyltransferase octamer, regardless of whether the two domains are covalently linked and regardless of whether this interaction is transient or locked in place.

Chemical Biology CryoEM and CryoET Christianson Group X-ray Crystallography Marmorstein Lab

Chemical Versatility in Catalysis and Inhibition of the Class IIb Histone Deacetylases

Christianson DW. Chemical Versatility in Catalysis and Inhibition of the Class IIb Histone Deacetylases. Acc Chem Res. 2024 Mar 26. doi: 10.1021/acs.accounts.3c00801. Epub ahead of print. PMID: 38530703.

Abstract

ConspectusThe zinc-dependent histone deacetylases (HDACs 1-11) belong to the arginase-deacetylase superfamily of proteins, members of which share a common α/β fold and catalytic metal binding site. While several HDACs play a role in epigenetic regulation by catalyzing acetyllysine hydrolysis in histone proteins, the biological activities of HDACs extend far beyond histones. HDACs also deacetylate nonhistone proteins in the nucleus as well as the cytosol to regulate myriad cellular processes. The substrate pool is even more diverse in that certain HDACs can hydrolyze other covalent modifications. For example, HDAC6 is also a lysine decrotonylase, and HDAC11 is a lysine-fatty acid deacylase. Surprisingly, HDAC10 is not a lysine deacetylase but instead is a polyamine deacetylase. Thus, the HDACs are biologically and chemically versatile catalysts as they regulate the function of diverse protein and nonprotein substrates throughout the cell.Owing to their critical regulatory functions, HDACs serve as prominent targets for drug design. At present, four HDAC inhibitors are FDA-approved for cancer chemotherapy. However, these inhibitors are active against multiple HDAC isozymes, and a lack of selectivity is thought to contribute to undesirable side effects. Current medicinal chemistry campaigns focus on the development of isozyme-selective inhibitors, and many such studies largely focus on HDAC6 and HDAC10. HDAC6 is a target for therapeutic intervention due to its cellular role as a tubulin deacetylase and tau deacetylase, and selective inhibitors are being studied in cancer chemotherapy and the treatment of peripheral neuropathy. Crystal structures of enzyme-inhibitor complexes reveal how various features of inhibitor design, such as zinc-coordinating groups, bifurcated capping groups, and aromatic fluorination patterns, contribute to affinity and isozyme selectivity. The polyamine deacetylase HDAC10 is also an emerging target for cancer chemotherapy.

CryoEM and CryoET Christianson Group X-ray Crystallography

Ultrastructure of human brain tissue vitrified from autopsy revealed by cryo-ET with cryo-plasma FIB milling

Creekmore BC, Kixmoeller K, Black BE, Lee EB, Chang YW. Ultrastructure of human brain tissue vitrified from autopsy revealed by cryo-ET with cryo-plasma FIB milling. Nat Commun. 2024 Mar 26;15(1):2660. doi: 10.1038/s41467-024-47066-1. PMID: 38531877; PMCID: PMC10965902.

Abstract

Ultrastructure of human brain tissue has traditionally been examined using electron microscopy (EM) following fixation, staining, and sectioning, which limit resolution and introduce artifacts. Alternatively, cryo-electron tomography (cryo-ET) allows higher resolution imaging of unfixed cellular samples while preserving architecture, but it requires samples to be vitreous and thin enough for transmission EM. Due to these requirements, cryo-ET has yet to be employed to investigate unfixed, never previously frozen human brain tissue. Here we present a method for generating lamellae in human brain tissue obtained at time of autopsy that can be imaged via cryo-ET. We vitrify the tissue via plunge-freezing and use xenon plasma focused ion beam (FIB) milling to generate lamellae directly on-grid at variable depth inside the tissue. Lamellae generated in Alzheimer’s disease brain tissue reveal intact subcellular structures including components of autophagy and potential pathologic tau fibrils. Furthermore, we reveal intact compact myelin and functional cytoplasmic expansions. These images indicate that plasma FIB milling with cryo-ET may be used to elucidate nanoscale structures within the human brain.

CryoEM and CryoET Mass Spectrometry Yi-Wei Chang Lab

Optical Control of G-Actin with a Photoswitchable Latrunculin

Vepřek NA, Cooper MH, Laprell L, Yang EJ, Folkerts S, Bao R, Boczkowska M, Palmer NJ, Dominguez R, Oertner TG, Pon LA, Zuchero JB, Trauner DH. Optical Control of G-Actin with a Photoswitchable Latrunculin. J Am Chem Soc. 2024 Mar 21. doi: 10.1021/jacs.3c10776. Epub ahead of print. PMID: 38511265.

Abstract

Actin is one of the most abundant proteins in eukaryotic cells and is a key component of the cytoskeleton. A range of small molecules has emerged that interfere with actin dynamics by either binding to polymeric F-actin or monomeric G-actin to stabilize or destabilize filaments or prevent their formation and growth, respectively. Among these, the latrunculins, which bind to G-actin and affect polymerization, are widely used as tools to investigate actin-dependent cellular processes. Here, we report a photoswitchable version of latrunculin, termed opto-latrunculin (OptoLat), which binds to G-actin in a light-dependent fashion and affords optical control over actin polymerization. OptoLat can be activated with 390-490 nm pulsed light and rapidly relaxes to its inactive form in the dark. Light activated OptoLat induced depolymerization of F-actin networks in oligodendrocytes and budding yeast, as shown by fluorescence microscopy. Subcellular control of actin dynamics in human cancer cell lines was demonstrated via live cell imaging. Light-activated OptoLat also reduced microglia surveillance in organotypic mouse brain slices while ramification was not affected. Incubation in the dark did not alter the structural and functional integrity of the microglia. Together, our data demonstrate that OptoLat is a useful tool for the elucidation of G-actin dependent dynamic processes in cells and tissues.

Chemical Biology CryoEM and CryoET Dominguez Lab Trauner Group

Involvement of ArlI, ArlJ, and CirA in Archaeal Type-IV Pilin-Mediated Motility Regulation

Chatterjee P, Garcia MA, Cote JA, Yun K, Legerme GP, Habib R, Tripepi M, Young C, Kulp D, Dyall-Smith M, Pohlschroder M. Involvement of ArlI, ArlJ, and CirA in Archaeal Type-IV Pilin-Mediated Motility Regulation. bioRxiv [Preprint]. 2024 Mar 20:2024.03.04.583388. doi: 10.1101/2024.03.04.583388. PMID: 38562816; PMCID: PMC10983859.

Abstract

Many prokaryotes use swimming motility to move toward favorable conditions and escape adverse surroundings. Regulatory mechanisms governing bacterial flagella-driven motility are well-established, however, little is yet known about the regulation underlying swimming motility propelled by the archaeal cell surface structure, the archaella. Previous research showed that deletion of the adhesion pilins (PilA1-6), subunits of the type IV pili cell surface structure, renders the model archaeon Haloferax volcanii non-motile. In this study, we used EMS mutagenesis and a motility assay to identify motile suppressors of the ΔpilA[1-6] strain. Of the eight suppressors identified, six contain missense mutations in archaella biosynthesis genes, arlI and arlJ. Overexpression of these arlI and arlJ mutant constructs in the respective multi-deletion strains ΔpilA[1-6arlI and ΔpilA[1-6arlJ confirmed their role in suppressing the ΔpilA[1-6] motility defect. Additionally, three suppressors harbor co-occurring disruptive missense and nonsense mutations in cirA, a gene encoding a proposed regulatory protein. A deletion of cirA resulted in hypermotility, while cirA overexpression in wild-type cells led to decreased motility. Moreover, qRT-PCR analysis revealed that in wild-type cells, higher expression levels of arlIarlJ, and the archaellin gene arlA1 were observed in motile early-log phase rod-shaped cells compared to non-motile mid-log phase disk-shaped cells.

Computational Biology CryoEM and CryoET Kulp Lab

Novel modifications of PARP inhibitor veliparib increase PARP1 binding to DNA breaks

Velagapudi UK, Rouleau-Turcotte É, Billur R, Shao X, Patil M, Black BE, Pascal JM, Talele TT. Novel modifications of PARP inhibitor veliparib increase PARP1 binding to DNA breaks. Biochem J. 2024 Mar 20;481(6):437-460. doi: 10.1042/BCJ20230406. PMID: 38372302.

Abstract

Catalytic poly(ADP-ribose) production by PARP1 is allosterically activated through interaction with DNA breaks, and PARP inhibitor compounds have the potential to influence PARP1 allostery in addition to preventing catalytic activity. Using the benzimidazole-4-carboxamide pharmacophore present in the first generation PARP1 inhibitor veliparib, a series of 11 derivatives was designed, synthesized, and evaluated as allosteric PARP1 inhibitors, with the premise that bulky substituents would engage the regulatory helical domain (HD) and thereby promote PARP1 retention on DNA breaks. We found that core scaffold modifications could indeed increase PARP1 affinity for DNA; however, the bulk of the modification alone was insufficient to trigger PARP1 allosteric retention on DNA breaks. Rather, compounds eliciting PARP1 retention on DNA breaks were found to be rigidly held in a position that interferes with a specific region of the HD domain, a region that is not targeted by current clinical PARP inhibitors. Collectively, these compounds highlight a unique way to trigger PARP1 retention on DNA breaks and open a path to unveil the pharmacological benefits of such inhibitors with novel properties.

Black Lab Chemical Biology CryoEM and CryoET

Structure-Based Engineering of a Sesquiterpene Cyclase to Generate an Alcohol Product: Conversion of epi-Isozizaene Synthase into α-Bisabolol Synthase

Eaton SA, Christianson DW. Structure-Based Engineering of a Sesquiterpene Cyclase to Generate an Alcohol Product: Conversion of epi-Isozizaene Synthase into α-Bisabolol Synthase. Biochemistry. 2024 Mar 19;63(6):797-805. doi: 10.1021/acs.biochem.3c00681. Epub 2024 Feb 29. PMID: 38420671; PMCID: PMC10961106.

Abstract

The sesquiterpene cyclase epi-isozizaene synthase (EIZS) from Streptomyces coelicolor catalyzes the metal-dependent conversion of farnesyl diphosphate (FPP) into the complex tricyclic product epi-isozizaene. This remarkable transformation is governed by an active site contour that serves as a template for catalysis, directing the conformations of multiple carbocation intermediates leading to the final product. Mutagenesis of residues defining the active site contour remolds its three-dimensional shape and reprograms the cyclization cascade to generate alternative cyclization products. In some cases, mutagenesis enables alternative chemistry to quench carbocation intermediates, e.g., through hydroxylation. Here, we combine structural and biochemical data from previously characterized EIZS mutants to design and prepare F95S-F198S EIZS, which converts EIZS into an α-bisabolol synthase with moderate fidelity (65% at 18 °C, 74% at 4 °C). We report the complete biochemical characterization of this double mutant as well as the 1.47 Å resolution X-ray crystal structure of its complex with three Mg2+ ions, inorganic pyrophosphate, and the benzyltriethylammonium cation, which partially mimics a carbocation intermediate. Most notably, the two mutations together create an active site contour that stabilizes the bisabolyl carbocation intermediate and positions a water molecule for the hydroxylation reaction. Structural comparison with a naturally occurring α-bisabolol synthase reveals common active site features that direct α-bisabolol generation. In showing that EIZS can be redesigned to generate a sesquiterpene alcohol product instead of a sesquiterpene hydrocarbon product, we have expanded the potential of EIZS as a platform for the development of designer cyclases that could be utilized in synthetic biology applications.

CryoEM and CryoET Christianson Group X-ray Crystallography

Multiplexed DNA-PAINT Imaging of the Heterogeneity of Late Endosome/Lysosome Protein Composition

Bond C, Hugelier S, Xing J, Sorokina EM, Lakadamyali M. Multiplexed DNA-PAINT Imaging of the Heterogeneity of Late Endosome/Lysosome Protein Composition. bioRxiv [Preprint]. 2024 Mar 19:2024.03.18.585634. doi: 10.1101/2024.03.18.585634. PMID: 38562776; PMCID: PMC10983937.

Abstract

Late endosomes/lysosomes (LELs) are crucial for numerous physiological processes and their dysfunction is linked to many diseases. Proteomic analyses have identified hundreds of LEL proteins, however, whether these proteins are uniformly present on each LEL, or if there are cell-type dependent LEL sub-populations with unique protein compositions is unclear. We employed a quantitative, multiplexed DNA-PAINT super-resolution approach to examine the distribution of six key LEL proteins (LAMP1, LAMP2, CD63, TMEM192, NPC1 and LAMTOR4) on individual LELs. While LAMP1 and LAMP2 were abundant across LELs, marking a common population, most analyzed proteins were associated with specific LEL subpopulations. Our multiplexed imaging approach identified up to eight different LEL subpopulations based on their unique membrane protein composition. Additionally, our analysis of the spatial relationships between these subpopulations and mitochondria revealed a cell-type specific tendency for NPC1-positive LELs to be closely positioned to mitochondria. Our approach will be broadly applicable to determining organelle heterogeneity with single organelle resolution in many biological contexts.

Single Molecule Imaging Lakadamyali Lab

Identification of the growth cone as a probe and driver of neuronal migration in the injured brain

Nakajima C, Sawada M, Umeda E, Takagi Y, Nakashima N, Kuboyama K, Kaneko N, Yamamoto S, Nakamura H, Shimada N, Nakamura K, Matsuno K, Uesugi S, Vepřek NA, Küllmer F, Nasufović V, Uchiyama H, Nakada M, Otsuka Y, Ito Y, Herranz-Pérez V, García-Verdugo JM, Ohno N, Arndt HD, Trauner D, Tabata Y, Igarashi M, Sawamoto K. Identification of the growth cone as a probe and driver of neuronal migration in the injured brain. Nat Commun. 2024 Mar 9;15(1):1877. doi: 10.1038/s41467-024-45825-8. PMID: 38461182; PMCID: PMC10924819.

Abstract

Axonal growth cones mediate axonal guidance and growth regulation. We show that migrating neurons in mice possess a growth cone at the tip of their leading process, similar to that of axons, in terms of the cytoskeletal dynamics and functional responsivity through protein tyrosine phosphatase receptor type sigma (PTPσ). Migrating-neuron growth cones respond to chondroitin sulfate (CS) through PTPσ and collapse, which leads to inhibition of neuronal migration. In the presence of CS, the growth cones can revert to their extended morphology when their leading filopodia interact with heparan sulfate (HS), thus re-enabling neuronal migration. Implantation of an HS-containing biomaterial in the CS-rich injured cortex promotes the extension of the growth cone and improve the migration and regeneration of neurons, thereby enabling functional recovery. Thus, the growth cone of migrating neurons is responsive to extracellular environments and acts as a primary regulator of neuronal migration.

Chemical Biology CryoEM and CryoET Trauner Group

A Multidisciplinary Hyper-Modeling Scheme in Personalized In Silico Oncology: Coupling Cell Kinetics With Metabolism, Signaling Networks and Biomechanics As Plug-In Component Models of a Cancer Digital Twin

Kolokotroni, E.; Abler, D.; Ghosh, A.; Tzamali, E.; Grogan, J.; Georgiadi, E.; Büchler, P.; Radhakrishnan, R.; Byrne, H.; Sakkalis, V.; Nikiforaki, K.; Karatzanis, I.; McFarlane, N.J.; Kaba, D.; Dong, F.; Bohle, R.M.; Meese, E.; Graf, N.; Stamatakos, G. A Multidisciplinary Hyper-Modeling Scheme in Personalized In Silico Oncology: Coupling Cell Kinetics With Metabolism, Signaling Networks and Biomechanics As Plug-In Component Models of a Cancer Digital Twin. Preprints 2024, 2024030490. https://doi.org/10.20944/preprints202403.0490.v1

Abstract The massive amount of human biological, imaging and clinical data produced by multiple and diverse sources, necessitates integrative modeling approaches able to summarize all this information into answers to specific clinical questions. In this paper, we present a hypermodeling scheme able to combine models of diverse cancer aspects regardless of their underlying method or scale. Describing tissue-scale cancer cell proliferation, biomechanical tumor growth, nutrient transport, genomic-scale aberrant cancer cell metabolism, and cell signaling pathways that regulate the cellular response to therapy, the hypermodel integrates mutation, miRNA expression, imaging, and clinical data. The constituting hypomodels as well as their orchestration and links are described. Two specific cancer types, Wilms tumor (nephroblastoma) and non-small cell lung cancer, are addressed as proof of concept study cases. Personalized simulations over the actual anatomy of a patient have been carried out. The hypermodel has also applied to predict tumor control after radiotherapy and the relationship between tumor proliferative activity and response to neoadjuvant chemotherapy. Our innovative hypermodel holds promise as a digital twin based clinical decision support system and the core of future in silico trial platforms, although additional retrospective adaptation and validation is necessary.
Computational Biology Radhakrishnan Lab

Toward physics-based precision medicine: Exploiting protein dynamics to design new therapeutics and interpret variants

Meller A, Kelly D, Smith LG, Bowman GR. Toward physics-based precision medicine: Exploiting protein dynamics to design new therapeutics and interpret variants. Protein Sci. 2024 Mar;33(3):e4902. doi: 10.1002/pro.4902. PMID: 38358129; PMCID: PMC10868452.

Abstract

The goal of precision medicine is to utilize our knowledge of the molecular causes of disease to better diagnose and treat patients. However, there is a substantial mismatch between the small number of food and drug administration (FDA)-approved drugs and annotated coding variants compared to the needs of precision medicine. This review introduces the concept of physics-based precision medicine, a scalable framework that promises to improve our understanding of sequence-function relationships and accelerate drug discovery. We show that accounting for the ensemble of structures a protein adopts in solution with computer simulations overcomes many of the limitations imposed by assuming a single protein structure. We highlight studies of protein dynamics and recent methods for the analysis of structural ensembles. These studies demonstrate that differences in conformational distributions predict functional differences within protein families and between variants. Thanks to new computational tools that are providing unprecedented access to protein structural ensembles, this insight may enable accurate predictions of variant pathogenicity for entire libraries of variants. We further show that explicitly accounting for protein ensembles, with methods like alchemical free energy calculations or docking to Markov state models, can uncover novel lead compounds. To conclude, we demonstrate that cryptic pockets, or cavities absent in experimental structures, provide an avenue to target proteins that are currently considered undruggable. Taken together, our review provides a roadmap for the field of protein science to accelerate precision medicine.

Bowman Lab Computational Biology

Bile acid metabolism mediates cholesterol homeostasis and promotes tumorigenesis in clear cell renal cell carcinoma

Riscal R, Gardner SM, Coffey NJ, Carens M, Mesaros C, Xu JP, Xue Y, Davis L, Demczyszyn S, Vogt A, Olia A, Finan JM, Godfrey J, Schultz DC, Blair IA, Keith B, Marmorstein R, Skuli N, Simon MC. Bile acid metabolism mediates cholesterol homeostasis and promotes tumorigenesis in clear cell renal cell carcinoma. Cancer Res. 2024 Feb 28. doi: 10.1158/0008-5472.CAN-23-0821. Epub ahead of print. PMID: 38417134.

Abstract

Clear cell renal cell carcinoma (ccRCC) incidence has risen steadily over the last decade. Elevated lipid uptake and storage is required for ccRCC cell viability. As stored cholesterol is the most abundant component in ccRCC intracellular lipid droplets, it may also play an important role in ccRCC cellular homeostasis. In support of this hypothesis, ccRCC cells acquire exogenous cholesterol through the HDL receptor SCARB1, inhibition or suppression of which induces apoptosis. Here, we showed that elevated expression of 3 beta-hydroxy steroid dehydrogenase type 7 (HSD3B7), which metabolizes cholesterol-derived oxysterols in the bile acid biosynthetic pathway, is also essential for ccRCC cell survival. Development of an HSD3B7 enzymatic assay and screening for small molecule inhibitors uncovered the compound celastrol as a potent HSD3B7 inhibitor with low micromolar activity. Repressing HSD3B7 expression genetically or treating ccRCC cells with celastrol resulted in toxic oxysterol accumulation, impaired proliferation, and increased apoptosis in vitro and in vivo. These data demonstrate that bile acid synthesis regulates cholesterol homeostasis in ccRCC and identifies HSD3B7 as a plausible therapeutic target.

Chemical Biology CryoEM and CryoET Protein Production Services X-ray Crystallography Marmorstein Lab Schultz Lab

SlumberNet: deep learning classification of sleep stages using residual neural networks

Jha PK, Valekunja UK, Reddy AB. SlumberNet: deep learning classification of sleep stages using residual neural networks. Sci Rep. 2024 Feb 27;14(1):4797. doi: 10.1038/s41598-024-54727-0. PMID: 38413666; PMCID: PMC10899258.

Abstract

Sleep research is fundamental to understanding health and well-being, as proper sleep is essential for maintaining optimal physiological function. Here we present SlumberNet, a novel deep learning model based on residual network (ResNet) architecture, designed to classify sleep states in mice using electroencephalogram (EEG) and electromyogram (EMG) signals. Our model was trained and tested on data from mice undergoing baseline sleep, sleep deprivation, and recovery sleep, enabling it to handle a wide range of sleep conditions. Employing k-fold cross-validation and data augmentation techniques, SlumberNet achieved high levels of overall performance (accuracy = 97%; F1 score = 96%) in predicting sleep stages and showed robust performance even with a small and diverse training dataset. Comparison of SlumberNet’s performance to manual sleep stage classification revealed a significant reduction in analysis time (~ 50 × faster), without sacrificing accuracy. Our study showcases the potential of deep learning to facilitate sleep research by providing a more efficient, accurate, and scalable method for sleep stage classification. Our work with SlumberNet further demonstrates the power of deep learning in mouse sleep research.

CryoEM and CryoET Mass Spectrometry Reddy Lab

Image Denoising of Low-Dose PET Mouse Scans with Deep Learning: Validation Study for Preclinical Imaging Applicability

Muller FM, Vervenne B, Maebe J, Blankemeyer E, Sellmyer MA, Zhou R, Karp JS, Vanhove C, Vandenberghe S. Image Denoising of Low-Dose PET Mouse Scans with Deep Learning: Validation Study for Preclinical Imaging Applicability. Mol Imaging Biol. 2024 Feb;26(1):101-113. doi: 10.1007/s11307-023-01866-x. Epub 2023 Oct 24. PMID: 37875748.

Abstract

Purpose: Positron emission tomography (PET) image quality can be improved by higher injected activity and/or longer acquisition time, but both may often not be practical in preclinical imaging. Common preclinical radioactive doses (10 MBq) have been shown to cause deterministic changes in biological pathways. Reducing the injected tracer activity and/or shortening the scan time inevitably results in low-count acquisitions which poses a challenge because of the inherent noise introduction. We present an image-based deep learning (DL) framework for denoising lower count micro-PET images.

Chemical Biology Sellmyer Lab

DDX3X and DDX3Y constitutively form nano-sized RNA-protein clusters that foster enzymatic activity

Yanas A, Shweta H, Owens MC, Liu KF, Goldman YE. DDX3X and DDX3Y constitutively form nano-sized RNA-protein clusters that foster enzymatic activity. bioRxiv [Preprint]. 2023 Nov 29:2023.11.29.569239. doi: 10.1101/2023.11.29.569239. PMID: 38077005; PMCID: PMC10705435.

Abstract

DEAD-box helicases, which are crucial for many aspects of RNA metabolism, often contain intrinsically disordered regions (IDRs), whose functions remain unclear. Using multiparameter confocal microscopy, we reveal that sex chromosome-encoded homologous RNA helicases, DDX3X and DDX3Y, form nano-sized RNA-protein clusters (RPCs) that foster their catalytic activities in vitro and in cells. The IDRs are critical for the formation of these RPCs. A thorough analysis of the catalytic cycle of DDX3X and DDX3Y by ensemble biochemistry and single molecule photon bursts in the confocal microscope showed that RNA release is a major step that differentiates the unwinding activities of DDX3X and DDX3Y. Our findings provide new insights that the nano-sized helicase RPCs may be the normal state of these helicases under non-stressed conditions that promote their RNA unwinding and act as nucleation points for liquid-liquid phase separation under stress. This mechanism may apply broadly among other members of the DEAD-box helicase family.

Mass Spectrometry X-ray Crystallography Liu Lab

Simple visualization of submicroscopic protein clusters with a phase-separation-based fluorescent reporter

Mumford TR, Rae D, Brackhahn E, Idris A, Gonzalez-Martinez D, Pal AA, Chung MC, Guan J, Rhoades E, Bugaj LJ. Simple visualization of submicroscopic protein clusters with a phase-separation-based fluorescent reporter. Cell Syst. 2024 Feb 21;15(2):166-179.e7. doi: 10.1016/j.cels.2024.01.005. Epub 2024 Feb 8. PMID: 38335954.

Abstract

Protein clustering plays numerous roles in cell physiology and disease. However, protein oligomers can be difficult to detect because they are often too small to appear as puncta in conventional fluorescence microscopy. Here, we describe a fluorescent reporter strategy that detects protein clusters with high sensitivity called CluMPS (clusters magnified by phase separation). A CluMPS reporter detects and visually amplifies even small clusters of a binding partner, generating large, quantifiable fluorescence condensates. We use computational modeling and optogenetic clustering to demonstrate that CluMPS can detect small oligomers and behaves rationally according to key system parameters. CluMPS detected small aggregates of pathological proteins where the corresponding GFP fusions appeared diffuse. CluMPS also detected and tracked clusters of unmodified and tagged endogenous proteins, and orthogonal CluMPS probes could be multiplexed in cells. CluMPS provides a powerful yet straightforward approach to observe higher-order protein assembly in its native cellular context. A record of this paper’s transparent peer review process is included in the supplemental information.

Single Molecule Imaging Rhoades Lab

Homologous mutations in human β, embryonic, and perinatal muscle myosins have divergent effects on molecular power generation

Liu C, Karabina A, Meller A, Bhattacharjee A, Agostino CJ, Bowman GR, Ruppel KM, Spudich JA, Leinwand LA. Homologous mutations in human β, embryonic, and perinatal muscle myosins have divergent effects on molecular power generation. Proc Natl Acad Sci U S A. 2024 Feb 27;121(9):e2315472121. doi: 10.1073/pnas.2315472121. Epub 2024 Feb 20. PMID: 38377203; PMCID: PMC10907259.

Abstract

Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in β-cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman-Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus-pseudocamptodactyly syndrome. It is not known whether their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human β, embryonic, and perinatal myosin subfragment-1. We found large effects in the developmental myosins but minimal effects in β myosin, and magnitude of changes correlated partially with clinical severity. The mutations in the developmental myosins dramatically decreased the step size and load-sensitive actin-detachment rate of single molecules measured by optical tweezers, in addition to decreasing overall enzymatic (ATPase) cycle rate. In contrast, the only measured effect of R671C in β myosin was a larger step size. Our measurements of step size and bound times predicted velocities consistent with those measured in an in vitro motility assay. Finally, molecular dynamics simulations predicted that the arginine to cysteine mutation in embryonic, but not β, myosin may reduce pre-powerstroke lever arm priming and ADP pocket opening, providing a possible structural mechanism consistent with the experimental observations. This paper presents direct comparisons of homologous mutations in several different myosin isoforms, whose divergent functional effects are a testament to myosin’s highly allosteric nature.

Bowman Lab Computational Biology

Computational Design of Peptides for Biomaterials Applications

Wang Y, Stebe KJ, de la Fuente-Nunez C, Radhakrishnan R. Computational Design of Peptides for Biomaterials Applications. ACS Appl Bio Mater. 2024 Feb 19;7(2):617-625. doi: 10.1021/acsabm.2c01023. Epub 2023 Mar 27. PMID: 36971822.

Abstract

Computer-aided molecular design and protein engineering emerge as promising and active subjects in bioengineering and biotechnological applications. On one hand, due to the advancing computing power in the past decade, modeling toolkits and force fields have been put to use for accurate multiscale modeling of biomolecules including lipid, protein, carbohydrate, and nucleic acids. On the other hand, machine learning emerges as a revolutionary data analysis tool that promises to leverage physicochemical properties and structural information obtained from modeling in order to build quantitative protein structure-function relationships. We review recent computational works that utilize state-of-the-art computational methods to engineer peptides and proteins for various emerging biomedical, antimicrobial, and antifreeze applications. We also discuss challenges and possible future directions toward developing a roadmap for efficient biomolecular design and engineering.

Computational Biology Radhakrishnan Lab

Optical Control of Proteasomal Protein Degradation with a Photoswitchable Lipopeptide

Morstein J, Amatuni A, Shuster A, Kuttenlochner W, Ko T, Abegg D, Groll M, Adibekian A, Renata H, Trauner DH. Optical Control of Proteasomal Protein Degradation with a Photoswitchable Lipopeptide. Angew Chem Int Ed Engl. 2024 Feb 19;63(8):e202314791. doi: 10.1002/anie.202314791. Epub 2024 Jan 16. PMID: 38109686.

Abstract

Photolipids have emerged as attractive tools for the optical control of lipid functions. They often contain an azobenzene photoswitch that imparts a cis double-bond upon irradiation. Herein, we present the application of photoswitching to a lipidated natural product, the potent proteasome inhibitor cepafungin I. Several azobenzene-containing lipids were attached to the cyclopeptide core, yielding photoswitchable derivatives. Most notably, PhotoCep4 exhibited a 10-fold higher cellular potency in its light-induced cis-form, matching the potency of natural cepafungin I. The length of the photolipid tail and distal positioning of the azobenzene photoswitch with respect to the macrocycle is critical for this activity. In a proteome-wide experiment, light-triggered PhotoCep4 modulation showed high overlap with constitutively active cepafungin I. The mode of action was studied using crystallography and revealed an identical binding of the cyclopeptide in comparison to cepafungin I, suggesting that differences in their cellular activity originate from switching the tail structure. The photopharmacological approach described herein could be applicable to many other natural products as lipid conjugation is common and often necessary for potent activity. Such lipids are often introduced late in synthetic routes, enabling facile chemical modifications.

Chemical Biology CryoEM and CryoET Trauner Group

Enterovirus evolution reveals the mechanism of an RNA-targeted antiviral and determinants of viral replication

Davila-Calderon J, Li ML, Penumutchu SR, Haddad C, Malcolm L, King J, Hargrove AE, Brewer G, Tolbert BS. Enterovirus evolution reveals the mechanism of an RNA-targeted antiviral and determinants of viral replication. Sci Adv. 2024 Feb 16;10(7):eadg3060. doi: 10.1126/sciadv.adg3060. Epub 2024 Feb 16. PMID: 38363831; PMCID: PMC10871541.

Abstract

Selective pressures on viruses provide opportunities to establish target site specificity and mechanisms of antivirals. Enterovirus (EV)-A71 with resistant mutations in the stem loop (SL) II internal ribosome entry site (IRES) (SLIIresist) were selected at low doses of the antiviral dimethylamiloride (DMA)-135. The EV-A71 mutants were resistant to DMA-135 at concentrations that inhibit replication of wild-type virus. EV-A71 IRES structures harboring resistant mutations induced efficient expression of Luciferase messenger RNA in the presence of noncytotoxic doses of DMA-135. Nuclear magnetic resonance indicates that the mutations change the structure of SLII at the binding site of DMA-135 and at the surface recognized by the host protein AU-rich element/poly(U)-binding/degradation factor 1 (AUF1). Biophysical studies of complexes formed between AUF1, DMA-135, and either SLII or SLIIresist show that DMA-135 stabilizes a ternary complex with AUF1-SLII but not AUF1-SLIIresist. This work demonstrates how viral evolution elucidates the (DMA-135)-RNA binding site specificity in cells and provides insights into the viral pathways inhibited by the antiviral.

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CryoEM and CryoET NMR Tolbert Lab

PopShift: A Thermodynamically Sound Approach to Estimate Binding Free Energies by Accounting for Ligand-Induced Population Shifts from a Ligand-Free Markov State Model

Smith LG, Novak B, Osato M, Mobley DL, Bowman GR. PopShift: A Thermodynamically Sound Approach to Estimate Binding Free Energies by Accounting for Ligand-Induced Population Shifts from a Ligand-Free Markov State Model. J Chem Theory Comput. 2024 Feb 13;20(3):1036-1050. doi: 10.1021/acs.jctc.3c00870. Epub 2024 Jan 31. PMID: 38291966; PMCID: PMC10867841.

Abstract

Obtaining accurate binding free energies from in silico screens has been a long-standing goal for the computational chemistry community. However, accuracy and computational cost are at odds with one another, limiting the utility of methods that perform this type of calculation. Many methods achieve massive scale by explicitly or implicitly assuming that the target protein adopts a single structure, or undergoes limited fluctuations around that structure, to minimize computational cost. Others simulate each protein-ligand complex of interest, accepting lower throughput in exchange for better predictions of binding affinities. Here, we present the PopShift framework for accounting for the ensemble of structures a protein adopts and their relative probabilities. Protein degrees of freedom are enumerated once, and then arbitrarily many molecules can be screened against this ensemble. Specifically, we use Markov state models (MSMs) as a compressed representation of a protein’s thermodynamic ensemble. We start with a ligand-free MSM and then calculate how addition of a ligand shifts the populations of each protein conformational state based on the strength of the interaction between that protein conformation and the ligand. In this work we use docking to estimate the affinity between a given protein structure and ligand, but any estimator of binding affinities could be used in the PopShift framework. We test PopShift on the classic benchmark pocket T4 Lysozyme L99A. We find that PopShift is more accurate than common strategies, such as docking to a single structure and traditional ensemble docking─producing results that compare favorably with alchemical binding free energy calculations in terms of RMSE but not correlation─and may have a more favorable computational cost profile in some applications.

Bowman Lab Computational Biology

Cancer cell mechanotypes dependent on ECM properties drive tumor progression through extracellular vesicle production

Cancer cell mechanotypes dependent on ECM properties drive tumor progression through extracellular vesicle production

Kshitiz Parihar, Jonathan Nukpezah, Phyoe K Myint, Di-Ao Liu, Ghmkin Hassan, Tobias Baumgart, Valerie M Weaver, Wei Guo, Paul A Janmey, Ravi Radhakrishnan

 

Computational Biology Radhakrishnan Lab

Myosin-I Synergizes with Arp2/3 Complex to Enhance Pushing Forces of Branched Actin Networks

Xu M, Rutkowski DM, Rebowski G, Boczkowska M, Pollard LW, Dominguez R, Vavylonis D, Ostap EM. Myosin-I Synergizes with Arp2/3 Complex to Enhance Pushing Forces of Branched Actin Networks. bioRxiv [Preprint]. 2024 Feb 12:2024.02.09.579714. doi: 10.1101/2024.02.09.579714. PMID: 38405741; PMCID: PMC10888859.

Abstract

Myosin-Is colocalize with Arp2/3 complex-nucleated actin networks at sites of membrane protrusion and invagination, but the mechanisms by which myosin-I motor activity coordinates with branched actin assembly to generate force are unknown. We mimicked the interplay of these proteins using the “comet tail” bead motility assay, where branched actin networks are nucleated by Arp2/3 complex on the surface of beads coated with myosin-I and the WCA domain of N-WASP. We observed that myosin-I increased bead movement efficiency by thinning actin networks without affecting growth rates. Remarkably, myosin-I triggered symmetry breaking and comet-tail formation in dense networks resistant to spontaneous fracturing. Even with arrested actin assembly, myosin-I alone could break the network. Computational modeling recapitulated these observations suggesting myosin-I acts as a repulsive force shaping the network’s architecture and boosting its force-generating capacity. We propose that myosin-I leverages its power stroke to amplify the forces generated by Arp2/3 complex-nucleated actin networks.

CryoEM and CryoET Mass Spectrometry Dominguez Lab X-ray Crystallography Ostap Lab

Sequential CRISPR screening reveals partial NatB inhibition as a strategy to mitigate alpha-synuclein levels in human neurons

Santhosh Kumar S, Naseri NN, Pather SR, Hallacli E, Ndayisaba A, Buenaventura C, Acosta K, Roof J, Fazelinia H, Spruce LA, Luk K, Khurana V, Rhoades E, Shalem O. Sequential CRISPR screening reveals partial NatB inhibition as a strategy to mitigate alpha-synuclein levels in human neurons. Sci Adv. 2024 Feb 9;10(6):eadj4767. doi: 10.1126/sciadv.adj4767. Epub 2024 Feb 9. PMID: 38335281; PMCID: PMC10857481.

Abstract

Alpha-synuclein (αSyn) protein levels correlate with the risk and severity of Parkinson’s disease and related neurodegenerative diseases. Lowering αSyn is being actively investigated as a therapeutic modality. Here, we systematically map the regulatory network that controls endogenous αSyn using sequential CRISPR-knockout and -interference screens in an αSyn gene (SNCA)-tagged cell line and induced pluripotent stem cell-derived neurons (iNeurons). We uncover αSyn modifiers at multiple regulatory layers, with amino-terminal acetyltransferase B (NatB) enzymes being the most potent endogenous αSyn modifiers in both cell lines. Amino-terminal acetylation protects the cytosolic αSyn from rapid degradation by the proteasome in a Ube2w-dependent manner. Moreover, we show that pharmacological inhibition of methionyl-aminopeptidase 2, a regulator of NatB complex formation, attenuates endogenous αSyn in iNeurons carrying SNCA triplication. Together, our study reveals several gene networks that control endogenous αSyn, identifies mechanisms mediating the degradation of nonacetylated αSyn, and illustrates potential therapeutic pathways for decreasing αSyn levels in synucleinopathies.

Single Molecule Imaging Rhoades Lab

Decoding the cell nucleus

Kimura H, Lakadamyali M. Decoding the cell nucleus. Curr Opin Cell Biol. 2024 Feb;86:102319. doi: 10.1016/j.ceb.2023.102319. Epub 2024 Jan 13. PMID: 38219526.

 

Live Organoid Cyclic Imaging

Abstract

Organoids are becoming increasingly relevant in biology and medicine for their physiological complexity and accuracy in modeling human disease. To fully assess their biological profile while preserving their spatial information, spatiotemporal imaging tools are warranted. While previously developed imaging techniques, such as four-dimensional (4D) live imaging and light-sheet imaging have yielded important clinical insights, these technologies lack the combination of cyclic and multiplexed analysis. To address these challenges, bioorthogonal click chemistry is applied to display the first demonstration of multiplexed cyclic imaging of live and fixed patient-derived glioblastoma tumor organoids. This technology exploits bioorthogonal click chemistry to quench fluorescent signals from the surface and intracellular of labeled cells across multiple cycles, allowing for more accurate and efficient molecular profiling of their complex phenotypes. Herein, the versatility of this technology is demonstrated for the screening of glioblastoma markers in patient-derived human glioblastoma organoids while conserving their viability. It is anticipated that the findings and applications of this work can be broadly translated into investigating physiological developments in other organoid systems.

Chemical Biology Sellmyer Lab

TXNRD1 drives the innate immune response in senescent cells with implications for age-associated inflammation

Hao X, Zhao B, Towers M, Liao L, Monteiro EL, Xu X, Freeman C, Peng H, Tang HY, Havas A, Kossenkov AV, Berger SL, Adams PD, Speicher DW, Schultz D, Marmorstein R, Zaret KS, Zhang R. TXNRD1 drives the innate immune response in senescent cells with implications for age-associated inflammation. Nat Aging. 2024 Feb;4(2):185-197. doi: 10.1038/s43587-023-00564-1. Epub 2024 Jan 24. PMID: 38267705.

Abstract

Sterile inflammation, also known as ‘inflammaging’, is a hallmark of tissue aging. Cellular senescence contributes to tissue aging, in part, through the secretion of proinflammatory factors collectively known as the senescence-associated secretory phenotype (SASP). The genetic variability of thioredoxin reductase 1 (TXNRD1) is associated with aging and age-associated phenotypes such as late-life survival, activity of daily living and physical performance in old age. TXNRD1’s role in regulating tissue aging has been attributed to its enzymatic role in cellular redox regulation. Here, we show that TXNRD1 drives the SASP and inflammaging through the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) innate immune response pathway independently of its enzymatic activity. TXNRD1 localizes to cytoplasmic chromatin fragments and interacts with cGAS in a senescence-status-dependent manner, which is necessary for the SASP. TXNRD1 enhances the enzymatic activity of cGAS. TXNRD1 is required for both the tumor-promoting and immune surveillance functions of senescent cells, which are mediated by the SASP in vivo in mouse models. Treatment of aged mice with a TXNRD1 inhibitor that disrupts its interaction with cGAS, but not with an inhibitor of its enzymatic activity alone, downregulated markers of inflammaging in several tissues. In summary, our results show that TXNRD1 promotes the SASP through the innate immune response, with implications for inflammaging. This suggests that the TXNRD1-cGAS interaction is a relevant target for selectively suppressing inflammaging.

Chemical Biology CryoEM and CryoET X-ray Crystallography Marmorstein Lab

Toward a comprehensive view of gene architecture during transcription

Woodworth MA, Lakadamyali M. Toward a comprehensive view of gene architecture during transcription. Curr Opin Genet Dev. 2024 Feb 2;85:102154. doi: 10.1016/j.gde.2024.102154. Epub ahead of print. PMID: 38309073.

Abstract

The activation of genes within the nucleus of eukaryotic cells is a tightly regulated process, orchestrated by a complex interplay of various physical properties and interacting factors. Studying the multitude of components and features that collectively contribute to gene activation has proven challenging due to the complexities of simultaneously visualizing the dynamic and transiently interacting elements that coalesce within the small space occupied by each individual gene. However, various labeling and imaging advances are now starting to overcome this challenge, enabling visualization of gene activation at different lengths and timescales. In this review, we aim to highlight these microscopy-based advances and suggest how they can be combined to provide a comprehensive view of the mechanisms regulating gene activation.

Single Molecule Imaging Lakadamyali Lab

Expanded palette of RNA base editors for comprehensive RBP-RNA interactome studies

Medina-Munoz HC, Kofman E, Jagannatha P, Boyle EA, Yu T, Jones KL, Mueller JR, Lykins GD, Doudna AT, Park SS, Blue SM, Ranzau BL, Kohli RM, Komor AC, Yeo GW. Expanded palette of RNA base editors for comprehensive RBP-RNA interactome studies. Nat Commun. 2024 Jan 29;15(1):875. doi: 10.1038/s41467-024-45009-4. PMID: 38287010; PMCID: PMC10825223.

Abstract

RNA binding proteins (RBPs) are key regulators of RNA processing and cellular function. Technologies to discover RNA targets of RBPs such as TRIBE (targets of RNA binding proteins identified by editing) and STAMP (surveying targets by APOBEC1 mediated profiling) utilize fusions of RNA base-editors (rBEs) to RBPs to circumvent the limitations of immunoprecipitation (CLIP)-based methods that require enzymatic digestion and large amounts of input material. To broaden the repertoire of rBEs suitable for editing-based RBP-RNA interaction studies, we have devised experimental and computational assays in a framework called PRINTER (protein-RNA interaction-based triaging of enzymes that edit RNA) to assess over thirty A-to-I and C-to-U rBEs, allowing us to identify rBEs that expand the characterization of binding patterns for both sequence-specific and broad-binding RBPs. We also propose specific rBEs suitable for dual-RBP applications. We show that the choice between single or multiple rBEs to fuse with a given RBP or pair of RBPs hinges on the editing biases of the rBEs and the binding preferences of the RBPs themselves. We believe our study streamlines and enhances the selection of rBEs for the next generation of RBP-RNA target discovery.

Chemical Biology CryoEM and CryoET Kohli Lab

Fumarate respiration of Fasciola flukes as a potential drug target

Tashibu A, Inaoka DK, Sakamoto K, Murakami K, Zannatul F, Kita K, Ichikawa-Seki M. Fumarate respiration of Fasciola flukes as a potential drug target. Front Cell Infect Microbiol. 2024 Jan 25;13:1302114. doi: 10.3389/fcimb.2023.1302114. PMID: 38332950; PMCID: PMC10850294.

Abstract

Fascioliasis is a neglected tropical zoonotic disease caused by liver flukes belonging to the genus Fasciola. The emergence of resistance to triclabendazole, the only World Health Organization-recommended drug for this disease, highlights the need for the development of new drugs. Helminths possess an anaerobic mitochondrial respiratory chain (fumarate respiration) which is considered a potential drug target. This study aimed to evaluate the occurrence of fumarate respiration in Fasciola flukes. We analyzed the properties of the respiratory chain of Fasciola flukes in both adults and newly excysted juveniles (NEJs). Fasciola flukes travel and mature through the stomach, bowel, and abdominal cavity to the liver, where oxygen levels gradually decline. High fumarate reductase activity was observed in the mitochondrial fraction of adult Fasciola flukes. Furthermore, rhodoquinone-10 (RQ10 Em’= -63 mV), a low-potential electron mediator used in fumarate respiration was found to be predominant in adults. In contrast, the activity of oxygen respiration was low in adults. Rotenone, atpenin A5, and ascochlorin, typical inhibitors of mitochondrial enzymes in complexes I, II, and III, respectively, inhibit the activity of each enzyme in the adult mitochondrial fraction. These inhibitors were then used for in vitro viability tests of NEJs. Under aerobic conditions, NEJs were killed by rotenone or ascochlorin, which inhibit aerobic respiration (complex I-III), whereas atpenin A5, which inhibits complex II involved in fumarate respiration, did not affect NEJs. Moreover, ubiquinone-10 (UQ10 Em’= +110 mV), which is used in oxidative respiration, was detected in NEJs, in addition to RQ10.

CryoEM and CryoET Mass Spectrometry Murakami Lab

Cooperativity between Cas9 and hyperactive AID establishes broad and diversifying mutational footprints in base editors

Berríos KN, Barka A, Gill J, Serrano JC, Bailer PF, Parker JB, Evitt NH, Gajula KS, Shi J, Kohli RM. Cooperativity between Cas9 and hyperactive AID establishes broad and diversifying mutational footprints in base editors. Nucleic Acids Res. 2024 Jan 23:gkae024. doi: 10.1093/nar/gkae024. Epub ahead of print. PMID: 38261989.

Abstract

The partnership of DNA deaminase enzymes with CRISPR-Cas nucleases is now a well-established method to enable targeted genomic base editing. However, an understanding of how Cas9 and DNA deaminases collaborate to shape base editor (BE) outcomes has been lacking. Here, we support a novel mechanistic model of base editing by deriving a range of hyperactive activation-induced deaminase (AID) base editors (hBEs) and exploiting their characteristic diversifying activity. Our model involves multiple layers of previously underappreciated cooperativity in BE steps including: (i) Cas9 binding can potentially expose both DNA strands for ‘capture’ by the deaminase, a feature that is enhanced by guide RNA mismatches; (ii) after strand capture, the intrinsic activity of the DNA deaminase can tune window size and base editing efficiency; (iii) Cas9 defines the boundaries of editing on each strand, with deamination blocked by Cas9 binding to either the PAM or the protospacer and (iv) non-canonical edits on the guide RNA bound strand can be further elicited by changing which strand is nicked by Cas9. Leveraging insights from our mechanistic model, we create novel hBEs that can remarkably generate simultaneous C > T and G > A transitions over >65 bp with significant potential for targeted gene diversification

Chemical Biology CryoEM and CryoET Kohli Lab

Structure of the prenyltransferase in bifunctional copalyl diphosphate synthase from Penicillium fellutanum reveals an open hexamer conformation

Gaynes MN, Ronnebaum TA, Schultz K, Faylo JL, Marmorstein R, Christianson DW. Structure of the prenyltransferase in bifunctional copalyl diphosphate synthase from Penicillium fellutanum reveals an open hexamer conformation. J Struct Biol. 2024 Jan 5;216(1):108060. doi: 10.1016/j.jsb.2023.108060. Epub ahead of print. PMID: 38184156.

Abstract

Copalyl diphosphate synthase from Penicillium fellutanum (PfCPS) is an assembly-line terpene synthase that contains both prenyltransferase and class II cyclase activities. The prenyltransferase catalyzes processive chain elongation reactions using dimethylallyl diphosphate and three equivalents of isopentenyl diphosphate to yield geranylgeranyl diphosphate, which is then utilized as a substrate by the class II cyclase domain to generate copalyl diphosphate. Here, we report the 2.81 Å-resolution cryo-EM structure of the hexameric prenyltransferase of full-length PfCPS, which is surrounded by randomly splayed-out class II cyclase domains connected by disordered polypeptide linkers. The hexamer can be described as a trimer of dimers; surprisingly, one of the three dimer-dimer interfaces is separated to yield an open hexamer conformation, thus breaking the D3 symmetry typically observed in crystal structures of other prenyltransferase hexamers such as wild-type human GGPP synthase (hGGPPS). Interestingly, however, an open hexamer conformation was previously observed in the crystal structure of D188Y hGGPPS, apparently facilitated by hexamer-hexamer packing in the crystal lattice. The cryo-EM structure of the PfCPS prenyltransferase hexamer is the first to reveal that an open conformation can be achieved even in the absence of a point mutation or interaction with another hexamer. Even though PfCPS octamers are not detected, we suggest that the open hexamer conformation represents an intermediate in the hexamer-octamer equilibrium for those prenyltransferases that do exhibit oligomeric heterogeneity.

Chemical Biology CryoEM and CryoET Christianson Group X-ray Crystallography Marmorstein Lab

Molecular details of ruthenium red pore block in TRPV channels

Pumroy RA, De Jesús-Pérez JJ, Protopopova AD, Rocereta JA, Fluck EC, Fricke T, Lee BH, Rohacs T, Leffler A, Moiseenkova-Bell V. Molecular details of ruthenium red pore block in TRPV channels. EMBO Rep. 2024 Jan 15. doi: 10.1038/s44319-023-00050-0. Epub ahead of print. PMID: 38225355.

Abstract

Transient receptor potential vanilloid (TRPV) channels play a critical role in calcium homeostasis, pain sensation, immunological response, and cancer progression. TRPV channels are blocked by ruthenium red (RR), a universal pore blocker for a wide array of cation channels. Here we use cryo-electron microscopy to reveal the molecular details of RR block in TRPV2 and TRPV5, members of the two TRPV subfamilies. In TRPV2 activated by 2-aminoethoxydiphenyl borate, RR is tightly coordinated in the open selectivity filter, blocking ion flow and preventing channel inactivation. In TRPV5 activated by phosphatidylinositol 4,5-bisphosphate, RR blocks the selectivity filter and closes the lower gate through an interaction with polar residues in the pore vestibule. Together, our results provide a detailed understanding of TRPV subfamily pore block, the dynamic nature of the selectivity filter and allosteric communication between the selectivity filter and lower gate.

CryoEM and CryoET Mass Spectrometry Vera Moiseenkova-Bell Lab

Characterization and genomic analysis of the Lyme disease spirochete bacteriophage ϕBB-1

Faith DR, Kinnersley M, Brooks DM, Drecktrah D, Hall LS, Luo E, Santiago-Frangos A, Wachter J, Samuels DS, Secor PR. Characterization and genomic analysis of the Lyme disease spirochete bacteriophage ϕBB-1. bioRxiv [Preprint]. 2024 Jan 13:2024.01.08.574763. doi: 10.1101/2024.01.08.574763. PMID: 38260690; PMCID: PMC10802411.

Abstract

Lyme disease is a tick-borne infection caused by the spirochete Borrelia (BorreliellaburgdorferiBorrelia species have highly fragmented genomes composed of a linear chromosome and a constellation of linear and circular plasmids some of which are required throughout the enzootic cycle. Included in this plasmid repertoire by almost all Lyme disease spirochetes are the 32-kb circular plasmid cp32 prophages that are capable of lytic replication to produce infectious virions called ϕBB-1. While the B. burgdorferi genome contains evidence of horizontal transfer, the mechanisms of gene transfer between strains remain unclear. While we know that ϕBB-1 transduces cp32 and shuttle vector DNA during in vitro cultivation, the extent of ϕBB-1 DNA transfer is not clear. Herein, we use proteomics and long-read sequencing to further characterize ϕBB-1 virions. Our studies identified the cp32 pac region and revealed that ϕBB-1 packages linear cp32s via a headful mechanism with preferentially packaging of plasmids containing the cp32 pac region. Additionally, we find ϕBB-1 packages fragments of the linear chromosome and full-length plasmids including lp54, cp26, and others. Furthermore, sequencing of ϕBB-1 packaged DNA allowed us to resolve the covalently closed hairpin telomeres for the linear B. burgdorferi chromosome and most linear plasmids in strain CA-11.2A. Collectively, our results shed light on the biology of the ubiquitous ϕBB-1 phage and further implicates ϕBB-1 in the generalized transduction of diverse genes and the maintenance of genetic diversity in Lyme disease spirochetes.

Computational Biology CryoEM and CryoET Santiago-Frangos Lab

Deep learning the structural determinants of protein biochemical properties by comparing structural ensembles with DiffNets

Ward MD, Zimmerman MI, Meller A, Chung M, Swamidass SJ, Bowman GR. Deep learning the structural determinants of protein biochemical properties by comparing structural ensembles with DiffNets. Nat Commun. 2021 May 21;12(1):3023. doi: 10.1038/s41467-021-23246-1. PMID: 34021153; PMCID: PMC8140102.

Abstract

Understanding the structural determinants of a protein’s biochemical properties, such as activity and stability, is a major challenge in biology and medicine. Comparing computer simulations of protein variants with different biochemical properties is an increasingly powerful means to drive progress. However, success often hinges on dimensionality reduction algorithms for simplifying the complex ensemble of structures each variant adopts. Unfortunately, common algorithms rely on potentially misleading assumptions about what structural features are important, such as emphasizing larger geometric changes over smaller ones. Here we present DiffNets, self-supervised autoencoders that avoid such assumptions, and automatically identify the relevant features, by requiring that the low-dimensional representations they learn are sufficient to predict the biochemical differences between protein variants. For example, DiffNets automatically identify subtle structural signatures that predict the relative stabilities of β-lactamase variants and duty ratios of myosin isoforms. DiffNets should also be applicable to understanding other perturbations, such as ligand binding.

Bowman Lab Computational Biology

Sustained rhoptry docking and discharge requires Toxoplasma gondii intraconoidal microtubule-associated proteins

Dos Santos Pacheco N, Tell I Puig A, Guérin A, Martinez M, Maco B, Tosetti N, Delgado-Betancourt E, Lunghi M, Striepen B, Chang YW, Soldati-Favre D. Sustained rhoptry docking and discharge requires Toxoplasma gondii intraconoidal microtubule-associated proteins. Nat Commun. 2024 Jan 9;15(1):379. doi: 10.1038/s41467-023-44631-y. PMID: 38191574; PMCID: PMC10774369.

Abstract

In Apicomplexa, rhoptry discharge is essential for invasion and involves an apical vesicle (AV) docking one or two rhoptries to a macromolecular secretory apparatus. Toxoplasma gondii is armed with 10-12 rhoptries and 5-6 microtubule-associated vesicles (MVs) presumably for iterative rhoptry discharge. Here, we have addressed the localization and functional significance of two intraconoidal microtubule (ICMT)-associated proteins instrumental for invasion. Mechanistically, depletion of ICMAP2 leads to a dissociation of the ICMTs, their detachment from the conoid and dispersion of MVs and rhoptries. ICMAP3 exists in two isoforms that contribute to the control of the ICMTs length and the docking of the two rhoptries at the AV, respectively. This study illuminates the central role ICMTs play in scaffolding the discharge of multiple rhoptries. This process is instrumental for virulence in the mouse model of infection and in addition promotes sterile protection against T. gondii via the release of key effectors inducing immunity.

CryoEM and CryoET Mass Spectrometry Yi-Wei Chang Lab

Viral proteins activate PARIS-mediated tRNA degradation and viral tRNAs rescue infection

Burman N, Belukhina S, Depardieu F, Wilkinson RA, Skutel M, Santiago-Frangos A, Graham AB, Livenskyi A, Chechenina A, Morozova N, Zahl T, Henriques WS, Buyukyoruk M, Rouillon C, Shyrokova L, Kurata T, Hauryliuk V, Severinov K, Groseille J, Thierry A, Koszul R, Tesson F, Bernheim A, Bikard D, Wiedenheft B, Isaev A. Viral proteins activate PARIS-mediated tRNA degradation and viral tRNAs rescue infection. bioRxiv [Preprint]. 2024 Jan 9:2024.01.02.573894. doi: 10.1101/2024.01.02.573894. PMID: 38260645; PMCID: PMC10802454.

Abstract

Viruses compete with each other for limited cellular resources, and some viruses deliver defense mechanisms that protect the host from competing genetic parasites. PARIS is a defense system, often encoded in viral genomes, that is composed of a 53 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB). Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-EM to determine the structure of this complex which explains how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving the host tRNALys. Phage T5 subverts PARIS immunity through expression of a tRNALys variant that prevents PARIS-mediated cleavage, and thereby restores viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids.

Computational Biology CryoEM and CryoET Santiago-Frangos Lab

Combining per-particle cryo-ET and cryo-EM single particle analysis to elucidate heterogeneous DNA-protein organization

Palao L 3rd, Murakami K, Chang YW. Combining per-particle cryo-ET and cryo-EM single particle analysis to elucidate heterogeneous DNA-protein organization. Curr Opin Struct Biol. 2024 Jan 4;84:102765. doi: 10.1016/j.sbi.2023.102765. Epub ahead of print. PMID: 38181688.

Abstract

Cryo-electron microscopy single particle analysis (cryo-EM SPA) and cryo-electron tomography (cryo-ET) have historically been employed as distinct approaches for investigating molecular structures of disparate sample types, focusing on highly purified biological macromolecules and in situ cellular contexts, respectively. However, these techniques offer inherently complementary structural insights that, when combined, provide a more comprehensive understanding of complex biological systems. For example, if both techniques are applied to the same purified biological macromolecules, cryo-ET has the ability to resolve highly flexible yet strong signal features on an individual target molecule which will not be preserved in the high-resolution cryo-EM SPA results. In this review, we highlight recent achievements utilizing such applications to unveil new insights into the chromatin assembly and activities of DNA-protein assemblies. This convergence of cryo-EM SPA and cryo-ET holds great promise for elucidating new structural aspects of these essential molecular processes.

CryoEM and CryoET Mass Spectrometry Murakami Lab Yi-Wei Chang Lab

In vivo delivery of engineered synthetic DNA-encoded SARS-CoV-2 monoclonal antibodies for pre-exposure prophylaxis in non-human primates

Patel A, Rosenke K, Parzych EM, Feldmann F, Bharti S, Griffin AJ, Schouest B, Lewis M, Choi J, Chokkalingam N, Machado V, Smith BJ, Frase D, Ali AR, Lovaglio J, Nguyen B, Hanley PW, Walker SN, Gary EN, Kulkarni A, Generotti A, Francica JR, Rosenthal K, Kulp DW, Esser MT, Smith TRF, Shaia C, Weiner DB, Feldmann H. In vivo delivery of engineered synthetic DNA-encoded SARS-CoV-2 monoclonal antibodies for pre-exposure prophylaxis in non-human primates. Emerg Microbes Infect. 2024 Jan 2:2294860. doi: 10.1080/22221751.2023.2294860. Epub ahead of print. PMID: 38165394.

Abstract

AbstractCOVID-19 remains a major public health concern. Monoclonal antibodies have received emergency use authorization (EUA) for pre-exposure prophylaxis against COVID-19 among high-risk groups for treatment of mild to moderate COVID-19. In addition to recombinant biologics, engineered synthetic DNA-encoded antibodies (DMAb) are an important strategy for direct in vivo delivery of protective mAb. A DMAb cocktail was synthetically engineered to encode the immunoglobulin heavy and light chains of two different two different Fc-engineered anti-SARS-CoV-2 antibodies. The DMAbs were designed to enhance in vivo expression and delivered intramuscularly to cynomolgus and rhesus macaques with a modified in vivo delivery regimen. Serum levels were detected in macaques, along with specific binding to SARS-CoV-2 spike receptor binding domain protein and neutralization of multiple SARS-CoV-2 variants of concern in pseudovirus and authentic live virus assays. Prophylactic administration was protective in rhesus macaques against signs of SARS-CoV-2 (USA-WA1/2020) associated disease in the lungs. Overall, the data support further study of DNA-encoded antibodies as an additional delivery mode for prevention of COVID-19 severe disease. These data have implications for human translation of gene-encoded mAbs for emerging infectious diseases and low dose mAb delivery against COVID-19.Trial registration: ClinicalTrials.gov identifier: NCT05648110..Trial registration: ClinicalTrials.gov identifier: NCT03831503..

Computational Biology CryoEM and CryoET Kulp Lab

Piggybacking functionalized DNA nanostructures into live cell nuclei

Roozbahani GM, Colosi P, Oravecz A, Sorokina EM, Pfeifer W, Shokri S, Wei Y, Didier P, DeLuca M, Arya G, Tora L, Lakadamyali M, Poirier MG, Castro CE. Piggybacking functionalized DNA nanostructures into live cell nuclei. bioRxiv [Preprint]. 2024 Jan 1:2023.12.30.573746. doi: 10.1101/2023.12.30.573746. PMID: 38260628; PMCID: PMC10802371.

Abstract

DNA origami (DO) are promising tools for in vitro or in vivo applications including drug delivery; biosensing, detecting biomolecules; and probing chromatin sub-structures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing visualizing and controlling important biological processes in live cells. Here we present an approach to deliver DO strucures into live cell nuclei. We show that labelled DOs do not undergo detectable structural degradation in cell culture media or human cell extracts for 24 hr. To deliver DO platforms into the nuclei of human U2OS cells, we conjugated 30 nm long DO nanorods with an antibody raised against the largest subunit of RNA Polymerase II (Pol II), a key enzyme involved in gene transcription. We find that DOs remain structurally intact in cells for 24hr, including within the nucleus. Using fluorescence microscopy we demonstrate that the electroporated anti-Pol II antibody conjugated DOs are efficiently piggybacked into nuclei and exihibit sub-diffusive motion inside the nucleus. Our results reveal that functionalizing DOs with an antibody raised against a nuclear factor is a highly effective method for the delivery of nanodevices into live cell nuclei.

Single Molecule Imaging Lakadamyali Lab

The Repurposing of Cellular Proteins during Enterovirus A71 Infection

Abedeera SM, Davila-Calderon J, Haddad C, Henry B, King J, Penumutchu S, Tolbert BS. The Repurposing of Cellular Proteins during Enterovirus A71 Infection. Viruses. 2023 Dec 31;16(1):75. doi: 10.3390/v16010075. PMID: 38257775; PMCID: PMC10821071.

Abstract

Viruses pose a great threat to people’s lives. Enterovirus A71 (EV-A71) infects children and infants all over the world with no FDA-approved treatment to date. Understanding the basic mechanisms of viral processes aids in selecting more efficient drug targets and designing more effective antivirals to thwart this virus. The 5′-untranslated region (5′-UTR) of the viral RNA genome is composed of a cloverleaf structure and an internal ribosome entry site (IRES). Cellular proteins that bind to the cloverleaf structure regulate viral RNA synthesis, while those that bind to the IRES also known as IRES trans-acting factors (ITAFs) regulate viral translation. In this review, we survey the cellular proteins currently known to bind the 5′-UTR and influence viral gene expression with emphasis on comparing proteins’ functions and localizations pre- and post-(EV-A71) infection. A comprehensive understanding of how the host cell’s machinery is hijacked and reprogrammed by the virus to facilitate its replication is crucial for developing effective antivirals.

CryoEM and CryoET NMR Tolbert Lab

Targeting the tissue factor coagulation initiation complex prevents antiphospholipid antibody development

Mueller-Calleja N, Grunz K, Nguyen TS, Posma J, Pedrosa D, Meineck M, Hollerbach A, Braun J, Muth S, Schild H, Saar K, Hübner N, Krishnaswamy S, Royce J, Teyton L, Lemmermann NA, Weinmann-Menke J, Lackner KJ, Ruf W. Targeting the tissue factor coagulation initiation complex prevents antiphospholipid antibody development. Blood. 2023 Dec 24:blood.2023022276. doi: 10.1182/blood.2023022276. Epub ahead of print. PMID: 38142429.

Abstract

Antiphospholipid antibodies (aPL) in primary or secondary antiphospholipid syndrome (APS) are a major cause for acquired thrombophilia, but specific interventions preventing autoimmune aPL development are an unmet clinical need. While autoimmune aPL cross-react with various coagulation regulatory proteins, lipid-reactive and COVID-19 patient-derived aPL recognize the endo-lysosomal phospholipid lysobisphosphatidic acid (LBPA) presented by the cell surface expressed endothelial protein C receptor (EPCR). This specific recognition leads to complement-mediated activation of tissue factor (TF) dependent proinflammatory signaling and thrombosis. Here we show that specific inhibition of the TF coagulation initiation complex with nematode anticoagulant protein c2 (NAPc2) prevents the prothrombotic effects of COVID-19 patient-derived aPL in mice and the aPL-induced proinflammatory and prothrombotic activation of monocytes. The induction of experimental APS is dependent on the NAPDH oxidase complex and NAPc2 suppresses monocyte endosomal reactive oxygen species production requiring the TF cytoplasmic domain and IFNa secretion from dendritic cells. Latent infection with murine cytomegalovirus virus causes TF cytoplasmic domain-dependent development of persistent aPL and circulating phospholipid-reactive B1 cells, which is prevented by short term intervention with NAPc2 during acute viral infection. In addition, treatment of lupus prone MRL-lpr mice with NAPc2, but not with heparin, suppresses dendritic cell activation in the spleen, aPL production and circulating phospholipid-reactive B1 cells and attenuates lupus pathology. These data demonstrate a convergent TF-dependent mechanism of aPL development in latent viral infection and autoimmune disease and provide initial evidence that specific targeting of the TF initiation complex has therapeutic benefits beyond currently used clinical anticoagulant strategies.

CryoEM and CryoET X-ray Crystallography Krishnaswamy Lab

Structural mechanism of TRPV5 inhibition by econazole

De Jesús-Pérez JJ, Gabrielle M, Raheem S, Fluck EC, Rohacs T, Moiseenkova-Bell VY. Structural mechanism of TRPV5 inhibition by econazole. Structure. 2023 Dec 19:S0969-2126(23)00438-0. doi: 10.1016/j.str.2023.11.012. Epub ahead of print. PMID: 38141613.

Abstract

The calcium-selective TRPV5 channel activated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is involved in calcium homeostasis. Recently, cryoelectron microscopy (cryo-EM) provided molecular details of TRPV5 modulation by exogenous and endogenous molecules. However, the details of TRPV5 inhibition by the antifungal agent econazole (ECN) remain elusive due to the low resolution of the currently available structure. In this study, we employ cryo-EM to comprehensively examine how the ECN inhibits TRPV5. By combining our structural findings with site-directed mutagenesis, calcium measurements, electrophysiology, and molecular dynamics simulations, we determined that residues F472 and L475 on the S4 helix, along with residue W495 on the S5 helix, collectively constitute the ECN-binding site. Additionally, the structure of TRPV5 in the presence of ECN and PI(4,5)P2, which does not show the bound activator, reveals a potential inhibition mechanism in which ECN competes with PI(4,5)P2, preventing the latter from binding, and ultimately pore closure.

 

CryoEM and CryoET Mass Spectrometry Vera Moiseenkova-Bell Lab

Delineation of DNA and mRNA COVID-19 vaccine-induced immune responses in preclinical animal models

Andrade VM, Maricic I, Kalia R, Jachimowicz L, Bedoya O, Kulp DW, Humeau L, Smith TRF. Delineation of DNA and mRNA COVID-19 vaccine-induced immune responses in preclinical animal models. Hum Vaccin Immunother. 2023 Dec 15;19(3):2281733. doi: 10.1080/21645515.2023.2281733. Epub 2023 Nov 27. PMID: 38012018; PMCID: PMC10760386.

Abstract

Nucleic acid vaccines are designed based on genetic sequences (DNA or mRNA) of a target antigen to be expressed in vivo to drive a host immune response. In response to the COVID-19 pandemic, mRNA and DNA vaccines based on the SARS-CoV-2 Spike antigen were developed. Surprisingly, head-to-head characterizations of the immune responses elicited by each vaccine type has not been performed to date. Here, we have employed a range of preclinical animal models including the hamster, guinea pig, rabbit, and mouse to compare and delineate the immune response raised by DNA, administered intradermally (ID) with electroporation (EP) and mRNA vaccines (BNT162b2 or mRNA-1273), administered intramuscularly (IM), expressing the SARS-CoV-2 WT spike antigen. The results revealed clear differences in the quality and magnitude of the immune response between the two vaccine platforms. The DNA vaccine immune response was characterized by strong T cell responses, while the mRNA vaccine elicited robust humoral responses. The results may assist in guiding the disease target each vaccine type may be best matched against and suggest mechanisms to further enhance the breadth of each platform’s immune response.

Computational Biology CryoEM and CryoET Kulp Lab

Conformational plasticity of RAS Q61 family of neoepitopes results in distinct features for targeted recognition

McShan AC, Flores-Solis D, Sun Y, Garfinkle SE, Toor JS, Young MC, Sgourakis NG. Nat Commun. 2023 Dec 11. doi: 10.1038/s41467-023-43654-9.

Abstract

The conformational landscapes of peptide/human leucocyte antigen (pHLA) protein complexes encompassing tumor neoantigens provide a rationale for target selection towards autologous T cell, vaccine, and antibody-based therapeutic modalities. Here, using complementary biophysical and computational methods, we characterize recurrent RAS55-64 Q61 neoepitopes presented by the common HLA-A*01:01 allotype. We integrate sparse NMR restraints with Rosetta docking to determine the solution structure of NRASQ61K/HLA-A*01:01, which enables modeling of other common RAS55-64 neoepitopes. Hydrogen/deuterium exchange mass spectrometry experiments alongside molecular dynamics simulations reveal differences in solvent accessibility and conformational plasticity across a panel of common Q61 neoepitopes that are relevant for recognition by immunoreceptors. Finally, we predict binding and provide structural models of NRASQ61K antigens spanning the entire HLA allelic landscape, together with in vitro validation for HLA-A*01:191, HLA-B*15:01, and HLA-C*08:02. Our work provides a basis to delineate the solution surface features and immunogenicity of clinically relevant neoepitope/HLA targets for cancer therapy.

Computational Biology NMR X-ray Crystallography SgourakisLab

A genetically encoded protein tag for control and quantitative imaging of CAR T cell therapy

Lee IK, Sharma N, Noguera-Ortega E, Liousia M, Baroja ML, Etersque JM, Pham J, Sarkar S, Carreno BM, Linette GP, Puré E, Albelda SM, Sellmyer MA. A genetically encoded protein tag for control and quantitative imaging of CAR T cell therapy. Mol Ther. 2023 Dec 6;31(12):3564-3578. doi: 10.1016/j.ymthe.2023.10.020. Epub 2023 Nov 3. PMID: 37919903; PMCID: PMC10727978.

Abstract

Chimeric antigen receptor (CAR) T cell therapy has been successful for hematological malignancies. Still, a lack of efficacy and potential toxicities have slowed its application for other indications. Furthermore, CAR T cells undergo dynamic expansion and contraction in vivo that cannot be easily predicted or controlled. Therefore, the safety and utility of such therapies could be enhanced by engineered mechanisms that engender reversible control and quantitative monitoring. Here, we use a genetic tag based on the enzyme Escherichia coli dihydrofolate reductase (eDHFR), and derivatives of trimethoprim (TMP) to modulate and monitor CAR expression and T cell activity. We fused eDHFR to the CAR C terminus, allowing regulation with TMP-based proteolysis-targeting chimeric small molecules (PROTACs). Fusion of eDHFR to the CAR does not interfere with cell signaling or its cytotoxic function, and the addition of TMP-based PROTACs results in a reversible and dose-dependent inhibition of CAR activity via the proteosome. We show the regulation of CAR expression in vivo and demonstrate imaging of the cells with TMP radiotracers. In vitro immunogenicity assays using primary human immune cells and overlapping peptide fragments of eDHFR showed no memory immune repertoire for eDHFR. Overall, this translationally-orientied approach allows for temporal monitoring and image-guided control of cell-based therapies.

Chemical Biology Sellmyer Lab

Interplay between stochastic enzyme activity and microtubule stability drives detyrosination enrichment on microtubule subsets

Tang Q, Sensale S, Bond C, Xing J, Qiao A, Hugelier S, Arab A, Arya G, Lakadamyali M. Interplay between stochastic enzyme activity and microtubule stability drives detyrosination enrichment on microtubule subsets. Curr Biol. 2023 Dec 4;33(23):5169-5184.e8. doi: 10.1016/j.cub.2023.10.068. Epub 2023 Nov 17. PMID: 37979580; PMCID: PMC10843832.

Abstract

Microtubules in cells consist of functionally diverse subpopulations carrying distinct post-translational modifications (PTMs). Akin to the histone code, the tubulin code regulates a myriad of microtubule functions, ranging from intracellular transport to chromosome segregation. However, how individual PTMs only occur on subsets of microtubules to contribute to microtubule specialization is not well understood. In particular, microtubule detyrosination, the removal of the C-terminal tyrosine on α-tubulin subunits, marks the stable population of microtubules and modifies how microtubules interact with other microtubule-associated proteins to regulate a wide range of cellular processes. Previously, we found that in certain cell types, only ∼30% of microtubules are highly enriched with the detyrosination mark and that detyrosination spans most of the length of a microtubule, often adjacent to a completely tyrosinated microtubule. How the activity of a cytosolic detyrosinase, vasohibin (VASH), leads to only a small subpopulation of highly detyrosinated microtubules is unclear. Here, using quantitative super-resolution microscopy, we visualized nascent microtubule detyrosination events in cells consisting of 1-3 detyrosinated α-tubulin subunits after nocodazole washout. Microtubule detyrosination accumulates slowly and in a dispersed pattern across the microtubule length. By visualizing single molecules of VASH in live cells, we found that VASH engages with microtubules stochastically on a short timescale, suggesting limited removal of tyrosine per interaction, consistent with the super-resolution results. Combining these quantitative imaging results with simulations incorporating parameters from our experiments, we provide evidence for a stochastic model for cells to establish a subset of detyrosinated microtubules via a detyrosination-stabilization feedback mechanism.

Single Molecule Imaging Lakadamyali Lab

Axonal transport of autophagosomes is regulated by dynein activators JIP3/JIP4 and ARF/RAB GTPases

Cason SE, Holzbaur ELF. Axonal transport of autophagosomes is regulated by dynein activators JIP3/JIP4 and ARF/RAB GTPases. J Cell Biol. 2023 Dec 4;222(12):e202301084. doi: 10.1083/jcb.202301084. Epub 2023 Nov 1. PMID: 37909920; PMCID: PMC10620608.

Abstract

Neuronal autophagosomes form and engulf cargos at presynaptic sites in the axon and are then transported to the soma to recycle their cargo. Autophagic vacuoles (AVs) mature en route via fusion with lysosomes to become degradatively competent organelles; transport is driven by the microtubule motor protein cytoplasmic dynein, with motor activity regulated by a sequential series of adaptors. Using lysate-based single-molecule motility assays and live-cell imaging in primary neurons, we show that JNK-interacting proteins 3 (JIP3) and 4 (JIP4) are activating adaptors for dynein that are regulated on autophagosomes and lysosomes by the small GTPases ARF6 and RAB10. GTP-bound ARF6 promotes formation of the JIP3/4-dynein-dynactin complex. Either knockdown or overexpression of RAB10 stalls transport, suggesting that this GTPase is also required to coordinate the opposing activities of bound dynein and kinesin motors. These findings highlight the complex coordination of motor regulation during organelle transport in neurons.

CryoEM and CryoET Holzbaur Lab Single Molecule Imaging

Interaction between the mitochondrial adaptor MIRO and the motor adaptor TRAK

Baltrusaitis EE, Ravitch EE, Fenton AR, Perez TA, Holzbaur ELF, Dominguez R. Interaction between the mitochondrial adaptor MIRO and the motor adaptor TRAK. J Biol Chem. 2023 Dec;299(12):105441. doi: 10.1016/j.jbc.2023.105441. Epub 2023 Nov 8. PMID: 37949220; PMCID: PMC10746525.

Abstract

MIRO (mitochondrial Rho GTPase) consists of two GTPase domains flanking two Ca2+-binding EF-hand domains. A C-terminal transmembrane helix anchors MIRO to the outer mitochondrial membrane, where it functions as a general adaptor for the recruitment of cytoskeletal proteins that control mitochondrial dynamics. One protein recruited by MIRO is TRAK (trafficking kinesin-binding protein), which in turn recruits the microtubule-based motors kinesin-1 and dynein-dynactin. The mechanism by which MIRO interacts with TRAK is not well understood. Here, we map and quantitatively characterize the interaction of human MIRO1 and TRAK1 and test its potential regulation by Ca2+ and/or GTP binding. TRAK1 binds MIRO1 with low micromolar affinity. The interaction was mapped to a fragment comprising MIRO1’s EF-hands and C-terminal GTPase domain and to a conserved sequence motif within TRAK1 residues 394 to 431, immediately C-terminal to the Spindly motif. This sequence is sufficient for MIRO1 binding in vitro and is necessary for MIRO1-dependent localization of TRAK1 to mitochondria in cells. MIRO1’s EF-hands bind Ca2+ with dissociation constants (KD) of 3.9 μM and 300 nM. This suggests that under cellular conditions one EF-hand may be constitutively bound to Ca2+ whereas the other EF-hand binds Ca2+ in a regulated manner, depending on its local concentration. Yet, the MIRO1-TRAK1 interaction is independent of Ca2+ binding to the EF-hands and of the nucleotide state (GDP or GTP) of the C-terminal GTPase. The interaction is also independent of TRAK1 dimerization, such that a TRAK1 dimer can be expected to bind two MIRO1 molecules on the mitochondrial surface.

CryoEM and CryoET Holzbaur Lab Single Molecule Imaging

Structural principles of peptide-centric chimeric antigen receptor recognition guide therapeutic expansion

Sun Y, Florio TJ, Gupta S, Young MC, Marshall QF, Garfinkle SE, Papadaki GF, Truong HV, Mycek E, Li P, Farrel A, Church NL, Jabar S, Beasley MD, Kiefel BR, Yarmarkovich M, Mallik L, Maris JM, Sgourakis NG. Sci Immunol. 2023 Dec 1. doi: 10.1126/sciimmunol.adj5792.

Abstract Peptide-centric chimeric antigen receptors (PC-CARs) recognize oncoprotein epitopes displayed by cell-surface human leukocyte antigens (HLAs) and offer a promising strategy for targeted cancer therapy. We have previously developed a PC-CAR targeting a neuroblastoma-associated PHOX2B peptide, leading to robust tumor cell lysis restricted by two common HLA allotypes. Here, we determine the 2.1-angstrom crystal structure of the PC-CAR–PHOX2B–HLA-A*24:02–β2m complex, which reveals the basis for antigen-specific recognition through interactions with CAR complementarity-determining regions (CDRs). This PC-CAR adopts a diagonal docking mode, where interactions with both conserved and polymorphic HLA framework residues permit recognition of multiple HLA allotypes from the A9 serological cross-reactive group, covering a combined global population frequency of up to 46.7%. Biochemical binding assays, molecular dynamics simulations, and structural and functional analyses demonstrate that high-affinity PC-CAR recognition of cross-reactive pHLAs necessitates the presentation of a specific peptide backbone, where subtle structural adaptations of the peptide are critical for high-affinity complex formation, and CAR T cell killing. Our results provide a molecular blueprint for engineering CARs with optimal recognition of tumor-associated antigens in the context of different HLAs, while minimizing cross-reactivity with self-epitopes.
Computational Biology NMR X-ray Crystallography SgourakisLab

The C-terminus of α-Synuclein Regulates its Dynamic Cellular Internalization by Neurexin 1β

Birol M, Muñoz IID, Rhoades E. The C-terminus of α-Synuclein Regulates its Dynamic Cellular Internalization by Neurexin 1β. Mol Biol Cell. 2023 Dec 1;34(13):br21. doi: 10.1091/mbc.E22-11-0496. Epub 2023 Sep 20. PMID: 37729016; PMCID: PMC10848939.

Abstract

The aggregation of the disordered neuronal protein, α-Synuclein (αS), is the primary pathological feature of Parkinson’s disease. Current hypotheses favor cell-to-cell spread of αS species as underlying disease progression, driving interest in identifying the molecular species and cellular processes involved in cellular internalization of αS. Prior work from our lab identified the chemically specific interaction between αS and the presynaptic adhesion protein neurexin-1β (N1β) to be capable of driving cellular internalization of both monomer and aggregated forms of αS. Here we explore the physical basis of N1β-driven internalization of αS. Specifically, we show that spontaneous internalization of αS by SH-SY5Y and HEK293 cells expressing N1β requires essentially all of the membrane-binding domain of αS; αS constructs truncated beyond residue 90 bind to N1β in the plasma membrane of HEK cells, but are not internalized. Interestingly, before internalization, αS and N1β codiffuse rapidly in the plasma membrane. αS constructs that are not internalized show very slow mobility themselves, as well as slow N1β diffusion. Finally, we find that truncated αS is capable of blocking internalization of full-length αS. Our results draw attention to the potential therapeutic value of blocking αS-N1β interactions.

Single Molecule Imaging Rhoades Lab

Lead-oriented synthesis of epigenetic relevant scaffolds

Maujean T, Kannaboina P, Green AI, Burslem GM. Lead-oriented synthesis of epigenetic relevant scaffolds. Chem Commun (Camb). 2023 Dec 7;59(98):14555-14558. doi: 10.1039/d3cc04317g. PMID: 37991354.

Abstract

A simple and rational method to rank lead-likeness of molecules using continuous evaluation functions was hereby developed. This strategy proved to be competitive against known methods and finally helped in driving synthetic efforts towards candidates of interest for epigenetic applications against HDAC6, BRD4 and EZH2.

Chemical Biology Burslem Lab Mass Spectrometry

Structure reveals why genome folding is necessary for site-specific integration of foreign DNA into CRISPR arrays

Santiago-Frangos A, Henriques WS, Wiegand T, Gauvin CC, Buyukyoruk M, Graham AB, Wilkinson RA, Triem L, Neselu K, Eng ET, Lander GC, Wiedenheft B. Structure reveals why genome folding is necessary for site-specific integration of foreign DNA into CRISPR arrays. Nat Struct Mol Biol. 2023 Nov;30(11):1675-1685. doi: 10.1038/s41594-023-01097-2. Epub 2023 Sep 14. PMID: 37710013; PMCID: PMC10872659.

Abstract

Bacteria and archaea acquire resistance to viruses and plasmids by integrating fragments of foreign DNA into the first repeat of a CRISPR array. However, the mechanism of site-specific integration remains poorly understood. Here, we determine a 560-kDa integration complex structure that explains how Pseudomonas aeruginosa Cas (Cas1-Cas2/3) and non-Cas proteins (for example, integration host factor) fold 150 base pairs of host DNA into a U-shaped bend and a loop that protrude from Cas1-2/3 at right angles. The U-shaped bend traps foreign DNA on one face of the Cas1-2/3 integrase, while the loop places the first CRISPR repeat in the Cas1 active site. Both Cas3 proteins rotate 100 degrees to expose DNA-binding sites on either side of the Cas2 homodimer, which each bind an inverted repeat motif in the leader. Leader sequence motifs direct Cas1-2/3-mediated integration to diverse repeat sequences that have a 5′-GT. Collectively, this work reveals new DNA-binding surfaces on Cas2 that are critical for DNA folding and site-specific delivery of foreign DNA.

Computational Biology CryoEM and CryoET Santiago-Frangos Lab

Mutant forms of DDX3X with diminished catalysis form hollow condensates that exhibit sex-specific regulation

Owens MC, Shen H, Yanas A, Mendoza-Figueroa MS, Lavorando E, Wei X, Shweta H, Tang HY, Goldman YE, Liu KF. Mutant forms of DDX3X with diminished catalysis form hollow condensates that exhibit sex-specific regulation. bioRxiv [Preprint]. 2023 Nov 29:2023.03.19.533240. doi: 10.1101/2023.03.19.533240. PMID: 38076929; PMCID: PMC10705264.

Abstract

Mutations in the RNA helicase DDX3X, implicated in various cancers and neurodevelopmental disorders, often impair RNA unwinding and translation. However, the mechanisms underlying this impairment and the differential interactions of DDX3X mutants with wild-type (WT) X-linked DDX3X and Y-linked homolog DDX3Y remain elusive. This study reveals that specific DDX3X mutants more frequently found in disease form distinct hollow condensates in cells. Using a combined structural, biochemical, and single-molecule microscopy study, we show that reduced ATPase and RNA release activities contribute to condensate formation and the catalytic deficits result from inhibiting the catalytic cycle at multiple steps. Proteomic investigations further demonstrate that these hollow condensates sequester WT DDX3X/DDX3Y and other proteins crucial for diverse signaling pathways. WT DDX3X enhances the dynamics of heterogeneous mutant/WT hollow condensates more effectively than DDX3Y. These findings offer valuable insights into the catalytic defects of specific DDX3X mutants and their differential interactions with wild-type DDX3X and DDX3Y, potentially explaining sex biases in disease.

 

 

Mass Spectrometry X-ray Crystallography Liu Lab

SQSTM1/P62 promotes lysophagy via formation of liquid-like condensates maintained by HSP27

Gallagher ER, Holzbaur ELF. SQSTM1/P62 promotes lysophagy via formation of liquid-like condensates maintained by HSP27. Autophagy. 2023 Nov;19(11):3029-3030. doi: 10.1080/15548627.2023.2210943. Epub 2023 May 16. PMID: 37194327; PMCID: PMC10548893.

Abstract

SQSTM1/p62: Sequestosome-1; HSP27: Heat shock protein 27; LLPS: liquid-liquid phase separation; iPSC: induced pluripotent stem cell; PB1: Phox and Bem1p; FRAP: fluorescence recovery after photo-bleaching; ATG: autophagy-related; ALS: amyotrophic lateral sclerosis.

 

CryoEM and CryoET Holzbaur Lab Single Molecule Imaging

Reversible histone deacetylase activity catalyzes lysine acylation

Tsusaka T, Najar MA, Schwarz B, Bohrnsen E, Oses-Prieto JA, Lee C, Burlingame AL, Bosio CM, Burslem GM, Goldberg EL. Reversible histone deacetylase activity catalyzes lysine acylation. bioRxiv [Preprint]. 2023 Nov 17:2023.11.17.567549. doi: 10.1101/2023.11.17.567549. PMID: 38014285; PMCID: PMC10680841.

Abstract

Starvation and low carbohydrate diets lead to the accumulation of the ketone body, β-hydroxybutyrate (BHB), whose blood concentrations increase more than 10-fold into the millimolar range. In addition to providing a carbon source, BHB accumulation triggers lysine β-hydroxybutyrylation (Kbhb) of proteins via unknown mechanisms. As with other lysine acylation events, Kbhb marks can be removed by histone deacetylases (HDACs). Here, we report that class I HDACs unexpectedly catalyze protein lysine modification with β-hydroxybutyrate (BHB). Mutational analyses of the HDAC2 active site reveal a shared reliance on key amino acids for classical deacetylation and non-canonical HDAC-catalyzed β-hydroxybutyrylation. Also consistent with reverse HDAC activity, Kbhb formation is driven by mass action and substrate availability. This reverse HDAC activity is not limited to BHB but also extends to multiple short-chain fatty acids. The reversible activity of class I HDACs described here represents a novel mechanism of PTM deposition relevant to metabolically-sensitive proteome modifications.

Chemical Biology Burslem Lab Mass Spectrometry

The RNA helicase DDX39A binds a conserved structure in chikungunya virus RNA to control infection

Tapescu I, Taschuk F, Pokharel SM, Zginnyk O, Ferretti M, Bailer PF, Whig K, Madden EA, Heise MT, Schultz DC, Cherry S. The RNA helicase DDX39A binds a conserved structure in chikungunya virus RNA to control infection. Mol Cell. 2023 Nov 16;83(22):4174-4189.e7. doi: 10.1016/j.molcel.2023.10.008. Epub 2023 Nov 9. PMID: 37949067; PMCID: PMC10722560.

Abstract

Alphaviruses are a large group of re-emerging arthropod-borne RNA viruses. The compact viral RNA genomes harbor diverse structures that facilitate replication. These structures can be recognized by antiviral cellular RNA-binding proteins, including DExD-box (DDX) helicases, that bind viral RNAs to control infection. The full spectrum of antiviral DDXs and the structures that are recognized remain unclear. Genetic screening identified DDX39A as antiviral against the alphavirus chikungunya virus (CHIKV) and other medically relevant alphaviruses. Upon infection, the predominantly nuclear DDX39A accumulates in the cytoplasm inhibiting alphavirus replication, independent of the canonical interferon pathway. Biochemically, DDX39A binds to CHIKV genomic RNA, interacting with the 5′ conserved sequence element (5’CSE), which is essential for the antiviral activity of DDX39A. Altogether, DDX39A relocalization and binding to a conserved structural element in the alphavirus genomic RNA attenuates infection, revealing a previously unknown layer to the cellular control of infection.

Chemical Biology Cherry Lab Protein Production Services Schultz Lab

Evolutionary arms race between SARS-CoV-2 and interferon signaling via dynamic interaction with autophagy

Lee JS, Dittmar M, Miller J, Li M, Ayyanathan K, Ferretti M, Hulahan J, Whig K, Etwebi Z, Griesman T, Schultz DC, Cherry S. Evolutionary arms race between SARS-CoV-2 and interferon signaling via dynamic interaction with autophagy. bioRxiv [Preprint]. 2023 Nov 16:2023.11.13.566859. doi: 10.1101/2023.11.13.566859. PMID: 38014114; PMCID: PMC10680587.

Abstract

SARS-CoV-2 emerged, and is evolving to efficiently infect humans worldwide. SARS-CoV-2 evades early innate recognition, interferon signaling activated only in bystander cells. This balance of innate activation and viral evasion has important consequences, but the pathways involved are incompletely understood. Here we find that autophagy genes regulate innate immune signaling, impacting the basal set point of interferons, and thus permissivity to infection. Mechanistically, autophagy genes negatively regulate MAVS, and this low basal level of MAVS is efficiently antagonized by SARS-CoV-2 ORF9b, blocking interferon activation in infected cells. However, upon loss of autophagy increased MAVS overcomes ORF9b-mediated antagonism suppressing infection. This has led to the evolution of SARS-CoV-2 variants to express higher levels of ORF9b, allowing SARS-CoV-2 to replicate under conditions of increased MAVS signaling. Altogether, we find a critical role of autophagy in the regulation of innate immunity and uncover an evolutionary trajectory of SARS-CoV-2 ORF9b to overcome host defenses.

Chemical Biology Protein Production Services Schultz Lab

Activating mutations drive human MEK1 kinase using a gear-shifting mechanism

Patil K, Wang Y, Chen Z, Suresh K, Radhakrishnan R. Activating mutations drive human MEK1 kinase using a gear-shifting mechanism. Biochem J. 2023 Nov 15;480(21):1733-1751. doi: 10.1042/BCJ20230281. PMID: 37869794; PMCID: PMC10872882.

Abstract

There is an unmet need to classify cancer-promoting kinase mutations in a mechanistically cognizant way. The challenge is to understand how mutations stabilize different kinase configurations to alter function, and how this influences pathogenic potential of the kinase and its responses to therapeutic inhibitors. This goal is made more challenging by the complexity of the mutational landscape of diseases, and is further compounded by the conformational plasticity of each variant where multiple conformations coexist. We focus here on the human MEK1 kinase, a vital component of the RAS/MAPK pathway in which mutations cause cancers and developmental disorders called RASopathies. We sought to explore how these mutations alter the human MEK1 kinase at atomic resolution by utilizing enhanced sampling simulations and free energy calculations. We computationally mapped the different conformational stabilities of individual mutated systems by delineating the free energy landscapes, and showed how this relates directly to experimentally quantified developmental transformation potentials of the mutations. We conclude that mutations leverage variations in the hydrogen bonding network associated with the conformational plasticity to progressively stabilize the active-like conformational state of the kinase while destabilizing the inactive-like state. The mutations alter residue-level internal molecular correlations by differentially prioritizing different conformational states, delineating the various modes of MEK1 activation reminiscent of a gear-shifting mechanism. We define the molecular basis of conversion of this kinase from its inactive to its active state, connecting structure, dynamics, and function by delineating the energy landscape and conformational plasticity, thus augmenting our understanding of MEK1 regulation.

Computational Biology Radhakrishnan Lab

Structure-Based Prediction of Kinase Activation amidst a Varied Mutational Landscape Using Privileged Learning

Yiming Wang, Fangping Wan, Zhangtao Chen, Jonathan Nukpezah, Cesar de la Fuente-Nunez, Ravi Radhakrishnan

Abstract

Post-translational modifications such as phosphorylation catalyzed by kinases are essential for cell signaling. The activation of mutated kinase in a cancer cell can profoundly impact disease progression and drug efficacy. However, numerous clinical mutations in the human kinome impose challenges in defining the quantitative structure-activity relationship. Previous work (Patil K. et al. PNAS 2021, 118(10), e2019132118.) shows that perturbation of structural properties such as hydrogen-bonding occupancy in the áC-helix and the activation loop domains computed from molecular dynamics (MD) simulation is a good indicator of the activation status of anaplastic ymphoma kinase (ALK) mutants with 2/3rd of the mutants utilizing this mechanism of activation.

Computational Biology Radhakrishnan Lab

Open science discovery of potent noncovalent SARS-CoV-2 main protease inhibitors

Boby ML, Fearon D, Ferla M, Filep M, Koekemoer L, Robinson MC; COVID Moonshot Consortium‡; Chodera JD, Lee AA, London N, von Delft A, von Delft F, Achdout H, Aimon A, Alonzi DS, Arbon R, Aschenbrenner JC, Balcomb BH, Bar-David E, Barr H, Ben-Shmuel A, Bennett J, Bilenko VA, Borden B, Boulet P, Bowman GR, Brewitz L, Brun J, Bvnbs S, Calmiano M, Carbery A, Carney DW, Cattermole E, Chang E, Chernyshenko E, Clyde A, Coffland JE, Cohen G, Cole JC, Contini A, Cox L, Croll TI, Cvitkovic M, De Jonghe S, Dias A, Donckers K, Dotson DL, Douangamath A, Duberstein S, Dudgeon T, Dunnett LE, Eastman P, Erez N, Eyermann CJ, Fairhead M, Fate G, Fedorov O, Fernandes RS, Ferrins L, Foster R, Foster H, Fraisse L, Gabizon R, García-Sastre A, Gawriljuk VO, Gehrtz P, Gileadi C, Giroud C, Glass WG, Glen RC, Glinert I, Godoy AS, Gorichko M, Gorrie-Stone T, Griffen EJ, Haneef A, Hassell Hart S, Heer J, Henry M, Hill M, Horrell S, Huang QYJ, Huliak VD, Hurley MFD, Israely T, Jajack A, Jansen J, Jnoff E, Jochmans D, John T, Kaminow B, Kang L, Kantsadi AL, Kenny PW, Kiappes JL, Kinakh SO, Kovar B, Krojer T, La VNT, Laghnimi-Hahn S, Lefker BA, Levy H, Lithgo RM, Logvinenko IG, Lukacik P, Macdonald HB, MacLean EM, Makower LL, Malla TR, Marples PG, Matviiuk T, McCorkindale W, McGovern BL, Melamed S, Melnykov KP, Michurin O, Miesen P, Mikolajek H, Milne BF, Minh D, Morris A, Morris GM, Morwitzer MJ, Moustakas D, Mowbray CE, Nakamura AM, Neto JB, Neyts J, Nguyen L, Noske GD, Oleinikovas V, Oliva G, Overheul GJ, Owen CD, Pai R, Pan J, Paran N, Payne AM, Perry B, Pingle M, Pinjari J, Politi B, Powell A, Pšenák V, Pulido I, Puni R, Rangel VL, Reddi RN,

Bowman Lab Computational Biology

Development of First-in-Class Dual Sirt2/HDAC6 Inhibitors as Molecular Tools for Dual Inhibition of Tubulin Deacetylation

Sinatra L, Vogelmann A, Friedrich F, Tararina MA, Neuwirt E, Colcerasa A, König P, Toy L, Yesiloglu TZ, Hilscher S, Gaitzsch L, Papenkordt N, Zhai S, Zhang L, Romier C, Einsle O, Sippl W, Schutkowski M, Gross O, Bendas G, Christianson DW, Hansen FK, Jung M, Schiedel M. Development of First-in-Class Dual Sirt2/HDAC6 Inhibitors as Molecular Tools for Dual Inhibition of Tubulin Deacetylation. J Med Chem. 2023 Nov 9;66(21):14787-14814. doi: 10.1021/acs.jmedchem.3c01385. Epub 2023 Oct 30. PMID: 37902787; PMCID: PMC10641818.

Abstract Dysregulation of both

Dysregulation of both tubulin deacetylases sirtuin 2 (Sirt2) and the histone deacetylase 6 (HDAC6) has been associated with the pathogenesis of cancer and neurodegeneration, thus making these two enzymes promising targets for pharmaceutical intervention. Herein, we report the design, synthesis, and biological characterization of the first-in-class dual Sirt2/HDAC6 inhibitors as molecular tools for dual inhibition of tubulin deacetylation. Using biochemical in vitro assays and cell-based methods for target engagement, we identified Mz325 (33) as a potent and selective inhibitor of both target enzymes. Inhibition of both targets was further confirmed by X-ray crystal structures of Sirt2 and HDAC6 in complex with building blocks of 33. In ovarian cancer cells, 33 evoked enhanced effects on cell viability compared to single or combination treatment with the unconjugated Sirt2 and HDAC6 inhibitors. Thus, our dual Sirt2/HDAC6 inhibitors are important new tools to study the consequences and the therapeutic potential of dual inhibition of tubulin deacetylation.

CryoEM and CryoET Christianson Group X-ray Crystallography

Targeting of intracellular oncoproteins with peptide-centric CARs

Yarmarkovich M, Marshall QF, Warrington JM, Premaratne R, Farrel A, Groff D, Li W, di Marco M, Runbeck E, Truong H, Toor JS, Tripathi S, Nguyen S, Shen H, Noel T, Church NL, Weiner A, Kendsersky N, Martinez D, Weisberg R, Christie M, Eisenlohr L, Bosse KR, Dimitrov DS, Stevanovic S, Sgourakis NG, Kiefel BR, Maris JM. Nature. 2023 Nov 8. doi: 10.1038/s41586-023-06706-0

Abstract

The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.

Interaction between the mitochondrial adaptor MIRO and the motor adaptor TRAK

Baltrusaitis EE, Ravitch EE, Fenton AR, Perez TA, Holzbaur ELF, Dominguez R. Interaction between the mitochondrial adaptor MIRO and the motor adaptor TRAK. J Biol Chem. 2023 Dec;299(12):105441. doi: 10.1016/j.jbc.2023.105441. Epub 2023 Nov 8. PMID: 37949220; PMCID: PMC10746525.

Abstract

MIRO (mitochondrial Rho GTPase) consists of two GTPase domains flanking two Ca2+-binding EF-hand domains. A C-terminal transmembrane helix anchors MIRO to the outer mitochondrial membrane, where it functions as a general adaptor for the recruitment of cytoskeletal proteins that control mitochondrial dynamics. One protein recruited by MIRO is TRAK (trafficking kinesin-binding protein), which in turn recruits the microtubule-based motors kinesin-1 and dynein-dynactin. The mechanism by which MIRO interacts with TRAK is not well understood. Here, we map and quantitatively characterize the interaction of human MIRO1 and TRAK1 and test its potential regulation by Ca2+ and/or GTP binding. TRAK1 binds MIRO1 with low micromolar affinity. The interaction was mapped to a fragment comprising MIRO1’s EF-hands and C-terminal GTPase domain and to a conserved sequence motif within TRAK1 residues 394 to 431, immediately C-terminal to the Spindly motif. This sequence is sufficient for MIRO1 binding in vitro and is necessary for MIRO1-dependent localization of TRAK1 to mitochondria in cells. MIRO1’s EF-hands bind Ca2+ with dissociation constants (KD) of 3.9 μM and 300 nM. This suggests that under cellular conditions one EF-hand may be constitutively bound to Ca2+ whereas the other EF-hand binds Ca2+ in a regulated manner, depending on its local concentration. Yet, the MIRO1-TRAK1 interaction is independent of Ca2+ binding to the EF-hands and of the nucleotide state (GDP or GTP) of the C-terminal GTPase. The interaction is also independent of TRAK1 dimerization, such that a TRAK1 dimer can be expected to bind two MIRO1 molecules on the mitochondrial surface.

CryoEM and CryoET Dominguez Lab X-ray Crystallography

Molecular Mechanisms behind Myc Regulation and Function in Cancer

Reshma Kalyan Sundaram, Ravi Radhakrishnan, Bomyi Lim

Abstract The transcription factor Myc is known to modulate a multitude of genes and cellular processes. Myc is deregulated in 70% of human cancers and is commonly known as an “undruggable” molecule. Factors such as Myc’s structure and primary nuclear localization make it difficult to directly target Myc in cancer treatments. This has led to an increased interest in identifying indirect ways to target Myc. Therefore, in this work, we sought to understand the molecular mechanisms behind 1) regulation (at the signaling level) and 2) function (at the transcription level) of Myc. In the first part of this work, we used an ODE-based kinetic modeling approach to study how extracellular mechanical and growth cues transduced through intracellular signaling pathways can drive Myc protein stabilization and accumulation in cells. The function of Myc in cancer is well-studied, however, the role of mechanotransduction in driving and sustaining cancers through Myc is yet to be uncovered. Understanding this is important as tissue stiffening is a precursor of various solid cancers. Therefore, we modeled Myc phosphorylation by MAPK, Rho/ROCK, and PI3K/Akt signaling pathways effected by EGF and integrin receptors. Our modeling results show that for normal cellular phenotypes, signaling through the EGF receptor strongly influences Myc phosphorylation. However, in cancerous phenotype, signaling through both EGF and integrin receptors play a combined role in modulating Myc. We, therefore, conclude that growth and mechanical signals play a synergistic role in driving and sustaining cancers by modulating Myc levels. Based on this, we propose that tissue stiffness must be considered as a factor while developing treatment strategies for Myc-driven solid cancers. Further, we systematically perturbed signaling interactions in the model to identify key processes governing Myc in cells. From this analysis, we identified intermediate nodes critical for Myc deregulation,
Computational Biology Radhakrishnan Lab

Integrated Computational and Experimental Design of Selective Lanthanide Binding Peptides

Yiming Wang, Jason Marmorstein, E James Petersson, Ivan J Dmochowski, Ravi Radhakrishnan, Kathleen J Stebe

Abstract Rare earth elements (REE), or lanthanides, are critical materials with a wide range of applications in clean energy industrysuch as battery, semiconductor, and electric vehicles, due to their unique luminescent, magnetic, and catalytic properties. Therefore, there is a growing demand for developing green industrial-scale extraction of lanthanides. However, efficient separation of heavy lanthanides (e.g. from Europium to Lutetium) with exceedingly similar chemical properties is technologically challenging, and is an active area of research. Here, we combine molecular dynamics (MD), enhanced sampling, and machine learning methods to design insilico short 17-residue peptides that selectively bind to trivalent REE cations in aqueous solution. The REE-peptide binding affinity are predicted by ML models that are trained on features that characterize the REE-peptide binding complex including hydration properties of REEs from literature studies, REE-peptide interaction energies obtained from MD simulations, and physicochemical properties of the peptide estimated from the DBAASP peptide database. Starting from the original lanthanide-binding tag (LBT) peptide, we computationally screen peptide candidates with mutations mainly on the six key ligating residues that improve predicted binding selectivity against 12 different REEs. Candidates with top selectivity scores selected from in silico discovery are verified by the fluorescence titration experiments. Moreover, we utilize a novel genetic algorithm framework to iteratively guide the design of new peptide sequences combining our model and experiments. Therefore, we demonstrate an integrated data-driven approach for quantitative sequence-structure-activity relationship as well as design of lanthanide binding peptides, paving the road for designing novel biomolecules for efficient rare earth elements recovery.
Computational Biology Radhakrishnan Lab

Targeting of intracellular oncoproteins with peptide-centric CARs

Yarmarkovich M, Marshall QF, Warrington JM, Premaratne R, Farrel A, Groff D, Li W, di Marco M, Runbeck E, Truong H, Toor JS, Tripathi S, Nguyen S, Shen H, Noel T, Church NL, Weiner A, Kendsersky N, Martinez D, Weisberg R, Christie M, Eisenlohr L, Bosse KR, Dimitrov DS, Stevanovic S, Sgourakis NG, Kiefel BR, Maris JM. Targeting of intracellular oncoproteins with peptide-centric CARs. Nature. 2023 Nov;623(7988):820-827. doi: 10.1038/s41586-023-06706-0. Epub 2023 Nov 8. PMID: 37938771; PMCID: PMC10665195.

Abstract

The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.

CryoEM and CryoET NMR SgourakisLab

Compare the effectiveness of extracorporeal shockwave and hyperbaric oxygen therapy on enhancing wound healing in a streptozotocin-induced diabetic rodent model

Chen RF, Lin YN, Liu KF, Lee CC, Hu CJ, Wang CT, Wang CJ, Kuo YR. Compare the effectiveness of extracorporeal shockwave and hyperbaric oxygen therapy on enhancing wound healing in a streptozotocin-induced diabetic rodent model. Kaohsiung J Med Sci. 2023 Nov;39(11):1135-1144. doi: 10.1002/kjm2.12746. Epub 2023 Sep 2. PMID: 37658698.

Abstract

Studies have revealed that both extracorporeal shock-wave therapy (ESWT) and hyperbaric oxygen therapy (HBOT) can accelerate wound healing. This study aimed to compare the effectiveness of ESWT and HBOT in enhancing diabetic wound healing. A dorsal skin defect in a streptozotocin-induced diabetes rodent model was used. Postoperative wound healing was assessed once every 3 days. Histologic examination was performed with hematoxylin and eosin staining. Proliferation marker protein Ki-67 (Ki-67), endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), and 8-hydroxy-2-deoxyguanosine (8-OHdG) were evaluated with immunohistochemical (IHC) staining. The wound area was significantly reduced in the ESWT and HBOT groups compared to that in the diabetic controls. However, the wound healing time was significantly increased in the HBOT group compared to the ESWT group. Histological findings showed a statistical increase in neovascularization and suppression of the inflammatory response by both HBOT and ESWT compared to the controls. IHC staining revealed a significant increase in Ki-67, VEGF, and eNOS but suppressed 8-OHdG expression in the ESWT group compared to the HBOT group. ESWT facilitated diabetic wound healing more effectively than HBOT by suppressing the inflammatory response and enhancing cellular proliferation and neovascularization and tissue regeneration.

 

Mass Spectrometry X-ray Crystallography Liu Lab

Regulation of eDHFR-tagged proteins with trimethoprim PROTACs

Etersque JM, Lee IK, Sharma N, Xu K, Ruff A, Northrup JD, Sarkar S, Nguyen T, Lauman R, Burslem GM, Sellmyer MA. Regulation of eDHFR-tagged proteins with trimethoprim PROTACs. Nat Commun. 2023 Nov 3;14(1):7071. doi: 10.1038/s41467-023-42820-3. PMID: 37923771; PMCID: PMC10624689.

Abstract

Temporal control of protein levels in cells and living animals can be used to improve our understanding of protein function. In addition, control of engineered proteins could be used in therapeutic applications. PRoteolysis-TArgeting Chimeras (PROTACs) have emerged as a small-molecule-driven strategy to achieve rapid, post-translational regulation of protein abundance via recruitment of an E3 ligase to the target protein of interest. Here, we develop several PROTAC molecules by covalently linking the antibiotic trimethoprim (TMP) to pomalidomide, a ligand for the E3 ligase, Cereblon. These molecules induce degradation of proteins of interest (POIs) genetically fused to a small protein domain, E. coli dihydrofolate reductase (eDHFR), the molecular target of TMP. We show that various eDHFR-tagged proteins can be robustly degraded to 95% of maximum expression with PROTAC molecule 7c. Moreover, TMP-based PROTACs minimally affect the expression of immunomodulatory imide drug (IMiD)-sensitive neosubstrates using proteomic and biochemical assays. Finally, we show multiplexed regulation with another known degron-PROTAC pair, as well as reversible protein regulation in a rodent model of metastatic cancer, demonstrating the formidable strength of this system. Altogether, TMP PROTACs are a robust approach for selective and reversible degradation of eDHFR-tagged proteins in vitro and in vivo.

Chemical Biology Burslem Lab Mass Spectrometry Sellmyer Lab

Pleiotropic roles of evolutionarily conserved signaling intermediate in toll pathway (ECSIT) in pathophysiology

Chaitanya NSN, Tammineni P, Nagaraju GP, Reddy AB. J Cell Physiol. 2022 Sep;237(9):3496-3504. doi: 10.1002/jcp.30832. Epub 2022 Jul 19.

Abstract

The evolutionarily conserved signaling intermediate in toll pathway (ECSIT) is a cytosolic adaptor protein associated with the toll-like receptor pathway. It has a distinct N-terminal mitochondrial targeting sequence, pentatricopeptide repeat motif, and a C-terminal pleckstrin homology domain. ECSIT regulates many biological processes like embryonic development, inflammation, cardiac function, and assembly of mitochondrial complex I. Besides, ECSIT also interacts with multiple signaling intermediates like tumor necrosis receptor associated factor 6 and retinoic acid inducible gene 1 as well as regulates various pathways in the microcellular environment. However, molecular details of ECSIT functions in pathophysiology remain elusive. This review summarizes the diverse functions of ECSIT and its involvement in pathophysiological conditions such as Alzheimer’s, oxidative stress, and infection.

CryoEM and CryoET Mass Spectrometry Reddy Lab

AMPK-induced novel phosphorylation of RUNX1 inhibits STAT3 activation and overcome imatinib resistance in chronic myelogenous leukemia (CML) subjects

Gayatri MB, Kancha RK, Behera A, Patchva D, Velugonda N, Gundeti S, Reddy ABM. Cell Death Discov. 2023 Oct 30;9(1):401. doi: 10.1038/s41420-023-01700-x.

Abstract

Imatinib resistance remains an unresolved problem in CML disease. Activation of JAK2/STAT3 pathway and increased expression of RUNX1 have become one reason for development of imatinib resistance in CML subjects. Metformin has gained attention as an antileukemic drug in recent times. However, the molecular mechanism remains elusive. The present study shows that RUNX1 is a novel substrate of AMP-activated kinase (AMPK), where AMPK phosphorylates RUNX1 at Ser 94 position. Activation of AMPK by metformin could lead to increased cytoplasmic retention of RUNX1 due to Ser 94 phosphorylation. RUNX1 Ser 94 phosphorylation resulted in increased interaction with STAT3, which was reflected in reduced transcriptional activity of both RUNX1 and STAT3 due to their cytoplasmic retention. The reduced transcriptional activity of STAT3 and RUNX1 resulted in the down-regulation of their signaling targets involved in proliferation and anti-apoptosis. Our cell proliferation assays using in vitro resistant cell line models and PBMCs isolated from CML clinical patients and normal subjects demonstrate that metformin treatment resulted in reduced growth and improved imatinib sensitivity of resistant subjects.

CryoEM and CryoET Mass Spectrometry Reddy Lab

Mechanism of synergistic activation of Arp2/3 complex by cortactin and WASP-family proteins

Fregoso FE, Boczkowska M, Rebowski G, Carman PJ, van Eeuwen T, Dominguez R. Mechanism of synergistic activation of Arp2/3 complex by cortactin and WASP-family proteins. Nat Commun. 2023 Oct 28;14(1):6894. doi: 10.1038/s41467-023-42229-y. PMID: 37898612; PMCID: PMC10613254.

Abstract

Cortactin coactivates Arp2/3 complex synergistically with WASP-family nucleation-promoting factors (NPFs) and stabilizes branched networks by linking Arp2/3 complex to F-actin. It is poorly understood how cortactin performs these functions. We describe the 2.89 Å resolution cryo-EM structure of cortactin’s N-terminal domain (Cort1-76) bound to Arp2/3 complex. Cortactin binds Arp2/3 complex through an inverted Acidic domain (D20-V29), which targets the same site on Arp3 as the Acidic domain of NPFs but with opposite polarity. Sequences N- and C-terminal to cortactin’s Acidic domain do not increase its affinity for Arp2/3 complex but contribute toward coactivation with NPFs. Coactivation further increases with NPF dimerization and for longer cortactin constructs with stronger binding to F-actin. The results suggest that cortactin contributes to Arp2/3 complex coactivation with NPFs in two ways, by helping recruit the complex to F-actin and by stabilizing the short-pitch (active) conformation, which are both byproducts of cortactin’s core function in branch stabilization.

 

CryoEM and CryoET Dominguez Lab X-ray Crystallography

Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution

Swanson NA, Lokareddy RK, Li F, Hou CD, Leptihn S, Pavlenok M, Niederweis M, Pumroy RA, Moiseenkova-Bell VY, Cingolani G. Mol Cell. 2021 Aug 5;81(15):3145-3159.e7. doi: 10.1016/j.molcel.2021.06.001. Epub 2021 Jul 1.

Summary

Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 Å resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 Å hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a ∼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysisDNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.

CryoEM and CryoET Mass Spectrometry Vera Moiseenkova-Bell Lab

Tuft cells utilize taste signaling molecules to respond to the pathobiont microbe Ruminococcus gnavus in the proximal colon

Lei H, Yu D, Xue YB, Li YH, Gong SM, Peng YY, Liu KF, Buratto D, Yang Y, Zhang SS, Wu M, Zhou R, Huang L. Tuft cells utilize taste signaling molecules to respond to the pathobiont microbe Ruminococcus gnavus in the proximal colon. Front Immunol. 2023 Oct 25;14:1259521. doi: 10.3389/fimmu.2023.1259521. PMID: 37954611; PMCID: PMC10634341.

Abstract

Tuft cells are a type of rare epithelial cells that have been recently found to utilize taste signal transduction pathways to detect and respond to various noxious stimuli and pathogens, including allergens, bacteria, protists and parasitic helminths. It is, however, not fully understood how many different types of pathogens they can sense or what exact molecular mechanisms they employ to initiate targeted responses. In this study, we found that an anaerobic pathobiont microbe, Ruminococcus gnavus (R. gnavus), can induce tuft cell proliferation in the proximal colon whereas the microbe’s lysate can stimulate these proximal colonic tuft cells to release interleukin-25 (IL-25). Nullification of the Gng13 and Trpm5 genes that encode the G protein subunit Gγ13 and transient receptor potential ion channel Trpm5, respectively, or application of the Tas2r inhibitor allyl isothiocyanate (AITC), G protein Gβγ subunit inhibitor Gallein or the phospholipase Cβ2 (PLCβ2) inhibitor U73122 reduces R. gnavus-elicited tuft cell proliferation or IL-25 release or both. Furthermore, Gng13 conditional knockout or Trpm5 knockout diminishes the expression of gasdermins C2, C3 and C4, and concomitantly increases the activated forms of caspases 3, 8 and 9 as well as the number of TUNEL-positive apoptotic cells in the proximal colon. Together, our data suggest that taste signal transduction pathways are not only involved in the detection of R. gnavus infection, but also contribute to helping maintain gasdermin expression and prevent apoptotic cell death in the proximal colon,

Mass Spectrometry X-ray Crystallography Liu Lab

SARS-CoV-2 reservoir in post-acute sequelae of COVID-19 (PASC)

Proal AD, VanElzakker MB, Aleman S, Bach K, Boribong BP, Buggert M, Cherry S, Chertow DS, Davies HE, Dupont CL, Deeks SG, Eimer W, Ely EW, Fasano A, Freire M, Geng LN, Griffin DE, Henrich TJ, Iwasaki A, Izquierdo-Garcia D, Locci M, Mehandru S, Painter MM, Peluso MJ, Pretorius E, Price DA, Putrino D, Scheuermann RH, Tan GS, Tanzi RE, VanBrocklin HF, Yonker LM, Wherry EJ. Author Correction: SARS-CoV-2 reservoir in post-acute sequelae of COVID-19 (PASC). Nat Immunol. 2023 Oct;24(10):1778. doi: 10.1038/s41590-023-01646-3. Erratum for: Nat Immunol. 2023 Oct;24(10):1616-1627. PMID: 37723351.

Abstract

Millions of people are suffering from Long COVID or post-acute sequelae of COVID-19 (PASC). Several biological factors have emerged as potential drivers of PASC pathology. Some individuals with PASC may not fully clear the coronavirus SARS-CoV-2 after acute infection. Instead, replicating virus and/or viral RNA-potentially capable of being translated to produce viral proteins-persist in tissue as a ‘reservoir’. This reservoir could modulate host immune responses or release viral proteins into the circulation. Here we review studies that have identified SARS-CoV-2 RNA/protein or immune responses indicative of a SARS-CoV-2 reservoir in PASC samples. Mechanisms by which a SARS-CoV-2 reservoir may contribute to PASC pathology, including coagulation, microbiome and neuroimmune abnormalities, are delineated. We identify research priorities to guide the further study of a SARS-CoV-2 reservoir in PASC, with the goal that clinical trials of antivirals or other therapeutics with potential to clear a SARS-CoV-2 reservoir are accelerated.

Chemical Biology Cherry Lab Protein Production Services

A Chicken Tapasin ortholog can chaperone empty HLA-B∗37:01 molecules independent of other peptide-loading components

Papadaki GF, Woodward CH, Young MC, Winters TJ, Burslem GM, Sgourakis NG. J. Biol. Chem. 2023 Oct 1. doi: 10.1016/j.jbc.2023.105136.

Human Tapasin (hTapasin) is the main chaperone of MHC-I molecules, enabling peptide loading and antigen repertoire optimization across HLA allotypes. However, it is restricted to the endoplasmic reticulum (ER) lumen as part of the protein loading complex (PLC), and therefore is highly unstable when expressed in recombinant form. Additional stabilizing co-factors such as ERp57 are required to catalyze peptide exchange in vitro, limiting uses for the generation of pMHC-I molecules of desired antigen specificities. Here, we show that the chicken Tapasin (chTapasin) ortholog can be expressed recombinantly at high yields in a stable form, independent of co-chaperones. chTapasin can bind the human HLA-B∗37:01 with low micromolar-range affinity to form a stable tertiary complex. Biophysical characterization by methyl-based NMR methods reveals that chTapasin recognizes a conserved β2m epitope on HLA-B∗37:01, consistent with previously solved X-ray structures of hTapasin. Finally, we provide evidence that the B∗37:01/chTapasin complex is peptide-receptive and can be dissociated upon binding of high-affinity peptides. Our results highlight the use of chTapasin as a stable scaffold for protein engineering applications aiming to expand the ligand exchange function on human MHC-I and MHC-like molecules.

Computational Biology NMR X-ray Crystallography SgourakisLab

Revealing Drivers for Carboxy- S-adenosyl-l-methionine Use by Neomorphic Variants of a DNA Methyltransferase

Loo CE, Hix MA, Wang T, Cisneros GA, Kohli RM. Revealing Drivers for Carboxy-S-adenosyl-l-methionine Use by Neomorphic Variants of a DNA Methyltransferase. ACS Chem Biol. 2023 Oct 20;18(10):2224-2232. doi: 10.1021/acschembio.3c00184. Epub 2023 Jun 28. PMID: 37379458; PMCID: PMC10592258.

Abstract

Methylation of DNA plays a key role in diverse biological processes spanning from bacteria to mammals. DNA methyltransferases (MTases) typically employ S-adenosyl-l-methionine (SAM) as a critical cosubstrate and the relevant methyl donor for modification of the C5 position of cytosine. Recently, work on the CpG-specific bacterial MTase, M.MpeI, has shown that a single N374K point mutation can confer the enzyme with the neomorphic ability to use the sparse, naturally occurring metabolite carboxy-S-adenosyl-l-methionine (CxSAM) in order to generate the unnatural DNA modification, 5-carboxymethylcytosine (5cxmC). Here, we aimed to investigate the mechanistic basis for this DNA carboxymethyltransferase (CxMTase) activity by employing a combination of computational modeling and in vitro characterization. Modeling of substrate interactions with the enzyme variant allowed us to identify a favorable salt bridge between CxSAM and N374K that helps to rationalize selectivity of the CxMTase. Unexpectedly, we also discovered a potential role for a key active site E45 residue that makes a bidentate interaction with the ribosyl sugar of CxSAM, located on the opposite face of the CxMTase active site. Prompted by these modeling results, we further explored the space-opening E45D mutation and found that the E45D/N374K double mutant in fact inverts selectivity, preferring CxSAM over SAM in biochemical assays. These findings provide new insight into CxMTase active site architecture and may offer broader utility given the numerous opportunities offered by using SAM analogs for selective molecular labeling in concert with nucleic acid or even protein-modifying MTases.

Chemical Biology CryoEM and CryoET Kohli Lab

Molecular hybridization strategy for tuning bioactive peptide function

Pedron CN, Torres MT, Oliveira CS, Silva AF, Andrade GP, Wang Y, Pinhal MAS, Cerchiaro G, da Silva Junior PI, da Silva FD, Radhakrishnan R, de la Fuente-Nunez C, Oliveira Junior VX. Molecular hybridization strategy for tuning bioactive peptide function. Commun Biol. 2023 Oct 19;6(1):1067. doi: 10.1038/s42003-023-05254-7. PMID: 37857855; PMCID: PMC10587126.

Abstract

The physicochemical and structural properties of antimicrobial peptides (AMPs) determine their mechanism of action and biological function. However, the development of AMPs as therapeutic drugs has been traditionally limited by their toxicity for human cells. Tuning the physicochemical properties of such molecules may abolish toxicity and yield synthetic molecules displaying optimal safety profiles and enhanced antimicrobial activity. Here, natural peptides were modified to improve their activity by the hybridization of sequences from two different active peptide sequences. Hybrid AMPs (hAMPs) were generated by combining the amphipathic faces of the highly toxic peptide VmCT1, derived from scorpion venom, with parts of four other naturally occurring peptides having high antimicrobial activity and low toxicity against human cells. This strategy led to the design of seven synthetic bioactive variants, all of which preserved their structure and presented increased antimicrobial activity (3.1-128 μmol L-1). Five of the peptides (three being hAMPs) presented high antiplasmodial at 0.8 μmol L-1, and virtually no undesired toxic effects against red blood cells. In sum, we demonstrate that peptide hybridization is an effective strategy for redirecting biological activity to generate novel bioactive molecules with desired properties.

Computational Biology Radhakrishnan Lab

Biological Variation in Biochemistry Analytes in Laboratory Guinea Pigs ( Cavia porcellus)

Rossi G, Liu KF, Kershaw H, Riddell D, Hyndman TH, Monks D, Musk GC. Biological Variation in Biochemistry Analytes in Laboratory Guinea Pigs (Cavia porcellus). Vet Sci. 2023 Oct 17;10(10):621. doi: 10.3390/vetsci10100621. PMID: 37888573; PMCID: PMC10610888.

Abstract

Biological variation (BV) describes the physiological random fluctuation around a homeostatic set point, which is a characteristic of all blood measurands (analytes). That variation may impact the clinical relevance of the changes that are observed in the serial results for an individual. Biological variation is represented mathematically by the coefficient of variation (CV) and occurs within each individual (CVI) and between individuals in a population (CVG). Biological variation data can be used to assess whether population-based reference or subject-based reference intervals should be used for the interpretation of laboratory results through the calculation of the index of individuality (IoI). This study aimed to determine the biological variations, calculate the IoI and reference change values (RCV) of clinical chemistry analytes in an outbred strain colony of Hartley guinea pigs (GPs), and set the quality specifications for clinical chemistry analytes. Blood was collected from 16 healthy adult laboratory colony GPs via jugular venipuncture at weekly intervals over six weeks. All the samples were frozen and analyzed in a single run. Analytical, CVI, and CVG biological variations, together with the IoI and RCV, were calculated for each measurand. Based on the estimated BV, the calculated IoI was low for glucose, so individual reference intervals (RCV) should be used. The majority of the measurands should be interpreted using both population-based and subject-based reference intervals as the IoIs were intermediate.

Mass Spectrometry X-ray Crystallography Liu Lab

Serotonin reduction in post-acute sequelae of viral infection

Wong AC, Devason AS, Umana IC, Cox TO, Dohnalová L, Litichevskiy L, Perla J, Lundgren P, Etwebi Z, Izzo LT, Kim J, Tetlak M, Descamps HC, Park SL, Wisser S, McKnight AD, Pardy RD, Kim J, Blank N, Patel S, Thum K, Mason S, Beltra JC, Michieletto MF, Ngiow SF, Miller BM, Liou MJ, Madhu B, Dmitrieva-Posocco O, Huber AS, Hewins P, Petucci C, Chu CP, Baraniecki-Zwil G, Giron LB, Baxter AE, Greenplate AR, Kearns C, Montone K, Litzky LA, Feldman M, Henao-Mejia J, Striepen B, Ramage H, Jurado KA, Wellen KE, O’Doherty U, Abdel-Mohsen M, Landay AL, Keshavarzian A, Henrich TJ, Deeks SG, Peluso MJ, Meyer NJ, Wherry EJ, Abramoff BA, Cherry S, Thaiss CA, Levy M. Serotonin reduction in post-acute sequelae of viral infection. Cell. 2023 Oct 26;186(22):4851-4867.e20. doi: 10.1016/j.cell.2023.09.013. Epub 2023 Oct 16. PMID: 37848036.

Abstract Post-acute sequelae of

Post-acute sequelae of COVID-19 (PASC, “Long COVID”) pose a significant global health challenge. The pathophysiology is unknown, and no effective treatments have been found to date. Several hypotheses have been formulated to explain the etiology of PASC, including viral persistence, chronic inflammation, hypercoagulability, and autonomic dysfunction. Here, we propose a mechanism that links all four hypotheses in a single pathway and provides actionable insights for therapeutic interventions. We find that PASC are associated with serotonin reduction. Viral infection and type I interferon-driven inflammation reduce serotonin through three mechanisms: diminished intestinal absorption of the serotonin precursor tryptophan; platelet hyperactivation and thrombocytopenia, which impacts serotonin storage; and enhanced MAO-mediated serotonin turnover. Peripheral serotonin reduction, in turn, impedes the activity of the vagus nerve and thereby impairs hippocampal responses and memory. These findings provide a possible explanation for neurocognitive symptoms associated with viral persistence in Long COVID, which may extend to other post-viral syndromes.

Chemical Biology Cherry Lab Protein Production Services

Difluoromethyl-1,3,4-oxadiazoles Are Selective, Mechanism-Based, and Essentially Irreversible Inhibitors of Histone Deacetylase 6

König B, Watson PR, Reßing N, Cragin AD, Schäker-Hübner L, Christianson DW, Hansen FK. Difluoromethyl-1,3,4-oxadiazoles Are Selective, Mechanism-Based, and Essentially Irreversible Inhibitors of Histone Deacetylase 6. J Med Chem. 2023 Oct 12;66(19):13821-13837. doi: 10.1021/acs.jmedchem.3c01345. Epub 2023 Oct 2. PMID: 37782298; PMCID: PMC10591924.

Abstract Histone deacetylase

Histone deacetylase 6 (HDAC6) is an important drug target in oncological and non-oncological diseases. Most available HDAC6 inhibitors (HDAC6i) utilize hydroxamic acids as a zinc-binding group, which limits therapeutic opportunities due to its genotoxic potential. Recently, difluoromethyl-1,3,4-oxadiazoles (DFMOs) were reported as potent and selective HDAC6i but their mode of inhibition remained enigmatic. Herein, we report that DFMOs act as mechanism-based and essentially irreversible HDAC6i. Biochemical data confirm that DFMO 6 is a tight-binding HDAC6i capable of inhibiting HDAC6 via a two-step slow-binding mechanism. Crystallographic and mechanistic experiments suggest that the attack of 6 by the zinc-bound water at the sp2 carbon closest to the difluoromethyl moiety followed by a subsequent ring opening of the oxadiazole yields deprotonated difluoroacetylhydrazide 13 as active species. The strong anionic zinc coordination of 13 and the binding of the difluoromethyl moiety in the P571 pocket finally result in an essentially irreversible inhibition of HDAC6.

CryoEM and CryoET Christianson Group X-ray Crystallography

The synergistic effect of full-spectrum light therapy and transient immunosuppressants prolonged allotransplant survival

Liu KF, Ramachandran S, Chang CW, Chen RF, Huang CH, Huang HT, Lee CC, Li YT, Kuo YR. The synergistic effect of full-spectrum light therapy and transient immunosuppressants prolonged allotransplant survival. Plast Reconstr Surg. 2023 Oct 10. doi: 10.1097/PRS.0000000000011135. Epub ahead of print. PMID: 37815307.

Abstract

Background: The lifelong administration of immunosuppressants remains its largest drawback in vascularized composite allotransplantation (VCA). Therefore, developing alternative strategies to minimize the long-term use of immunosuppressive agents is crucial. This study investigated whether full-spectrum bright light therapy (FBLT) combined with short-term immunosuppressant therapy could prolong VCA survival in a rodent hindlimb model.

Mass Spectrometry X-ray Crystallography Liu Lab

HLA3DB: comprehensive annotation of peptide/HLA complexes enables blind structure prediction of T cell epitopes

Gupta S, Nerli S, Kutti Kandy S, Mersky GL, Sgourakis NG. Nat. Commun. 2023 Oct 10. doi: 10.1038/s41467-023-42163-z.

Abstract

The class I proteins of the major histocompatibility complex (MHC-I) display epitopic peptides derived from endogenous proteins on the cell surface for immune surveillance. Accurate modeling of peptides bound to the human MHC, HLA, has been mired by conformational diversity of the central peptide residues, which are critical for recognition by T cell receptors. Here, analysis of X-ray crystal structures within our curated database (HLA3DB) shows that pHLA complexes encompassing multiple HLA allotypes present a discrete set of peptide backbone conformations. Leveraging these backbones, we employ a regression model trained on terms of a physically relevant energy function to develop a comparative modeling approach for nonamer pHLA structures named RepPred. Our method outperforms the top pHLA modeling approach by up to 19% in structural accuracy, and consistently predicts blind targets not included in our training set. Insights from our work may be applied towards predicting antigen immunogenicity, and receptor cross-reactivity.

Computational Biology NMR X-ray Crystallography SgourakisLab

A kinesin-1 adaptor complex controls bimodal slow axonal transport of spectrin in Caenorhabditis elegans

Glomb O, Swaim G, Munoz LLancao P, Lovejoy C, Sutradhar S, Park J, Wu Y, Cason SE, Holzbaur ELF, Hammarlund M, Howard J, Ferguson SM, Gramlich MW, Yogev S. A kinesin-1 adaptor complex controls bimodal slow axonal transport of spectrin in Caenorhabditis elegans. Dev Cell. 2023 Oct 9;58(19):1847-1863.e12. doi: 10.1016/j.devcel.2023.08.031. Epub 2023 Sep 25. PMID: 37751746; PMCID: PMC10574138.

Abstract

An actin-spectrin lattice, the membrane periodic skeleton (MPS), protects axons from breakage. MPS integrity relies on spectrin delivery via slow axonal transport, a process that remains poorly understood. We designed a probe to visualize endogenous spectrin dynamics at single-axon resolution in vivo. Surprisingly, spectrin transport is bimodal, comprising fast runs and movements that are 100-fold slower than previously reported. Modeling and genetic analysis suggest that the two rates are independent, yet both require kinesin-1 and the coiled-coil proteins UNC-76/FEZ1 and UNC-69/SCOC, which we identify as spectrin-kinesin adaptors. Knockdown of either protein led to disrupted spectrin motility and reduced distal MPS, and UNC-76 overexpression instructed excessive transport of spectrin. Artificially linking spectrin to kinesin-1 drove robust motility but inefficient MPS assembly, whereas impairing MPS assembly led to excessive spectrin transport, suggesting a balance between transport and assembly. These results provide insight into slow axonal transport and MPS integrity.

CryoEM and CryoET Single Molecule Imaging Holzbaur Lab

Conformation of actin subunits at the barbed and pointed ends of F-actin with and without capping proteins

Barrie KR, Carman PJ, Dominguez R. Conformation of actin subunits at the barbed and pointed ends of F-actin with and without capping proteins. Cytoskeleton (Hoboken). 2023 Sep-Oct;80(9-10):309-312. doi: 10.1002/cm.21770. Epub 2023 Aug 26. PMID: 37632366; PMCID: PMC10592188.

Abstract

Advances in cryo-electron microscopy have made possible the determination of structures of the barbed and pointed ends of F-actin, both in the absence and the presence of capping proteins that block subunit exchange. The conformation of the two exposed protomers at the barbed end resembles the “flat” conformation of protomers in the middle of F-actin. The barbed end changes little upon binding of CapZ, which in turn undergoes a major conformational change. At the pointed end, however, protomers have the “twisted” conformation characteristic of G-actin, whereas tropomodulin binding forces a flat conformation upon the second subunit. The structures provide a mechanistic understanding for the asymmetric addition/dissociation of actin subunits at the ends of F-actin and open the way to future studies of other regulators of filament end dynamics.

CryoEM and CryoET Dominguez Lab X-ray Crystallography

Photoactivated Protein Degrader for Optical Control of Synaptic Function

Ko T, Jou C, Grau-Perales AB, Reynders M, Fenton AA, Trauner D. Photoactivated Protein Degrader for Optical Control of Synaptic Function. ACS Chem Neurosci. 2023 Oct 4;14(19):3704-3713. doi: 10.1021/acschemneuro.3c00390. Epub 2023 Sep 15. PMID: 37712589; PMCID: PMC10557063.

Abstract

Hundreds of proteins determine the function of synapses, and synapses define the neuronal circuits that subserve myriad brain, cognitive, and behavioral functions. It is thus necessary to precisely manipulate specific proteins at specific sub-cellular locations and times to elucidate the roles of particular proteins and synapses in brain function. We developed PHOtochemically TArgeting Chimeras (PHOTACs) as a strategy to optically degrade specific proteins with high spatial and temporal precision. PHOTACs are small molecules that, upon wavelength-selective illumination, catalyze ubiquitylation and degradation of target proteins through endogenous proteasomes. Here, we describe the design and chemical properties of a PHOTAC that targets Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα), which is abundant and crucial for the baseline synaptic function of excitatory neurons. We validate the PHOTAC strategy, showing that the CaMKIIα-PHOTAC is effective in mouse brain tissue. Light activation of CaMKIIα-PHOTAC removed CaMKIIα from regions of the mouse hippocampus only within 25 μm of the illuminated brain surface. The optically controlled degradation decreases synaptic function within minutes of light activation, measured by the light-initiated attenuation of evoked field excitatory postsynaptic potential (fEPSP) responses to physiological stimulation. The PHOTACs methodology should be broadly applicable to other key proteins implicated in synaptic function, especially for evaluating their precise roles in the maintenance of long-term potentiation and memory within subcellular dendritic domains.

Chemical Biology CryoEM and CryoET Trauner Group

Report of the Assay Guidance Workshop on 3-Dimensional Tissue Models for Antiviral Drug Development

Jordan R, Ford-Scheimer SL, Alarcon RM, Atala A, Borenstein JT, Brimacombe KR, Cherry S, Clevers H, Davis MI, Funnell SGP, Gehrke L, Griffith LG, Grossman AC, Hartung T, Ingber DE, Kleinstreuer NC, Kuo CJ, Lee EM, Mummery CL, Pickett TE, Ramani S, Rosado-Olivieri EA, Struble EB, Wan Z, Williams MS, Hall MD, Ferrer M, Markossian S. Report of the Assay Guidance Workshop on 3-Dimensional Tissue Models for Antiviral Drug Development. J Infect Dis. 2023 Oct 3;228(Suppl 5):S337-S354. doi: 10.1093/infdis/jiad334. PMID: 37669225; PMCID: PMC10547463.

Abstract The National Center

The National Center for Advancing Translational Sciences (NCATS) Assay Guidance Manual (AGM) Workshop on 3D Tissue Models for Antiviral Drug Development, held virtually on 7-8 June 2022, provided comprehensive coverage of critical concepts intended to help scientists establish robust, reproducible, and scalable 3D tissue models to study viruses with pandemic potential. This workshop was organized by NCATS, the National Institute of Allergy and Infectious Diseases, and the Bill and Melinda Gates Foundation. During the workshop, scientific experts from academia, industry, and government provided an overview of 3D tissue models’ utility and limitations, use of existing 3D tissue models for antiviral drug development, practical advice, best practices, and case studies about the application of available 3D tissue models to infectious disease modeling. This report includes a summary of each workshop session as well as a discussion of perspectives and challenges related to the use of 3D tissues in antiviral drug discovery.

Chemical Biology Cherry Lab Protein Production Services

Visualizing Bacterial Infections With Novel Targeted Molecular Imaging Approaches

Chen X, Gallagher F, Sellmyer MA, Ordonez AA, Kjaer A, Ohliger M, Wilson DM, Jain SK. Visualizing Bacterial Infections With Novel Targeted Molecular Imaging Approaches. J Infect Dis. 2023 Oct 3;228(Suppl 4):S249-S258. doi: 10.1093/infdis/jiad078. PMID: 37788506; PMCID: PMC10547462.

Abstract

Although nearly a century has elapsed since the discovery of penicillin, bacterial infections remain a major global threat. Global antibiotic use resulted in an astounding 42 billion doses of antibiotics administered in 2015 with 128 billion annual doses expected by 2030. This overuse of antibiotics has led to the selection of multidrug-resistant “super-bugs,” resulting in increasing numbers of patients being susceptible to life-threatening infections with few available therapeutic options. New clinical tools are therefore urgently needed to identify bacterial infections and monitor response to antibiotics, thereby limiting overuse of antibiotics and improving overall health. Next-generation molecular imaging affords unique opportunities to target and identify bacterial infections, enabling spatial characterization as well as noninvasive, temporal monitoring of the natural course of the disease and response to therapy. These emerging noninvasive imaging approaches could overcome several limitations of current tools in infectious disease, such as the need for biological samples for testing with their associated sampling bias. Imaging of living bacteria can also reveal basic biological insights about their behavior in vivo.

Chemical Biology Sellmyer Lab

Crystal structure of histone deacetylase 6 complexed with (R)-lipoic acid, an essential cofactor in central carbon metabolism

Watson PR, Stollmaier JG, Christianson DW. Crystal structure of histone deacetylase 6 complexed with (R)-lipoic acid, an essential cofactor in central carbon metabolism. J Biol Chem. 2023 Oct;299(10):105228. doi: 10.1016/j.jbc.2023.105228. Epub 2023 Sep 12. PMID: 37703993; PMCID: PMC10622836.

Abstract

The enzyme cofactor (R)-lipoic acid plays a critical role in central carbon metabolism due to its catalytic function in the generation of acetyl-CoA, which links glycolysis with the tricarboxylic acid cycle. This cofactor is also essential for the generation of succinyl CoA within the tricarboxylic acid cycle. However, the biological functions of (R)-lipoic acid extend beyond metabolism owing to its facile redox chemistry. Most recently, the reduced form of (R)-lipoic acid, (R)-dihydrolipoic acid, has been shown to inhibit histone deacetylases (HDACs) with selectivity for the inhibition of HDAC6. Here, we report the 2.4 Å-resolution X-ray crystal structure of the complex between (R)-dihydrolipoic acid and HDAC6 catalytic domain 2 from Danio rerio, and we report a dissociation constant (KD) of 350 nM for this complex as determined by isothermal titration calorimetry. The crystal structure illuminates key affinity determinants in the enzyme active site, including thiolate-Zn2+ coordination and S-π interactions in the F583-F643 aromatic crevice. This study provides the first visualization of the connection between HDAC function and the biological response to oxidative stress: the dithiol moiety of (R)-dihydrolipoic acid can serve as a redox-regulated pharmacophore capable of simultaneously targeting the catalytic Zn2+ ion and the aromatic crevice in the active site of HDAC6.

CryoEM and CryoET Christianson Group X-ray Crystallography

Combining non-canonical amino acid mutagenesis and native chemical ligation for multiply modifying proteins: A case study of α-synuclein post-translational modifications

Galesic A, Pan B, Ramirez J, Rhoades E, Pratt MR, Petersson EJ. Combining non-canonical amino acid mutagenesis and native chemical ligation for multiply modifying proteins: A case study of α-synuclein post-translational modifications. Methods. 2023 Oct;218:101-109. doi: 10.1016/j.ymeth.2023.08.002. Epub 2023 Aug 6. PMID: 37549799; PMCID: PMC10657485.

Abstract

The Parkinson’s disease associated protein α-synuclein (αS) has been found to contain numerous post-translational modifications (PTMs), in both physiological and pathological states. One PTM site of particular interest is serine 87, which is subject to both O-linked β-N-acetylglucosamine (gS) modification and phosphorylation (pS), with αS-pS87 enriched in Parkinson’s disease. An often-overlooked aspect of these PTMs is their effect on the membrane-binding properties of αS, which are important to its role in regulating neurotransmitter release. Here, we show how one can study these effects by synthesizing αS constructs containing authentic PTMs and labels for single molecule fluorescence correlation spectroscopy measurements. We synthesize αS-gS87 and αS-pS87 by combining native chemical ligation with genetic code expansion approaches. We introduce the fluorophore by a click reaction with a non-canonical amino acid. Beyond the specific problem of PTM effects on αS, our studies highlight the value of this combination of methods for multiply modifying proteins.

Single Molecule Imaging Rhoades Lab

The Effects of Lipids on α-Synuclein Aggregation In Vitro

Ramirez J, Pancoe SX, Rhoades E, Petersson EJ. The Effects of Lipids on α-Synuclein Aggregation In Vitro. Biomolecules. 2023 Oct 2;13(10):1476. doi: 10.3390/biom13101476. PMID: 37892158; PMCID: PMC10604467.

Abstract

The small neuronal protein α-synuclein (αS) is found in pre-synaptic terminals and plays a role in vesicle recycling and neurotransmission. Fibrillar aggregates of αS are the hallmark of Parkinson’s disease and related neurodegenerative disorders. In both health and disease, interactions with lipids influence αS’s structure and function, prompting much study of the effects of lipids on αS aggregation. A comprehensive collection (126 examples) of aggregation rate data for various αS/lipid combinations was presented, including combinations of lipid variations and mutations or post-translational modifications of αS. These data were interpreted in terms of lipid structure to identify general trends. These tabulated data serve as a resource for the community to help in the interpretation of aggregation experiments with lipids and to be potentially used as inputs for computational models of lipid effects on aggregation.

Single Molecule Imaging Rhoades Lab

A Chicken Tapasin ortholog can chaperone empty HLA-B∗37:01 molecules independent of other peptide-loading components

Papadaki GF, Woodward CH, Young MC, Winters TJ, Burslem GM, Sgourakis NG. A Chicken Tapasin ortholog can chaperone empty HLA-B∗37:01 molecules independent of other peptide-loading components. J Biol Chem. 2023 Oct;299(10):105136. doi: 10.1016/j.jbc.2023.105136. Epub 2023 Aug 4. PMID: 37543367; PMCID: PMC10534222.

Abstract

Human Tapasin (hTapasin) is the main chaperone of MHC-I molecules, enabling peptide loading and antigen repertoire optimization across HLA allotypes. However, it is restricted to the endoplasmic reticulum (ER) lumen as part of the protein loading complex (PLC), and therefore is highly unstable when expressed in recombinant form. Additional stabilizing co-factors such as ERp57 are required to catalyze peptide exchange in vitro, limiting uses for the generation of pMHC-I molecules of desired antigen specificities. Here, we show that the chicken Tapasin (chTapasin) ortholog can be expressed recombinantly at high yields in a stable form, independent of co-chaperones. chTapasin can bind the human HLA-B∗37:01 with low micromolar-range affinity to form a stable tertiary complex. Biophysical characterization by methyl-based NMR methods reveals that chTapasin recognizes a conserved β2m epitope on HLA-B∗37:01, consistent with previously solved X-ray structures of hTapasin. Finally, we provide evidence that the B∗37:01/chTapasin complex is peptide-receptive and can be dissociated upon binding of high-affinity peptides. Our results highlight the use of chTapasin as a stable scaffold for protein engineering applications aiming to expand the ligand exchange function on human MHC-I and MHC-like molecules.

Chemical Biology Burslem Lab Mass Spectrometry

Endocytic myosin-1 is a force-insensitive, power-generating motor

Pedersen RTA, Snoberger A, Pyrpassopoulos S, Safer D, Drubin DG, Ostap EM. Endocytic myosin-1 is a force-insensitive, power-generating motor. J Cell Biol. 2023 Oct 2;222(10):e202303095. doi: 10.1083/jcb.202303095. Epub 2023 Aug 7. PMID: 37549220; PMCID: PMC10406613.

Abstract

Myosins are required for clathrin-mediated endocytosis, but their precise molecular roles in this process are not known. This is, in part, because the biophysical properties of the relevant motors have not been investigated. Myosins have diverse mechanochemical activities, ranging from powerful contractility against mechanical loads to force-sensitive anchoring. To better understand the essential molecular contribution of myosin to endocytosis, we studied the in vitro force-dependent kinetics of the Saccharomyces cerevisiae endocytic type I myosin called Myo5, a motor whose role in clathrin-mediated endocytosis has been meticulously studied in vivo. We report that Myo5 is a low-duty-ratio motor that is activated ∼10-fold by phosphorylation and that its working stroke and actin-detachment kinetics are relatively force-insensitive. Strikingly, the in vitro mechanochemistry of Myo5 is more like that of cardiac myosin than that of slow anchoring myosin-1s found on endosomal membranes. We, therefore, propose that Myo5 generates power to augment actin assembly-based forces during endocytosis in cells.

CryoEM and CryoET Single Molecule Imaging Ostap Lab

Low-input and single-cell methods for Infinium DNA methylation BeadChips

Lee SM, Loo CE, Prasasya RD, Bartolomei MS, Kohli RM, Zhou W. Low-input and single-cell methods for Infinium DNA methylation BeadChips. bioRxiv [Preprint]. 2023 Sep 22:2023.09.18.558252. doi: 10.1101/2023.09.18.558252. PMID: 37786695; PMCID: PMC10541608.

Abstract

The Infinium BeadChip is the most widely used DNA methylome assay technology for population-scale epigenome profiling. However, the standard workflow requires over 200 ng of input DNA, hindering its application to small cell-number samples, such as primordial germ cells. We developed experimental and analysis workflows to extend this technology to suboptimal input DNA conditions, including ultra-low input down to single cells. DNA preamplification significantly enhanced detection rates to over 50% in five-cell samples and ∼25% in single cells. Enzymatic conversion also substantially improved data quality. Computationally, we developed a method to model the background signal’s influence on the DNA methylation level readings. The modified detection p -values calculation achieved higher sensitivities for low-input datasets and was validated in over 100,000 public datasets with diverse methylation profiles. We employed the optimized workflow to query the demethylation dynamics in mouse primordial germ cells available at low cell numbers. Our data revealed nuanced chromatin states, sex disparities, and the role of DNA methylation in transposable element regulation during germ cell development. Collectively, we present comprehensive experimental and computational solutions to extend this widely used methylation assay technology to applications with limited DNA.

Chemical Biology CryoEM and CryoET Kohli Lab

Intrinsically disordered regions in TRPV2 mediate protein-protein interactions

Raghavendar R Sanganna Gari 1Grigory Tagiltsev 1Ruth A Pumroy 2 3Yining Jiang 1 4Martin Blackledge 5Vera Y Moiseenkova-Bell 2 3Simon Scheuring 6 7 . Commun Biol. 2023 Sep 22;6(1):966. doi: 10.1038/s42003-023-05343-7.

Abstract

Transient receptor potential (TRP) ion channels are gated by diverse intra- and extracellular stimuli leading to cation inflow (Na+, Ca2+) regulating many cellular processes and initiating organismic somatosensation. Structures of most TRP channels have been solved. However, structural and sequence analysis showed that ~30% of the TRP channel sequences, mainly the N- and C-termini, are intrinsically disordered regions (IDRs). Unfortunately, very little is known about IDR ‘structure’, dynamics and function, though it has been shown that they are essential for native channel function. Here, we imaged TRPV2 channels in membranes using high-speed atomic force microscopy (HS-AFM). The dynamic single molecule imaging capability of HS-AFM allowed us to visualize IDRs and revealed that N-terminal IDRs were involved in intermolecular interactions. Our work provides evidence about the ‘structure’ of the TRPV2 IDRs, and that the IDRs may mediate protein-protein interactions.

Subject terms: Atomic force microscopy, Ion transport

 

 

 

CryoEM and CryoET Mass Spectrometry Vera Moiseenkova-Bell Lab

Structure and Function of Kdac1, a Class II Deacetylase from the Multidrug-Resistant Pathogen Acinetobacter baumannii

Watson PR, Christianson DW. Structure and Function of Kdac1, a Class II Deacetylase from the Multidrug-Resistant Pathogen Acinetobacter baumannii. Biochemistry. 2023 Sep 19;62(18):2689-2699. doi: 10.1021/acs.biochem.3c00288. Epub 2023 Aug 25. PMID: 37624144; PMCID: PMC10528293.

Abstract Proteomics studies indicate

Proteomics studies indicate that 10% of proteins in the opportunistic pathogen Acinetobacter baumannii are acetylated, suggesting that lysine acetyltransferases and deacetylases function to maintain and regulate a robust bacterial acetylome. As the first step in exploring these fascinating prokaryotic enzymes, we now report the preparation and characterization of the lysine deacetylase Kdac1. We show that Kdac1 catalyzes the deacetylation of free acetyllysine and acetyllysine tetrapeptide assay substrates, and we also report the X-ray crystal structures of unliganded Kdac1 as well as its complex with the hydroxamate inhibitor Citarinostat. Kdac1 is a tetramer in solution and in the crystal; the crystal structure reveals that the L1 loop functions to stabilize quaternary structure, forming inter-subunit hydrogen bonds and salt bridges around a central arginine residue (R30). Surprisingly, the L1 loop partially blocks entry to the active site, but it is sufficiently flexible to allow for the binding of two Citarinostat molecules in the active site. The L12 loop is also important for maintaining quaternary structure; here, a conserved arginine (R278) accepts hydrogen bonds from the backbone carbonyl groups of residues in an adjacent monomer. Structural comparisons with two other prokaryotic lysine deacetylases reveal conserved residues in the L1 and L12 loops that similarly support tetramer assembly. These studies provide a structural foundation for understanding enzymes that regulate protein function in bacteria through reversible lysine acetylation, serving as a first step in the exploration of these enzymes as possible targets for the development of new antibiotics.

CryoEM and CryoET Christianson Group X-ray Crystallography

Direct Measurements of FLASH-Induced Changes in Intracellular Oxygenation

El Khatib M, Motlagh AO, Beyer JN, Troxler T, Allu SR, Sun Q, Burslem GM, Vinogradov SA. Direct Measurements of FLASH-Induced Changes in Intracellular Oxygenation. Int J Radiat Oncol Biol Phys. 2023 Sep 18:S0360-3016(23)07931-2. doi: 10.1016/j.ijrobp.2023.09.019. Epub ahead of print. PMID: 37729972.

Abstract

Purpose: The goal of our study was to characterize the dynamics of intracellular oxygen during application of radiation at conventional (CONV) and FLASH dose rates and obtain evidence for or against the oxygen depletion hypothesis as a mechanism of the FLASH effect.

Chemical Biology Burslem Lab Mass Spectrometry

Native ultrastructure of fresh human brain vitrified directly from autopsy revealed by cryo-electron tomography with cryo-plasma focused ion beam milling

Creekmore BC, Kixmoeller K, Black BE, Lee EB, Chang YW. Native ultrastructure of fresh human brain vitrified directly from autopsy revealed by cryo-electron tomography with cryo-plasma focused ion beam milling. bioRxiv [Preprint]. 2023 Sep 17:2023.09.13.557623. doi: 10.1101/2023.09.13.557623. PMID: 37745569; PMCID: PMC10516044.

Abstract

Ultrastructure of human brain tissue has traditionally been examined using electron microscopy (EM) following chemical fixation, staining, and mechanical sectioning, which limit attainable resolution and introduce artifacts. Alternatively, cryo-electron tomography (cryo-ET) offers the potential to image unfixed cellular samples at higher resolution while preserving their native structures, but it requires samples to be frozen free from crystalline ice and thin enough to image via transmission EM. Due to these requirements, cryo-ET has yet to be employed to investigate the native ultrastructure of unfixed, never previously frozen human brain tissue. Here we present a method for generating lamellae in human brain tissue obtained at time of autopsy that can be imaged via cryo-ET. We vitrify the tissue directly on cryo-EM grids via plunge-freezing, as opposed to high pressure freezing which is generally used for thick samples. Following vitrification, we use xenon plasma focused ion beam (FIB) milling to generate lamellae directly on-grid. In comparison to gallium FIB, which is commonly used for biological samples, xenon plasma FIB is powerful enough to efficiently mill large volume samples, such as human brain tissue. Additionally, our approach allows for lamellae to be generated at variable depth inside the tissue as opposed to being limited to starting at the surface of the tissue. Lamellae generated in Alzheimer’s disease brain tissue and imaged by cryo-ET reveal intact subcellular structures including components of autophagy and potential tau fibrils. Furthermore, we visualize myelin revealing intact compact myelin and functional cytoplasmic expansions such as cytoplasmic channels and the inner tongue. From these images we also measure the dimensions of myelin membranes,

Black Lab CryoEM and CryoET Mass Spectrometry Yi-Wei Chang Lab

Pooled endogenous protein tagging and recruitment for scalable discovery of effectors for induced proximity therapeutics

Serebrenik YV, Mani D, Maujean T, Burslem GM, Shalem O. Pooled endogenous protein tagging and recruitment for scalable discovery of effectors for induced proximity therapeutics. Res Sq [Preprint]. 2023 Sep 13:rs.3.rs-3161717. doi: 10.21203/rs.3.rs-3161717/v1. PMID: 37790450; PMCID: PMC10543026.

Abstract

The field of induced proximity therapeutics is in its ascendancy but is limited by a lack of scalable tools to systematically explore effector-target protein pairs in an unbiased manner. Here, we combined Scalable POoled Targeting with a LIgandable Tag at Endogenous Sites (SPOTLITES) for the high-throughput tagging of endogenous proteins, with generic small molecule-based protein recruitment to screen for novel proximity-based effectors. We apply this methodology in two orthogonal screens for targeted protein degradation: the first using fluorescence to monitor target protein levels directly, and the second using a cellular growth phenotype that depends on the degradation of an essential protein. Our screens revealed a multitude of potential new effector proteins for degradation and converged on members of the CTLH complex which we demonstrate potently induce degradation. Altogether, we introduce a platform for pooled induction of endogenous protein-protein interactions that can be used to expand our t

Chemical Biology Burslem Lab Mass Spectrometry

Centromere-specifying nucleosomes persist in aging mouse oocytes in the absence of nascent assembly

Das A, Boese KG, Tachibana K, Baek SH, Lampson MA, Black BE. Centromere-specifying nucleosomes persist in aging mouse oocytes in the absence of nascent assembly. Curr Biol. 2023 Sep 11;33(17):3759-3765.e3. doi: 10.1016/j.cub.2023.07.032. Epub 2023 Aug 14. PMID: 37582374; PMCID: PMC10528140.

Abstract

Centromeres direct genetic inheritance but are not themselves genetically encoded. Instead, centromeres are defined epigenetically by the presence of a histone H3 variant, CENP-A.1 In cultured somatic cells, an established paradigm of cell-cycle-coupled propagation maintains centromere identity: CENP-A is partitioned between sisters during replication and replenished by new assembly, which is restricted to G1. The mammalian female germ line challenges this model because of the cell-cycle arrest between pre-meiotic S phase and the subsequent G1, which can last for the entire reproductive lifespan (months to decades). New CENP-A chromatin assembly maintains centromeres during prophase I in worm and starfish oocytes,2,3 suggesting that a similar process may be required for centromere inheritance in mammals. To test this hypothesis, we developed an oocyte-specific conditional knockout (cKO) mouse for Mis18α, an essential component of the assembly machinery. We find that embryos derived from Mis18α knockout oocytes fail to assemble CENP-A nucleosomes prior to zygotic genome activation (ZGA), validating the knockout model. We show that deletion of Mis18α in the female germ line at the time of birth has no impact on centromeric CENP-A nucleosome abundance, even after 6-8 months of aging. In addition, there is no detectable detriment to fertility. Thus, centromere chromatin is maintained long-term, independent of new assembly during the extended prophase I arrest in mouse oocytes.

Black Lab CryoEM and CryoET Mass Spectrometry

Mettl3-catalyzed m6A regulates histone modifier and modification expression in self-renewing somatic tissue

Maldonado López AM, Ko EK, Huang S, Pacella G, Kuprasertkul N, D’souza CA, Reyes Hueros RA, Shen H, Stoute J, Elashal H, Sinkfield M, Anderson A, Prouty S, Li HB, Seykora JT, Liu KF, Capell BC. Mettl3-catalyzed m6A regulates histone modifier and modification expression in self-renewing somatic tissue. Sci Adv. 2023 Sep;9(35):eadg5234. doi: 10.1126/sciadv.adg5234. Epub 2023 Sep 1. PMID: 37656787; PMCID: PMC10854438.

Abstract

N6-methyladenosine (m6A) is the most abundant modification on messenger RNAs (mRNAs) and is catalyzed by methyltransferase-like protein 3 (Mettl3). To understand the role of m6A in a self-renewing somatic tissue, we deleted Mettl3 in epidermal progenitors in vivo. Mice lacking Mettl3 demonstrate marked features of dysfunctional development and self-renewal, including a loss of hair follicle morphogenesis and impaired cell adhesion and polarity associated with oral ulcerations. We show that Mettl3 promotes the m6A-mediated degradation of mRNAs encoding critical histone modifying enzymes. Depletion of Mettl3 results in the loss of m6A on these mRNAs and increases their expression and associated modifications, resulting in widespread gene expression abnormalities that mirror the gross phenotypic abnormalities. Collectively, these results have identified an additional layer of gene regulation within epithelial tissues, revealing an essential role for m6A in the regulation of chromatin modifiers, and underscoring a critical role for Mettl3-catalyzed m6A in proper epithelial development and self-renewal.

Mass Spectrometry X-ray Crystallography Liu Lab

Damaged mitochondria recruit the effector NEMO to activate NF-κB signaling

Harding O, Holzer E, Riley JF, Martens S, Holzbaur ELF. Damaged mitochondria recruit the effector NEMO to activate NF-κB signaling. Mol Cell. 2023 Sep 7;83(17):3188-3204.e7. doi: 10.1016/j.molcel.2023.08.005. PMID: 37683611; PMCID: PMC10510730.

Abstract

Failure to clear damaged mitochondria via mitophagy disrupts physiological function and may initiate damage signaling via inflammatory cascades, although how these pathways intersect remains unclear. We discovered that nuclear factor kappa B (NF-κB) essential regulator NF-κB effector molecule (NEMO) is recruited to damaged mitochondria in a Parkin-dependent manner in a time course similar to recruitment of the structurally related mitophagy adaptor, optineurin (OPTN). Upon recruitment, NEMO partitions into phase-separated condensates distinct from OPTN but colocalizing with p62/SQSTM1. NEMO recruitment, in turn, recruits the active catalytic inhibitor of kappa B kinase (IKK) component phospho-IKKβ, initiating NF-κB signaling and the upregulation of inflammatory cytokines. Consistent with a potential neuroinflammatory role, NEMO is recruited to mitochondria in primary astrocytes upon oxidative stress. These findings suggest that damaged, ubiquitinated mitochondria serve as an intracellular platform to initiate innate immune signaling, promoting the formation of activated IKK complexes sufficient to activate NF-κB signaling. We propose that mitophagy and NF-κB signaling are initiated as parallel pathways in response to mitochondrial stress.

CryoEM and CryoET Holzbaur Lab Single Molecule Imaging

Visualizing the membrane disruption action of antimicrobial peptides by cryo-electron tomography

Chen EH, Wang CH, Liao YT, Chan FY, Kanaoka Y, Uchihashi T, Kato K, Lai L, Chang YW, Ho MC, Chen RP. Visualizing the membrane disruption action of antimicrobial peptides by cryo-electron tomography. Nat Commun. 2023 Sep 6;14(1):5464. doi: 10.1038/s41467-023-41156-2. PMID: 37673860; PMCID: PMC10482868.

Abstract

The abuse of antibiotics has led to the emergence of multidrug-resistant microbial pathogens, presenting a pressing challenge in global healthcare. Membrane-disrupting antimicrobial peptides (AMPs) combat so-called superbugs via mechanisms different than conventional antibiotics and have good application prospects in medicine, agriculture, and the food industry. However, the mechanism-of-action of AMPs has not been fully characterized at the cellular level due to a lack of high-resolution imaging technologies that can capture cellular-membrane disruption events in the hydrated state. Previously, we reported PepD2M, a de novo-designed AMP with potent and wide-spectrum bactericidal and fungicidal activity. In this study, we use cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM) to directly visualize the pepD2M-induced disruption of the outer and inner membranes of the Gram-negative bacterium Escherichia coli, and compared with a well-known pore-forming peptide, melittin. Our high-resolution cryo-ET images reveal how pepD2M disrupts the E. coli membrane using a carpet/detergent-like mechanism. Our studies reveal the direct membrane-disrupting consequence of AMPs on the bacterial membrane by cryo-ET, and this information provides critical insights into the mechanisms of this class of antimicrobial agents.

CryoEM and CryoET Mass Spectrometry Yi-Wei Chang Lab

Myo-differentiation reporter screen reveals NF-Y as an activator of PAX3-FOXO1 in rhabdomyosarcoma

Sroka MW, Skopelitis D, Vermunt MW, Preall JB, El Demerdash O, de Almeida LMN, Chang K, Utama R, Gryder B, Caligiuri G, Ren D, Nalbant B, Milazzo JP, Tuveson DA, Dobin A, Hiebert SW, Stengel KR, Mantovani R, Khan J, Kohli RM, Shi J, Blobel GA, Vakoc CR. Myo-differentiation reporter screen reveals NF-Y as an activator of PAX3-FOXO1 in rhabdomyosarcoma. Proc Natl Acad Sci U S A. 2023 Sep 5;120(36):e2303859120. doi: 10.1073/pnas.2303859120. Epub 2023 Aug 28. PMID: 37639593; PMCID: PMC10483665.

Abstract

Recurrent chromosomal rearrangements found in rhabdomyosarcoma (RMS) produce the PAX3-FOXO1 fusion protein, which is an oncogenic driver and a dependency in this disease. One important function of PAX3-FOXO1 is to arrest myogenic differentiation, which is linked to the ability of RMS cells to gain an unlimited proliferation potential. Here, we developed a phenotypic screening strategy for identifying factors that collaborate with PAX3-FOXO1 to block myo-differentiation in RMS. Unlike most genes evaluated in our screen, we found that loss of any of the three subunits of the Nuclear Factor Y (NF-Y) complex leads to a myo-differentiation phenotype that resembles the effect of inactivating PAX3-FOXO1. While the transcriptomes of NF-Y- and PAX3-FOXO1-deficient RMS cells bear remarkable similarity to one another, we found that these two transcription factors occupy nonoverlapping sites along the genome: NF-Y preferentially occupies promoters, whereas PAX3-FOXO1 primarily binds to distal enhancers. By integrating multiple functional approaches, we map the PAX3 promoter as the point of intersection between these two regulators. We show that NF-Y occupies CCAAT motifs present upstream of PAX3 to function as a transcriptional activator of PAX3-FOXO1 expression in RMS. These findings reveal a critical upstream role of NF-Y in the oncogenic PAX3-FOXO1 pathway, highlighting how a broadly essential transcription factor can perform tumor-specific roles in governing cellular state.

Chemical Biology CryoEM and CryoET Kohli Lab

CRISPR tiling screen reveals cancer epigenetic ‘Goldilocks’ state

Barka A, Kohli RM, Shi J. CRISPR tiling screen reveals cancer epigenetic ‘Goldilocks’ state. Trends Pharmacol Sci. 2023 Sep;44(9):555-557. doi: 10.1016/j.tips.2023.05.007. Epub 2023 Jun 14. PMID: 37328396.

Abstract

CRISPR tiling screens offer an efficient way to identify gain-of-function mutations in targets of cancer therapy. Recently, by utilizing these screens, Kwok et al. unexpectedly discovered mutations conferring drug addiction in lymphoma, revealing a requirement for a ‘just right’ window of histone methylation crucial for cancer survival.

Chemical Biology CryoEM and CryoET Kohli Lab

SARS-CoV-2 ORF6 protein does not antagonize interferon signaling in respiratory epithelial Calu-3 cells during infection

Li M, Ayyanathan K, Dittmar M, Miller J, Tapescu I, Lee JS, McGrath ME, Xue Y, Vashee S, Schultz DC, Frieman MB, Cherry S. SARS-CoV-2 ORF6 protein does not antagonize interferon signaling in respiratory epithelial Calu-3 cells during infection. mBio. 2023 Aug 31;14(4):e0119423. doi: 10.1128/mbio.01194-23. Epub 2023 Jun 28. PMID: 37377442; PMCID: PMC10470815.

Abstract Severe acute respiratory

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of deaths, posing a substantial threat to global public health. Viruses evolve different strategies to antagonize or evade host immune responses. While ectopic expression of SARS-CoV-2 accessory protein ORF6 blocks interferon (IFN) production and downstream IFN signaling, the role of ORF6 in IFN signaling during bona fide viral infection of respiratory cells is unclear. By comparing wild-type (WT) and ORF6-deleted (ΔORF6) SARS-CoV-2 infection and IFN signaling in respiratory cells, we found that ΔORF6 SARS-CoV-2 replicates more efficiently than WT virus and, thus, stimulates more robust immune signaling. Loss of ORF6 does not alter innate signaling in infected cells: both WT and ΔORF6 virus induce delayed IFN responses only in bystander cells. Moreover, expression of ORF6 in the context of SARS-CoV-2 infection has no effect on Sendai virus-stimulated IFN induction: robust translocation of IRF3 is observed in both SARS-CoV-2 infected and bystander cells. Furthermore, IFN pretreatment potently blocks WT and ΔORF6 virus replication similarly, and both viruses fail to suppress the induction of interferon-stimulated genes (ISGs) upon IFN-β treatment. However, upon treatment with IFN-β, only bystander cells induce STAT1 translocation during infection with WT virus, whereas ΔORF6 virus-infected cells now show translocation. This suggests that under conditions of high IFN activation, ORF6 can attenuate STAT1 activation. These data provide evidence that ORF6 is not sufficient to antagonize IFN production or IFN signaling in SARS-CoV-2-infected respiratory cells but may impact the efficacy of therapeutics that stimulate innate immune pathways.

Chemical Biology Cherry Lab Protein Production Services Schultz Lab

Optical Control of Dopamine D2-like Receptors with Cell-Specific Fast-Relaxing Photoswitches

Hetzler BE, Donthamsetti P, Peitsinis Z, Stanley C, Trauner D, Isacoff EY. Optical Control of Dopamine D2-like Receptors with Cell-Specific Fast-Relaxing Photoswitches. J Am Chem Soc. 2023 Aug 30;145(34):18778-18788. doi: 10.1021/jacs.3c02735. Epub 2023 Aug 16. PMID: 37586061; PMCID: PMC10472511.

Abstract

Dopamine D2-like receptors (D2R, D3R, and D4R) control diverse physiological and behavioral functions and are important targets for the treatment of a variety of neuropsychiatric disorders. Their complex distribution and activation kinetics in the brain make it difficult to target specific receptor populations with sufficient precision. We describe a new toolkit of light-activatable, fast-relaxing, covalently taggable chemical photoswitches that fully activate, partially activate, or block D2-like receptors. This technology combines the spatiotemporal precision of a photoswitchable ligand (P) with cell type and spatial specificity of a genetically encoded membrane anchoring protein (M) to which the P tethers. These tools set the stage for targeting endogenous D2-like receptor signaling with molecular, cellular, and spatiotemporal precision using only one wavelength of light.

Chemical Biology CryoEM and CryoET Trauner Group

Joint single-cell profiling resolves 5mC and 5hmC and reveals their distinct gene regulatory effects

Fabyanic EB, Hu P, Qiu Q, Berríos KN, Connolly DR, Wang T, Flournoy J, Zhou Z, Kohli RM, Wu H. Joint single-cell profiling resolves 5mC and 5hmC and reveals their distinct gene regulatory effects. Nat Biotechnol. 2023 Aug 28. doi: 10.1038/s41587-023-01909-2. Epub ahead of print. PMID: 37640946.

Abstract

Oxidative modification of 5-methylcytosine (5mC) by ten-eleven translocation (TET) DNA dioxygenases generates 5-hydroxymethylcytosine (5hmC), the most abundant form of oxidized 5mC. Existing single-cell bisulfite sequencing methods cannot resolve 5mC and 5hmC, leaving the cell-type-specific regulatory mechanisms of TET and 5hmC largely unknown. Here, we present joint single-nucleus (hydroxy)methylcytosine sequencing (Joint-snhmC-seq), a scalable and quantitative approach that simultaneously profiles 5hmC and true 5mC in single cells by harnessing differential deaminase activity of APOBEC3A toward 5mC and chemically protected 5hmC. Joint-snhmC-seq profiling of single nuclei from mouse brains reveals an unprecedented level of epigenetic heterogeneity of both 5hmC and true 5mC at single-cell resolution. We show that cell-type-specific profiles of 5hmC or true 5mC improve multimodal single-cell data integration, enable accurate identification of neuronal subtypes and uncover context-specific regulatory effects on cell-type-specific genes by TET enzymes.

Chemical Biology CryoEM and CryoET Kohli Lab

Individual phosphatidylinositol transfer proteins have distinct functions that do not involve lipid transfer activity

Zhao L, Thorsheim CL, Suzuki A, Stalker TJ, Min SH, Krishnaswamy S, Cockcroft S, Anderson KE, Weiderhold B, Abrams CS. Individual phosphatidylinositol transfer proteins have distinct functions that do not involve lipid transfer activity. Blood Adv. 2023 Aug 22;7(16):4233-4246. doi: 10.1182/bloodadvances.2022008735. PMID: 36930803; PMCID: PMC10424146.

Abstract

Platelets use signal transduction pathways facilitated by class I phosphatidylinositol transfer proteins (PITPs). The 2 mammalian class I PITPs, PITPα and PITPβ, are single PITP domain soluble proteins that are encoded by different genes and share 77% sequence identity, although their individual roles in mammalian biology remain uncharacterized. These proteins are believed to shuttle phosphatidylinositol and phosphatidylcholine between separate intracellular membrane compartments, thereby regulating phosphoinositide synthesis and second messenger formation. Previously, we observed that platelet-specific deletion of PITPα, the predominantly expressed murine PITP isoform, had no effect on hemostasis but impaired tumor metastasis formation and disrupted phosphoinositide signaling. Here, we found that mice lacking the less expressed PITPβ in their platelets exhibited a similar phenotype. However, in contrast to PITPα-null platelet lysates, which have impaired lipid transfer activity, PITPβ-null platelet lysates have essentially normal lipid transfer activity, although both isoforms contribute to phosphoinositide synthesis in vitro. Moreover, we found that platelet-specific deletion of both PITPs led to ex vivo platelet aggregation/secretion and spreading defects, impaired tail bleeding, and profound tumor dissemination. Our study also demonstrated that PITP isoforms are required to maintain endogenous phosphoinositide PtdInsP2 levels and agonist-stimulated second messenger formation. The data shown here demonstrate that the 2 isoforms are functionally overlapping and that a single isoform is able to maintain the homeostasis of platelets. However, both class I PITP isoforms contribute to phosphoinositide signaling in platelets through distinct biochemical mechanisms or different subcellular domains.

CryoEM and CryoET X-ray Crystallography Krishnaswamy Lab

Phospho-signaling couples polar asymmetry and proteolysis within a membraneless microdomain in C. crescentus

Ahmed YM, Bowman GR. Phospho-signaling couples polar asymmetry and proteolysis within a membraneless microdomain in C. crescentus. bioRxiv [Preprint]. 2023 Aug 20:2023.08.19.553945. doi: 10.1101/2023.08.19.553945. PMID: 37645878; PMCID: PMC10462113.

Abstract

Asymmetric cell division in bacteria is achieved through cell polarization, where regulatory proteins are directed to specific cell poles. Curiously, both poles contain a membraneless microdomain, established by the polar assembly hub PopZ, through most of the cell cycle, yet many PopZ clients are unipolar and transiently localized. We find that PopZ’s interaction with the response regulator CpdR is controlled by phosphorylation, via the histidine kinase CckA. Phosphorylated CpdR does not interact with PopZ and is not localized to cell poles. At poles where CckA acts as a phosphatase, de-phosphorylated CpdR binds directly with PopZ and subsequently recruits ClpX, substrates, and other members of a protease complex to the cell pole. We also find that co-recruitment of protease components and substrates to polar microdomains enhances their coordinated activity. This study connects phosphosignaling with polar assembly and the activity of a protease that triggers cell cycle progression and cell differentiation.

Bowman Lab Computational Biology

Direct enzymatic sequencing of 5-methylcytosine at single-base resolution

Wang T, Fowler JM, Liu L, Loo CE, Luo M, Schutsky EK, Berríos KN, DeNizio JE, Dvorak A, Downey N, Montermoso S, Pingul BY, Nasrallah M, Gosal WS, Wu H, Kohli RM. Direct enzymatic sequencing of 5-methylcytosine at single-base resolution. Nat Chem Biol. 2023 Aug;19(8):1004-1012. doi: 10.1038/s41589-023-01318-1. Epub 2023 Jun 15. PMID: 37322153; PMCID: PMC10763687.

Abstract

5-methylcytosine (5mC) is the most important DNA modification in mammalian genomes. The ideal method for 5mC localization would be both nondestructive of DNA and direct, without requiring inference based on detection of unmodified cytosines. Here we present direct methylation sequencing (DM-Seq), a bisulfite-free method for profiling 5mC at single-base resolution using nanogram quantities of DNA. DM-Seq employs two key DNA-modifying enzymes: a neomorphic DNA methyltransferase and a DNA deaminase capable of precise discrimination between cytosine modification states. Coupling these activities with deaminase-resistant adapters enables accurate detection of only 5mC via a C-to-T transition in sequencing. By comparison, we uncover a PCR-related underdetection bias with the hybrid enzymatic-chemical TET-assisted pyridine borane sequencing approach. Importantly, we show that DM-Seq, unlike bisulfite sequencing, unmasks prognostically important CpGs in a clinical tumor sample by not confounding 5mC with 5-hydroxymethylcytosine. DM-Seq thus offers an all-enzymatic, nondestructive, faithful and direct method for the reading of 5mC alone.

Chemical Biology Kohli Lab

LNS8801 inhibits Acute Myeloid Leukemia by Inducing the Production of Reactive Oxygen Species and Activating the Endoplasmic Reticulum Stress Pathway

Lee I, Doepner M, Weissenrieder J, Majer AD, Mercado S, Estell A, Natale CA, Sung PJ, Foskett JK, Carroll MP, Ridky TW. LNS8801 inhibits Acute Myeloid Leukemia by Inducing the Production of Reactive Oxygen Species and Activating the Endoplasmic Reticulum Stress Pathway. Cancer Res Commun. 2023 Aug 18;3(8):1594-1606. doi: 10.1158/2767-9764.CRC-22-0478. PMID: 37599786; PMCID: PMC10438922.

Abstract

Despite recent therapeutic advances, the 5-year survival rate for adults with acute myeloid leukemia (AML) is poor and standard-of-care chemotherapy is associated with significant toxicity, highlighting the need for new therapeutic approaches. Recent work from our group and others established that the G protein-coupled estrogen receptor (GPER) is tumor suppressive in melanoma and other solid tumors. We performed a preliminary screen of human cancer cell lines from multiple malignancies and found that LNS8801, a synthetic pharmacologic agonist of GPER currently in early phase clinical trials, promoted apoptosis in human AML cells. Using human AML cell lines and primary cells, we show that LNS8801 inhibits human AML in preclinical in vitro models, while not affecting normal mononuclear cells. Although GPER is broadly expressed in normal and malignant myeloid cells, this cancer-specific LNS8801-induced inhibition appeared to be independent of GPER signaling. LNS8801 induced AML cell death primarily through a caspase-dependent apoptosis pathway. This was independent of secreted classical death receptor ligands, and instead required induction of reactive oxygen species (ROS) and activation of endoplasmic reticulum (ER) stress response pathways including IRE1α. These studies demonstrate a novel activity of LNS8801 in AML cells and show that targeting ER stress with LNS8801 may be a useful therapeutic approach for AML.

CryoEM and CryoET Foskett

Thermodynamic Coupling of the tandem RRM domains of hnRNP A1 underlie its Pleiotropic RNA Binding Functions

Levengood JD, Potoyan D, Penumutchu S, Kumar A, Wang Y, Hansen AL, Kutluay S, Roche J, Tolbert BS. Thermodynamic Coupling of the tandem RRM domains of hnRNP A1 underlie its Pleiotropic RNA Binding Functions. bioRxiv [Preprint]. 2023 Aug 17:2023.08.17.553700. doi: 10.1101/2023.08.17.553700. PMID: 37645738; PMCID: PMC10462124.

Abstract

The functional properties of RNA-binding proteins (RBPs) require allosteric regulation through inter-domain communication. Despite the foundational importance of allostery to biological regulation, almost no studies have been conducted to describe the biophysical nature by which inter-domain communication manifests in RBPs. Here, we show through high-pressure studies with hnRNP A1 that inter-domain communication is vital for the unique stability of its N- terminal domain containing a tandem of RNA Recognition Motifs (RRMs). Despite high sequence similarity and nearly identical tertiary structures, the two RRMs exhibit drastically different stability under pressure. RRM2 unfolds completely under high-pressure as an individual domain, but when appended to RRM1, it remains stable. Variants in which inter-domain communication is disrupted between the tandem RRMs show a large decrease in stability under pressure. Carrying these mutations over to the full-length protein for in vivo experiments revealed that the mutations affected the ability of the disordered C-terminus to engage in protein-protein interactions and more importantly, they also influenced the RNA binding capacity. Collectively, this work reveals that thermodynamic coupling between the tandem RRMs of hnRNP A1 accounts for its allosteric regulatory functions.

CryoEM and CryoET NMR Tolbert Lab

Transition State of Arp2/3 Complex Activation by Actin-Bound Dimeric Nucleation-Promoting Factor

van Eeuwen T, Boczkowska M, Rebowski G, Carman PJ, Fregoso FE, Dominguez R. Transition State of Arp2/3 Complex Activation by Actin-Bound Dimeric Nucleation-Promoting Factor. Proc Natl Acad Sci U S A. 2023 Aug 15;120(33):e2306165120. doi: 10.1073/pnas.2306165120. Epub 2023 Aug 7. PMID: 37549294; PMCID: PMC10434305.

Abstract

Arp2/3 complex generates branched actin networks that drive fundamental processes such as cell motility and cytokinesis. The complex comprises seven proteins, including actin-related proteins (Arps) 2 and 3 and five scaffolding proteins (ArpC1-ArpC5) that mediate interactions with a pre-existing (mother) actin filament at the branch junction. Arp2/3 complex exists in two main conformations, inactive with the Arps interacting end-to-end and active with the Arps interacting side-by-side like subunits of the short-pitch helix of the actin filament. Several cofactors drive the transition toward the active state, including ATP binding to the Arps, WASP-family nucleation-promoting factors (NPFs), actin monomers, and binding of Arp2/3 complex to the mother filament. The precise contribution of each cofactor to activation is poorly understood. We report the 3.32-Å resolution cryo-electron microscopy structure of a transition state of Arp2/3 complex activation with bound constitutively dimeric NPF. Arp2/3 complex-binding region of the NPF N-WASP was fused C-terminally to the α and β subunits of the CapZ heterodimer. One arm of the NPF dimer binds Arp2 and the other binds actin and Arp3. The conformation of the complex is intermediate between those of inactive and active Arp2/3 complex. Arp2, Arp3, and actin also adopt intermediate conformations between monomeric (G-actin) and filamentous (F-actin) states, but only actin hydrolyzes ATP. In solution, the transition complex is kinetically shifted toward the short-pitch conformation and has higher affinity for F-actin than inactive Arp2/3 complex. The results reveal how all the activating cofactors contribute in a coordinated manner toward Arp2/3 complex activation.

CryoEM and CryoET Dominguez Lab X-ray Crystallography

Mechanisms and physiological function of daily haemoglobin oxidation rhythms in red blood cells

Beale AD, Hayter EA, Crosby P, Valekunja UK, Edgar RS, Chesham JE, Maywood ES, Labeed FH, Reddy AB, Wright KP Jr, Lilley KS, Bechtold DA, Hastings MH, O’Neill JS. EMBO J. 2023 Oct 4;42(19):e114164. doi: 10.15252/embj.2023114164. Epub 2023 Aug 9.

Abstract

Cellular circadian rhythms confer temporal organisation upon physiology that is fundamental to human health. Rhythms are present in red blood cells (RBCs), the most abundant cell type in the body, but their physiological function is poorly understood. Here, we present a novel biochemical assay for haemoglobin (Hb) oxidation status which relies on a redox-sensitive covalent haem-Hb linkage that forms during SDS-mediated cell lysis. Formation of this linkage is lowest when ferrous Hb is oxidised, in the form of ferric metHb. Daily haemoglobin oxidation rhythms are observed in mouse and human RBCs cultured in vitro, or taken from humans in vivo, and are unaffected by mutations that affect circadian rhythms in nucleated cells. These rhythms correlate with daily rhythms in core body temperature, with temperature lowest when metHb levels are highest. Raising metHb levels with dietary sodium nitrite can further decrease daytime core body temperature in mice via nitric oxide (NO) signalling. These results extend our molecular understanding of RBC circadian rhythms and suggest they contribute to the regulation of body temperature.

CryoEM and CryoET Mass Spectrometry Reddy Lab

Spastin locally amplifies microtubule dynamics to pattern the axon for presynaptic cargo delivery

Aiken J, Holzbaur ELF. Spastin locally amplifies microtubule dynamics to pattern the axon for presynaptic cargo delivery. bioRxiv [Preprint]. 2023 Aug 9:2023.08.08.552320. doi: 10.1101/2023.08.08.552320. PMID: 37609249; PMCID: PMC10441300.

Abstract

Neurons rely on long-range trafficking of synaptic components to form and maintain the complex neural networks that encode the human experience. With a single neuron capable of forming thousands of distinct en passant synapses along its axon, spatially precise delivery of the necessary synaptic components is paramount. How these synapses are patterned, and how efficient delivery of synaptic components is regulated, remains largely unknown. Here, we reveal a novel role for the microtubule severing enzyme spastin in locally enhancing microtubule polymerization to influence presynaptic cargo pausing and retention along the axon. In human neurons derived from induced pluripotent stem cells (iPSCs), we identify sites stably enriched for presynaptic components, termed ‘protosynapses’, which are distributed along the axon prior to the robust assembly of mature presynapses apposed by postsynaptic contacts. These sites are capable of cycling synaptic vesicles, are enriched with spastin, and are hotspots for new microtubule growth and synaptic vesicle precursor (SVP) pausing/retention. Disruption of neuronal spastin, either by CRISPRi-mediated depletion or transient overexpression, interrupts the localized enrichment of dynamic microtubule plus ends and diminishes SVP accumulation. Using an innovative human heterologous synapse model, where microfluidically isolated human axons recognize and form presynaptic connections with neuroligin-expressing non-neuronal cells, we reveal that neurons deficient for spastin do not achieve the same level of presynaptic component accumulation as control neurons. We propose a model where spastin acts locally as an amplifier of microtubule polymerization to pattern specific regions of the axon for synaptogenesis and guide synaptic cargo delivery.

CryoEM and CryoET Holzbaur Lab Single Molecule Imaging

Crystal Structure of Histone Deacetylase 6 Complexed with ( R )-Lipoic Acid, an Essential Cofactor in Central Carbon Metabolism

Watson PR, Stollmaier JG, Christianson DW. Crystal Structure of Histone Deacetylase 6 Complexed with ( R )-Lipoic Acid, an Essential Cofactor in Central Carbon Metabolism. bioRxiv [Preprint]. 2023 Aug 9:2023.08.08.552419. doi: 10.1101/2023.08.08.552419. Update in: J Biol Chem. 2023 Sep 11;:105228. PMID: 37609266; PMCID: PMC10441330.

Abstract

The enzyme cofactor ( R )-lipoic acid plays a critical role in central carbon metabolism due to its catalytic function in the generation of acetyl-CoA, which links glycolysis with the tricarboxylic acid cycle. This cofactor is also essential for the generation of succinyl CoA within the tricarboxylic acid cycle. However, the biological functions of ( R )-lipoic acid extend beyond metabolism owing to its facile redox chemistry. Most recently, the reduced form of ( R )-lipoic acid, ( R )-dihydrolipoic acid, has been shown to inhibit histone deacetylases (HDACs) with selectivity for the inhibition of HDAC6. Here, we report the 2.4 Å-resolution X-ray crystal structure of the HDAC6-( R )-dihydrolipoic acid complex, and we report a dissociation constant (K D ) of 350 nM for this complex as determined by isothermal titration calorimetry. The crystal structure illuminates key affinity determinants in the enzyme active site, including thiolate-Zn 2+ coordination and S-π interactions in the F583-F643 aromatic crevice. This study provides the first visualization of the connection between HDAC function and the biological response to oxidative stress: the dithiol moiety of ( R )-dihydrolipoic acid can serve as a redox-regulated pharmacophore capable of simultaneously targeting the catalytic Zn 2+ ion and the aromatic crevice in the active site of HDAC6.

CryoEM and CryoET Christianson Group X-ray Crystallography

Origin and arrangement of actin filaments for gliding motility in apicomplexan parasites revealed by cryo-electron tomography

Martinez M, Mageswaran SK, Guérin A, Chen WD, Thompson CP, Chavin S, Soldati-Favre D, Striepen B, Chang YW. Origin and arrangement of actin filaments for gliding motility in apicomplexan parasites revealed by cryo-electron tomography. Nat Commun. 2023 Aug 9;14(1):4800. doi: 10.1038/s41467-023-40520-6. PMID: 37558667; PMCID: PMC10412601.

Abstract

The phylum Apicomplexa comprises important eukaryotic parasites that invade host tissues and cells using a unique mechanism of gliding motility. Gliding is powered by actomyosin motors that translocate host-attached surface adhesins along the parasite cell body. Actin filaments (F-actin) generated by Formin1 play a central role in this critical parasitic activity. However, their subcellular origin, path and ultrastructural arrangement are poorly understood. Here we used cryo-electron tomography to image motile Cryptosporidium parvum sporozoites and reveal the cellular architecture of F-actin at nanometer-scale resolution. We demonstrate that F-actin nucleates at the apically positioned preconoidal rings and is channeled into the pellicular space between the parasite plasma membrane and the inner membrane complex in a conoid extrusion-dependent manner. Within the pellicular space, filaments on the inner membrane complex surface appear to guide the apico-basal flux of F-actin. F-actin concordantly accumulates at the basal end of the parasite. Finally, analyzing a Formin1-depleted Toxoplasma gondii mutant pinpoints the upper preconoidal ring as the conserved nucleation hub for F-actin in Cryptosporidium and Toxoplasma. Together, we provide an ultrastructural model for the life cycle of F-actin for apicomplexan gliding motility.

CryoEM and CryoET Mass Spectrometry Yi-Wei Chang Lab

A Multifunctional Polyethylene Glycol/Triethoxysilane-Modified Polyurethane Foam Dressing with High Absorbency and Antiadhesion Properties Promotes Diabetic Wound Healing

Chen CF, Chen SH, Chen RF, Liu KF, Kuo YR, Wang CK, Lee TM, Wang YH. A Multifunctional Polyethylene Glycol/Triethoxysilane-Modified Polyurethane Foam Dressing with High Absorbency and Antiadhesion Properties Promotes Diabetic Wound Healing. Int J Mol Sci. 2023 Aug 7;24(15):12506. doi: 10.3390/ijms241512506. PMID: 37569881; PMCID: PMC10419382.

Abstract

The delayed healing of chronic wounds, such as diabetic foot ulcers (DFUs), is a clinical problem. Few dressings can promote wound healing by satisfying the demands of chronic wound exudate management and tissue granulation. Therefore, the aim of this study was to prepare a high-absorption polyurethane (PU) foam dressing modified by polyethylene glycol (PEG) and triethoxysilane (APTES) to promote wound healing. PEG-modified (PUE) and PEG/APTES-modified (PUESi) dressings were prepared by self-foaming reactions. Gauze and PolyMem were used as controls. Next, Fourier transform-infrared spectroscopy, thermomechanical analyses, scanning electron microscopy and tensile strength, water absorption, anti-protein absorption, surface dryness and biocompatibility tests were performed for in vitro characterization. Wound healing effects were further investigated in nondiabetic (non-DM) and diabetes mellitus (DM) rat models. The PUE and PUESi groups exhibited better physicochemical properties than the gauze and PolyMem groups. Moreover, PUESi dressing showed better anti-adhesion properties and absorption capacity with deformation. Furthermore, the PUESi dressing shortened the inflammatory phase and enhanced collagen deposition in both the non-DM and DM animal models. To conclude, the PUESi dressing not only was fabricated with a simple and effective strategy but also enhanced wound healing via micronegative-pressure generation by its high absorption compacity with deformation.

Mass Spectrometry X-ray Crystallography Liu Lab

Concise Synthesis of Glycerophospholipids

Mukhopadhyay TK, Trauner D. Concise Synthesis of Glycerophospholipids. J Org Chem. 2023 Aug 4;88(15):11253-11257. doi: 10.1021/acs.joc.2c02096. Epub 2022 Nov 30. PMID: 36449029.

Abstract

Glycerophospholipids are major components of cellular membranes and provide important signaling molecules. Besides shaping membrane properties, some bind to specific receptors to activate biological pathways. Untangling the roles of individual glycerophospholipids requires clearly defined molecular species, a challenge that can be best addressed through chemical synthesis. However, glycerophospholipid syntheses are often lengthy due to the contrasting polarities found within these lipids. We now report a general strategy to quickly access glycerophospholipids via opening of a phosphate triester epoxide with carboxylic acids catalyzed by Jacobsen’s Co(salen) complex. We show that this method can be applied to a variety of commercially available fatty acids, photoswitchable fatty acids, and other carboxylic acids to provide the corresponding glycerophosphate derivatives.

Chemical Biology CryoEM and CryoET Trauner Group

Nucleoside analogs NM107 and AT-527 are antiviral against rubella virus

Dittmar M, Whig K, Miller J, Kamalia B, Suppiah S, Perelygina L, Sullivan KE, Schultz DC, Cherry S. Nucleoside analogs NM107 and AT-527 are antiviral against rubella virus. PNAS Nexus. 2023 Aug 3;2(9):pgad256. doi: 10.1093/pnasnexus/pgad256. PMID: 37674858; PMCID: PMC10479830.

Abstract Rubella is a

Rubella is a highly contagious viral infection that usually causes a mild disease in children and adults. However, infection during pregnancy can result in a fetal or newborn death or congenital rubella syndrome (CRS), a constellation of permanent birth defects including cataracts, heart defects, and sensorineural deafness. The live-attenuated rubella vaccine has been highly effective, with the Americas declared free of endemic rubella transmission in 2015. However, rubella remains a significant problem worldwide and the leading cause of vaccine-preventable birth defects globally. Thus, elimination of rubella and CRS is a goal of the World Health Organization. No specific therapeutics are approved for the rubella virus. Therefore, we set out to identify whether existing small molecules may be repurposed for use against rubella virus infection. Thus, we performed a high-throughput screen for small molecules active against rubella virus in human respiratory cells and identified two nucleoside analogs, NM107 and AT-527, with potent antiviral activity. Furthermore, we found that combining these nucleoside analogs with inhibitors of host nucleoside biosynthesis had synergistic antiviral activity. These studies open the door to new potential approaches to treat rubella infections.

Chemical Biology Cherry Lab Protein Production Services Schultz Lab

Drosophila class-I myosins that can impact left-right asymmetry have distinct ATPase kinetics

Báez-Cruz FA, Ostap EM. Drosophila class-I myosins that can impact left-right asymmetry have distinct ATPase kinetics. J Biol Chem. 2023 Aug;299(8):104961. doi: 10.1016/j.jbc.2023.104961. Epub 2023 Jun 26. PMID: 37380077; PMCID: PMC10374968.

Abstract

Myosin-1D (myo1D) is important for Drosophila left-right asymmetry, and its effects are modulated by myosin-1C (myo1C). De novo expression of these myosins in nonchiral Drosophila tissues promotes cell and tissue chirality, with handedness depending on the paralog expressed. Remarkably, the identity of the motor domain determines the direction of organ chirality, rather than the regulatory or tail domains. Myo1D, but not myo1C, propels actin filaments in leftward circles in in vitro experiments, but it is not known if this property contributes to establishing cell and organ chirality. To further explore if there are differences in the mechanochemistry of these motors, we determined the ATPase mechanisms of myo1C and myo1D. We found that myo1D has a 12.5-fold higher actin-activated steady-state ATPase rate, and transient kinetic experiments revealed myo1D has an 8-fold higher MgADP release rate compared to myo1C. Actin-activated phosphate release is rate limiting for myo1C, whereas MgADP release is the rate-limiting step for myo1D. Notably, both myosins have among the tightest MgADP affinities measured for any myosin. Consistent with ATPase kinetics, myo1D propels actin filaments at higher speeds compared to myo1C in in vitro gliding assays. Finally, we tested the ability of both paralogs to transport 50 nm unilamellar vesicles along immobilized actin filaments and found robust transport by myo1D and actin binding but no transport by myo1C. Our findings support a model where myo1C is a slow transporter with long-lived actin attachments, whereas myo1D has kinetic properties associated with a transport motor.

CryoEM and CryoET Single Molecule Imaging Ostap Lab

Nanoscale details of mitochondrial constriction revealed by cryoelectron tomography

Mageswaran SK, Grotjahn DA, Zeng X, Barad BA, Medina M, Hoang MH, Dobro MJ, Chang YW, Xu M, Yang WY, Jensen GJ. Nanoscale details of mitochondrial constriction revealed by cryoelectron tomography. Biophys J. 2023 Sep 19;122(18):3768-3782. doi: 10.1016/j.bpj.2023.07.030. Epub 2023 Aug 1. PMID: 37533259; PMCID: PMC10541493.

Abstract

Mitochondria adapt to changing cellular environments, stress stimuli, and metabolic demands through dramatic morphological remodeling of their shape, and thus function. Such mitochondrial dynamics is often dependent on cytoskeletal filament interactions. However, the precise organization of these filamentous assemblies remains speculative. Here, we apply cryogenic electron tomography to directly image the nanoscale architecture of the cytoskeletal-membrane interactions involved in mitochondrial dynamics in response to damage. We induced mitochondrial damage via membrane depolarization, a cellular stress associated with mitochondrial fragmentation and mitophagy. We find that, in response to acute membrane depolarization, mammalian mitochondria predominantly organize into tubular morphology that abundantly displays constrictions. We observe long bundles of both unbranched actin and septin filaments enriched at these constrictions. We also observed septin-microtubule interactions at these sites and elsewhere, suggesting that these two filaments guide each other in the cytosolic space. Together, our results provide empirical parameters for the architecture of mitochondrial constriction factors to validate/refine existing models and inform the development of new ones.

CryoEM and CryoET Mass Spectrometry Yi-Wei Chang Lab

Reprogramming the Cyclization Cascade of epi-Isozizaene Synthase to Generate Alternative Terpene Products

Eaton SA, Christianson DW. Reprogramming the Cyclization Cascade of epi-Isozizaene Synthase to Generate Alternative Terpene Products. Biochemistry. 2023 Aug 1;62(15):2301-2313. doi: 10.1021/acs.biochem.3c00247. Epub 2023 Jul 14. PMID: 37449555; PMCID: PMC10527993.

Abstract

The class I sesquiterpene cyclase epi-isozizaene synthase from Streptomyces coelicolor (EIZS) catalyzes the transformation of linear farnesyl diphosphate (FPP) into the tricyclic hydrocarbon epi-isozizaene in the biosynthesis of albaflavenone antibiotics. The active site cavity of EIZS is largely framed by four aromatic residues – F95, F96, F198, and W203 – that form a product-shaped contour, serving as a template to chaperone conformations of the flexible substrate and multiple carbocation intermediates leading to epi-isozizaene. Remolding the active site contour by mutagenesis can redirect the cyclization cascade away from epi-isozizaene biosynthesis to generate alternative sesquiterpene products. Here, we present the biochemical and structural characterization of four EIZS mutants in which aromatic residues have been substituted with polar residues (F95S, F96H, F198S, and F198T) to generate alternative cyclization products. Most notably, F95S EIZS generates a mixture of monocyclic sesquiterpene precursors of bisabolane, a D2 diesel fuel substitute. X-ray crystal structures of the characterized mutants reveal subtle changes in the active site contour showing how each aromatic residue influences the chemistry of a different carbocation intermediate in the cyclization cascade. We advance that EIZS may serve as a robust platform for the development of designer cyclases for the generation of high-value sesquiterpene products ranging from pharmaceuticals to biofuels in synthetic biology approaches.

CryoEM and CryoET Christianson Group X-ray Crystallography

Expanding the phenotypic spectrum of NAA10-related neurodevelopmental syndrome and NAA15-related neurodevelopmental syndrome

Lyon GJ, Vedaie M, Beisheim T, Park A, Marchi E, Gottlieb L, Hsieh TC, Klinkhammer H, Sandomirsky K, Cheng H, Starr LJ, Preddy I, Tseng M, Li Q, Hu Y, Wang K, Carvalho A, Martinez F, Caro-Llopis A, Gavin M, Amble K, Krawitz P, Marmorstein R, Herr-Israel E. Expanding the phenotypic spectrum of NAA10-related neurodevelopmental syndrome and NAA15-related neurodevelopmental syndrome. Eur J Hum Genet. 2023 Jul;31(7):824-833. doi: 10.1038/s41431-023-01368-y. Epub 2023 May 2. PMID: 37130971; PMCID: PMC10325952.

Abstract

Amino-terminal (Nt-) acetylation (NTA) is a common protein modification, affecting 80% of cytosolic proteins in humans. The human essential gene, NAA10, encodes for the enzyme NAA10, which is the catalytic subunit in the N-terminal acetyltransferase A (NatA) complex, also including the accessory protein, NAA15. The full spectrum of human genetic variation in this pathway is currently unknown. Here we reveal the genetic landscape of variation in NAA10 and NAA15 in humans. Through a genotype-first approach, one clinician interviewed the parents of 56 individuals with NAA10 variants and 19 individuals with NAA15 variants, which were added to all known cases (N = 106 for NAA10 and N = 66 for NAA15). Although there is clinical overlap between the two syndromes, functional assessment demonstrates that the overall level of functioning for the probands with NAA10 variants is significantly lower than the probands with NAA15 variants. The phenotypic spectrum includes variable levels of intellectual disability, delayed milestones, autism spectrum disorder, craniofacial dysmorphology, cardiac anomalies, seizures, and visual abnormalities (including cortical visual impairment and microphthalmia). One female with the p.Arg83Cys variant and one female with an NAA15 frameshift variant both have microphthalmia. The frameshift variants located toward the C-terminal end of NAA10 have much less impact on overall functioning, whereas the females with the p.Arg83Cys missense in NAA10 have substantial impairment. The overall data are consistent with a phenotypic spectrum for these alleles,

Chemical Biology CryoEM and CryoET X-ray Crystallography Marmorstein Lab

Coordination of RNA modifications in the brain and beyond

Chen AY, Owens MC, Liu KF. Coordination of RNA modifications in the brain and beyond. Mol Psychiatry. 2023 Jul;28(7):2737-2749. doi: 10.1038/s41380-023-02083-2. Epub 2023 May 3. PMID: 37138184.

Abstract

Gene expression regulation is a critical process throughout the body, especially in the nervous system. One mechanism by which biological systems regulate gene expression is via enzyme-mediated RNA modifications, also known as epitranscriptomic regulation. RNA modifications, which have been found on nearly all RNA species across all domains of life, are chemically diverse covalent modifications of RNA nucleotides and represent a robust and rapid mechanism for the regulation of gene expression. Although numerous studies have been conducted regarding the impact that single modifications in single RNA molecules have on gene expression, emerging evidence highlights potential crosstalk between and coordination of modifications across RNA species. These potential coordination axes of RNA modifications have emerged as a new direction in the field of epitranscriptomic research. In this review, we will highlight several examples of gene regulation via RNA modification in the nervous system, followed by a summary of the current state of the field of RNA modification coordination axes. In doing so, we aim to inspire the field to gain a deeper understanding of the roles of RNA modifications and coordination of these modifications in the nervous system.

Mass Spectrometry X-ray Crystallography Liu Lab

Folding@home: Achievements from over 20 years of citizen science herald the exascale era

Voelz VA, Pande VS, Bowman GR. Folding@home: Achievements from over 20 years of citizen science herald the exascale era. Biophys J. 2023 Jul 25;122(14):2852-2863. doi: 10.1016/j.bpj.2023.03.028. Epub 2023 Mar 21. PMID: 36945779; PMCID: PMC10398258.

Abstract Simulations of biomolecules

Simulations of biomolecules have enormous potential to inform our understanding of biology but require extremely demanding calculations. For over 20 years, the Folding@home distributed computing project has pioneered a massively parallel approach to biomolecular simulation, harnessing the resources of citizen scientists across the globe. Here, we summarize the scientific and technical advances this perspective has enabled. As the project’s name implies, the early years of Folding@home focused on driving advances in our understanding of protein folding by developing statistical methods for capturing long-timescale processes and facilitating insight into complex dynamical processes. Success laid a foundation for broadening the scope of Folding@home to address other functionally relevant conformational changes, such as receptor signaling, enzyme dynamics, and ligand binding. Continued algorithmic advances, hardware developments such as graphics processing unit (GPU)-based computing, and the growing scale of Folding@home have enabled the project to focus on new areas where massively parallel sampling can be impactful. While previous work sought to expand toward larger proteins with slower conformational changes, new work focuses on large-scale comparative studies of different protein sequences and chemical compounds to better understand biology and inform the development of small-molecule drugs. Progress on these fronts enabled the community to pivot quickly in response to the COVID-19 pandemic, expanding to become the world’s first exascale computer and deploying this massive resource to provide insight into the inner workings of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus and aid the development of new antivirals. This success provides a glimpse of what is to come as exascale supercomputers come online and as Folding@home continues its work.

Bowman Lab Computational Biology

RAB3 phosphorylation by pathogenic LRRK2 impairs trafficking of synaptic vesicle precursors

Dou D, Aiken J, Holzbaur ELF. RAB3 phosphorylation by pathogenic LRRK2 impairs trafficking of synaptic vesicle precursors. bioRxiv [Preprint]. 2023 Jul 25:2023.07.25.550521. doi: 10.1101/2023.07.25.550521. PMID: 37546777; PMCID: PMC10402060.

Abstract

Gain-of-function mutations in the LRRK2 gene cause Parkinson’s disease (PD), characterized by debilitating motor and non-motor symptoms. Increased phosphorylation of a subset of RAB GTPases by LRRK2 is implicated in PD pathogenesis. We find that increased phosphorylation of RAB3A, a cardinal synaptic vesicle precursor (SVP) protein, disrupts anterograde axonal transport of SVPs in iPSC-derived human neurons (iNeurons) expressing hyperactive LRRK2-p.R1441H. Knockout of the opposing protein phosphatase 1H (PPM1H) in iNeurons phenocopies this effect. In these models, the compartmental distribution of synaptic proteins is altered; synaptophysin and synaptobrevin-2 become sequestered in the neuronal soma with decreased delivery to presynaptic sites along the axon. We find that RAB3A phosphorylation disrupts binding to the motor adapter MADD, potentially preventing formation of the RAB3A-MADD-KIF1A/1Bβ complex driving anterograde SVP transport. RAB3A hyperphosphorylation also disrupts interactions with RAB3GAP and RAB-GDI1. Our results reveal a mechanism by which pathogenic hyperactive LRRK2 may contribute to the altered synaptic homeostasis associated with characteristic non-motor and cognitive manifestations of PD.

CryoEM and CryoET Holzbaur Lab Single Molecule Imaging

Structural Basis for the Phase Separation of the Chromosome Passenger Complex

Bryan NW, Ali A, Niedzialkowska E, Mayne L, Stukenberg PT, Black BE. Structural Basis for the Phase Separation of the Chromosome Passenger Complex. bioRxiv [Preprint]. 2023 Jul 23:2023.05.22.541822. doi: 10.1101/2023.05.22.541822. PMID: 37292983; PMCID: PMC10245869.

Abstract

The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often referred to as ‘fuzzy’. Gaining clarity on the biogenesis of membraneless cellular compartments is one of the most demanding challenges in biology. Here, we focus on the chromosome passenger complex (CPC), that forms a chromatin body that regulates chromosome segregation in mitosis. Within the three regulatory subunits of the CPC implicated in phase separation – a heterotrimer of INCENP, Survivin, and Borealin – we identify the contact regions formed upon droplet formation using hydrogen/deuterium-exchange mass spectrometry (HXMS). These contact regions correspond to some of the interfaces seen between individual heterotrimers within the crystal lattice they form. A major contribution comes from specific electrostatic interactions that can be broken and reversed through initial and compensatory mutagenesis, respectively. Our findings reveal structural insight for interactions driving liquid-liquid demixing of the CPC. Moreover, we establish HXMS as an approach to define the structural basis for phase separation.

Black Lab CryoEM and CryoET Mass Spectrometry

Circadian regulation of lung repair and regeneration

Naik A, Forrest KM, Paul O, Issah Y, Valekunja UK, Tang SY, Reddy AB, Hennessy EJ, Brooks TG, Chaudhry F, Babu A, Morley M, Zepp JA, Grant GR, FitzGerald GA, Sehgal A, Worthen GS, Frank DB, Morrisey EE, Sengupta S. JCI Insight. 2023 Aug 22;8(16):e164720. doi: 10.1172/jci.insight.164720.

Abstract

Optimal lung repair and regeneration are essential for recovery from viral infections, including influenza A virus (IAV). We have previously demonstrated that acute inflammation and mortality induced by IAV is under circadian control. However, it is not known whether the influence of the circadian clock persists beyond the acute outcomes. Here, we utilize the UK Biobank to demonstrate an association between poor circadian rhythms and morbidity from lower respiratory tract infections, including the need for hospitalization and mortality after discharge; this persists even after adjusting for common confounding factors. Furthermore, we use a combination of lung organoid assays, single-cell RNA sequencing, and IAV infection in different models of clock disruption to investigate the role of the circadian clock in lung repair and regeneration. We show that lung organoids have a functional circadian clock and the disruption of this clock impairs regenerative capacity. Finally, we find that the circadian clock acts through distinct pathways in mediating lung regeneration — in tracheal cells via the Wnt/β-catenin pathway and through IL-1β in alveolar epithelial cells. We speculate that adding a circadian dimension to the critical process of lung repair and regeneration will lead to novel therapies and improve outcomes.

CryoEM and CryoET Mass Spectrometry Reddy Lab

Inflammasomes primarily restrict cytosolic Salmonella replication within human macrophages

Egan MS, O’Rourke EA, Mageswaran SK, Zuo B, Martynyuk I, Demissie T, Hunter EN, Bass AR, Chang YW, Brodsky IE, Shin S. Inflammasomes primarily restrict cytosolic Salmonella replication within human macrophages. bioRxiv [Preprint]. 2023 Jul 18:2023.07.17.549348. doi: 10.1101/2023.07.17.549348. PMID: 37503120; PMCID: PMC10370064.

Abstract

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that utilizes its type III secretion systems (T3SSs) to inject virulence factors into the host cell and colonize the host. In turn, a subset of cytosolic immune receptors respond to T3SS ligands by forming multimeric signaling complexes called inflammasomes, which activate caspases that induce interleukin-1 (IL-1) family cytokine release and an inflammatory form of cell death called pyroptosis. Human macrophages mount a multifaceted inflammasome response to Salmonella infection that ultimately restricts intracellular bacterial replication. However, how inflammasomes restrict Salmonella replication remains unknown. We find that caspase-1 is essential for mediating inflammasome responses to Salmonella and subsequent restriction of bacterial replication within human macrophages, with caspase-4 contributing as well. We also demonstrate that the downstream pore-forming protein gasdermin D (GSDMD) and ninjurin-1 (NINJ1), a mediator of terminal cell lysis, play a role in controlling Salmonella replication in human macrophages. Notably, in the absence of inflammasome responses, we observed hyperreplication of Salmonella within the cytosol of infected cells, and we also observed increased bacterial replication within vacuoles, suggesting that inflammasomes control Salmonella replication primarily within the cytosol and also within vacuoles. These findings reveal that inflammatory caspases and pyroptotic factors mediate inflammasome responses that restrict the subcellular localization of intracellular Salmonella replication within human macrophages.

CryoEM and CryoET Mass Spectrometry Yi-Wei Chang Lab

Gene-encoded nanoparticle vaccine platforms for in vivo assembly of multimeric antigen to promote adaptive immunity

Tursi NJ, Xu Z, Kulp DW, Weiner DB. Gene-encoded nanoparticle vaccine platforms for in vivo assembly of multimeric antigen to promote adaptive immunity. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2023 Jul-Aug;15(4):e1880. doi: 10.1002/wnan.1880. Epub 2023 Feb 20. PMID: 36807845; PMCID: PMC10665986.

Abstract

Nanoparticle vaccines are a diverse category of vaccines for the prophylaxis or treatment of various diseases. Several strategies have been employed for their optimization, especially to enhance vaccine immunogenicity and generate potent B-cell responses. Two major modalities utilized for particulate antigen vaccines include using nanoscale structures for antigen delivery and nanoparticles that are themselves vaccines due to antigen display or scaffolding-the latter of which we will define as “nanovaccines.” Multimeric antigen display has a variety of immunological benefits compared to monomeric vaccines mediated through potentiating antigen-presenting cell presentation and enhancing antigen-specific B-cell responses through B-cell activation. The majority of nanovaccine assembly is done in vitro using cell lines. However, in vivo assembly of scaffolded vaccines potentiated using nucleic acids or viral vectors is a burgeoning modality of nanovaccine delivery. Several advantages to in vivo assembly exist, including lower costs of production, fewer production barriers, as well as more rapid development of novel vaccine candidates for emerging diseases such as SARS-CoV-2. This review will characterize the methods for de novo assembly of nanovaccines in the host using methods of gene delivery including nucleic acid and viral vectored vaccines. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Therapeutic Approaches and Drug Discovery > Emerging Technologies.

Computational Biology CryoEM and CryoET Kulp Lab

Pooled tagging and hydrophobic targeting of endogenous proteins for unbiased mapping of unfolded protein responses

Sansbury SE, Serebrenik YV, Lapidot T, Burslem GM, Shalem O. Pooled tagging and hydrophobic targeting of endogenous proteins for unbiased mapping of unfolded protein responses. bioRxiv [Preprint]. 2023 Jul 14:2023.07.13.548611. doi: 10.1101/2023.07.13.548611. PMID: 37503003; PMCID: PMC10370017.

Abstract

System-level understanding of proteome organization and function requires methods for direct visualization and manipulation of proteins at scale. We developed an approach enabled by high-throughput gene tagging for the generation and analysis of complex cell pools with endogenously tagged proteins. Proteins are tagged with HaloTag to enable visualization or direct perturbation. Fluorescent labeling followed by in situ sequencing and deep learning-based image analysis identifies the localization pattern of each tag, providing a bird’s-eye-view of cellular organization. Next, we use a hydrophobic HaloTag ligand to misfold tagged proteins, inducing spatially restricted proteotoxic stress that is read out by single cell RNA sequencing. By integrating optical and perturbation data, we map compartment-specific responses to protein misfolding, revealing inter-compartment organization and direct crosstalk, and assigning proteostasis functions to uncharacterized genes. Altogether, we present a powerful and efficient method for large-scale studies of proteome dynamics, function, and homeostasis.

Chemical Biology Burslem Lab Mass Spectrometry

Bdellovibrio predation cycle characterized at nanometre-scale resolution with cryo-electron tomography

Kaplan M, Chang YW, Oikonomou CM, Nicolas WJ, Jewett AI, Kreida S, Dutka P, Rettberg LA, Maggi S, Jensen GJ. Bdellovibrio predation cycle characterized at nanometre-scale resolution with cryo-electron tomography. Nat Microbiol. 2023 Jul;8(7):1267-1279. doi: 10.1038/s41564-023-01401-2. Epub 2023 Jun 22. PMID: 37349588.

Abstract

Bdellovibrio bacteriovorus is a microbial predator that offers promise as a living antibiotic for its ability to kill Gram-negative bacteria, including human pathogens. Even after six decades of study, fundamental details of its predation cycle remain mysterious. Here we used cryo-electron tomography to comprehensively image the lifecycle of B. bacteriovorus at nanometre-scale resolution. With high-resolution images of predation in a native (hydrated, unstained) state, we discover several surprising features of the process, including macromolecular complexes involved in prey attachment/invasion and a flexible portal structure lining a hole in the prey peptidoglycan that tightly seals the prey outer membrane around the predator during entry. Unexpectedly, we find that B. bacteriovorus does not shed its flagellum during invasion, but rather resorbs it into its periplasm for degradation. Finally, following growth and division in the bdelloplast, we observe a transient and extensive ribosomal lattice on the condensed B. bacteriovorus nucleoid.

CryoEM and CryoET Mass Spectrometry Yi-Wei Chang Lab

mRNA expression of immune factors by milk somatic cells from healthy Holstein lactating cows

Murakami K, Fukuhara T, Kure S, Shimosakai T, Sato A, Murata R, Kosenda K, Ohtsuka H. mRNA expression of immune factors by milk somatic cells from healthy Holstein lactating cows. Can J Vet Res. 2023 Jul;87(3):231-236. PMID: 37397638; PMCID: PMC10291702.

Abstract in English, French

This study investigated the mRNA of immune factors expressed by milk somatic cells from 72 healthy lactating Holstein cows on 1 farm. Milk samples were collected aseptically from the right front mammary gland before milking. The milk samples that had a negative reaction to the California mastitis test were used to analyze the mRNA of immune factors. Cows were divided into 2 groups based on the detection of bacteria in milk samples: positive group (n = 22 cows), which showed bacteria in cultures, and negative group (n = 50 cows), which did not show bacteria in cultures. There were significant positive correlations among the relative mRNA levels of interleukin (IL)-6, IL-8, arginase 1, chemokine (C-C motif) ligand (CCL) 1, and chemokine (C-X-C motif) ligand (CXCL) 13, as well as among the relative mRNA levels of IL-10, pentraxin 3, CCL5, and CCL14. Significantly high levels of IL-1β, IL-6, IL-8, arginase 1, Batf, CCL1, CXCL14, and toll-like receptor 4 in the positive group were discovered compared to the negative group. These results suggest that the presence of bacteria in lactating healthy dairy cows may affect mRNA levels of inflammatory mediators expressed by somatic cells.

CryoEM and CryoET Mass Spectrometry Murakami Lab

Single cell susceptibility to SARS-CoV-2 infection is driven by variable cell states

Reffsin S, Miller J, Ayyanathan K, Dunagin MC, Jain N, Schultz DC, Cherry S, Raj A. Single cell susceptibility to SARS-CoV-2 infection is driven by variable cell states. bioRxiv [Preprint]. 2023 Jul 7:2023.07.06.547955. doi: 10.1101/2023.07.06.547955. PMID: 37461472; PMCID: PMC10350037.

Abstract

The ability of a virus to infect a cell type is at least in part determined by the presence of host factors required for the viral life cycle. However, even within cell types that express known factors needed for infection, not every cell is equally susceptible, suggesting that our knowledge of the full spectrum of factors that promote infection is incomplete. Profiling the most susceptible subsets of cells within a population may reveal additional factors that promote infection. However, because viral infection dramatically alters the state of the cell, new approaches are needed to reveal the state of these cells prior to infection with virus. Here, we used single-cell clone tracing to retrospectively identify and characterize lung epithelial cells that are highly susceptible to infection with SARS-CoV-2. The transcriptional state of these highly susceptible cells includes markers of retinoic acid signaling and epithelial differentiation. Loss of candidate factors identified by our approach revealed that many of these factors play roles in viral entry. Moreover, a subset of these factors exert control over the infectable cell state itself, regulating the expression of key factors associated with viral infection and entry. Analysis of patient samples revealed the heterogeneous expression of these factors across both cells and patients in vivo. Further, the expression of these factors is upregulated in particular inflammatory pathologies. Altogether, our results show that the variable expression of intrinsic cell states is a major determinant of whether a cell can be infected by SARS-CoV-2.

Chemical Biology Cherry Lab Protein Production Services Schultz Lab

PD-1 maintains CD8 T cell tolerance towards cutaneous neoantigens

Damo M, Hornick NI, Venkat A, William I, Clulo K, Venkatesan S, He J, Fagerberg E, Loza JL, Kwok D, Tal A, Buck J, Cui C, Singh J, Damsky WE, Leventhal JS, Krishnaswamy S, Joshi NS. PD-1 maintains CD8 T cell tolerance towards cutaneous neoantigens. Nature. 2023 Jul;619(7968):151-159. doi: 10.1038/s41586-023-06217-y. Epub 2023 Jun 21. PMID: 37344588.

Abstract

The peripheral T cell repertoire of healthy individuals contains self-reactive T cells1,2. Checkpoint receptors such as PD-1 are thought to enable the induction of peripheral tolerance by deletion or anergy of self-reactive CD8 T cells3-10. However, this model is challenged by the high frequency of immune-related adverse events in patients with cancer who have been treated with checkpoint inhibitors11. Here we developed a mouse model in which skin-specific expression of T cell antigens in the epidermis caused local infiltration of antigen-specific CD8 T cells with an effector gene-expression profile. In this setting, PD-1 enabled the maintenance of skin tolerance by preventing tissue-infiltrating antigen-specific effector CD8 T cells from (1) acquiring a fully functional, pathogenic differentiation state, (2) secreting significant amounts of effector molecules, and (3) gaining access to epidermal antigen-expressing cells. In the absence of PD-1, epidermal antigen-expressing cells were eliminated by antigen-specific CD8 T cells, resulting in local pathology. Transcriptomic analysis of skin biopsies from two patients with cutaneous lichenoid immune-related adverse events showed the presence of clonally expanded effector CD8 T cells in both lesional and non-lesional skin. Thus, our data support a model of peripheral T cell tolerance in which PD-1 allows antigen-specific effector CD8 T cells to co-exist with antigen-expressing cells in tissues without immunopathology.

CryoEM and CryoET X-ray Crystallography Krishnaswamy Lab

Divergent Molecular Phenotypes in Point Mutations at the Same Residue in Beta-Myosin Heavy Chain Lead to Distinct Cardiomyopathies

Lehman SJ, Meller A, Solieva SO, Lotthammer JM, Greenberg L, Langer SJ, Greenberg MJ, Tardiff JC, Bowman GR, Leinwand L. Divergent Molecular Phenotypes in Point Mutations at the Same Residue in Beta-Myosin Heavy Chain Lead to Distinct Cardiomyopathies. bioRxiv [Preprint]. 2023 Jul 3:2023.07.03.547580. doi: 10.1101/2023.07.03.547580. PMID: 37461648; PMCID: PMC10349964.

Abstract In genetic cardiomyopathies,

In genetic cardiomyopathies, a frequently described phenomenon is how similar mutations in one protein can lead to discrete clinical phenotypes. One example is illustrated by two mutations in beta myosin heavy chain (β-MHC) that are linked to hypertrophic cardiomyopathy (HCM) (Ile467Val, I467V) and left ventricular non-compaction (LVNC) (Ile467Thr, I467T). To investigate how these missense mutations lead to independent diseases, we studied the molecular effects of each mutation using recombinant human β-MHC Subfragment 1 (S1) in in vitro assays. Both HCM-I467V and LVNC-I467T S1 mutations exhibited similar mechanochemical function, including unchanged ATPase and enhanced actin velocity but had opposing effects on the super-relaxed (SRX) state of myosin. HCM-I467V S1 showed a small reduction in the SRX state, shifting myosin to a more actin-available state that may lead to the “gain-of-function” phenotype commonly described in HCM. In contrast, LVNC-I467T significantly increased the population of myosin in the ultra-slow SRX state. Interestingly, molecular dynamics simulations reveal that I467T allosterically disrupts interactions between ADP and the nucleotide-binding pocket, which may result in an increased ADP release rate. This predicted change in ADP release rate may define the enhanced actin velocity measured in LVNC-I467T, but also describe the uncoupled mechanochemical function for this mutation where the enhanced ADP release rate may be sufficient to offset the increased SRX population of myosin. These contrasting molecular effects may lead to contractile dysregulation that initiates LVNC-associated signaling pathways that progress the phenotype. Together, analysis of these mutations provides evidence that phenotypic complexity originates at the molecular level and is critical to understanding disease progression and developing therapies.

Bowman Lab Computational Biology

Effects of temperature on action potentials and ion conductances in type II taste-bud cells

Ma Z, Paudel U, Foskett JK. Effects of temperature on action potentials and ion conductances in type II taste-bud cells. Am J Physiol Cell Physiol. 2023 Jul 1;325(1):C155-C171. doi: 10.1152/ajpcell.00413.2022. Epub 2023 Jun 5. PMID: 37273235; PMCID: PMC10312327.

Abstract

Temperature strongly influences the intensity of taste, but it remains understudied despite its physiological, hedonic, and commercial implications. The relative roles of the peripheral gustatory and somatosensory systems innervating the oral cavity in mediating thermal effects on taste sensation and perception are poorly understood. Type II taste-bud cells, responsible for sensing sweet, bitter umami, and appetitive NaCl, release neurotransmitters to gustatory neurons by the generation of action potentials, but the effects of temperature on action potentials and the underlying voltage-gated conductances are unknown. Here, we used patch-clamp electrophysiology to explore the effects of temperature on acutely isolated type II taste-bud cell electrical excitability and whole cell conductances. Our data reveal that temperature strongly affects action potential generation, properties, and frequency and suggest that thermal sensitivities of underlying voltage-gated Na+ and K+ channel conductances provide a mechanism for how and whether voltage-gated Na+ and K+ channels in the peripheral gustatory system contribute to the influence of temperature on taste sensitivity and perception.NEW & NOTEWORTHY The temperature of food affects how it tastes. Nevertheless, the mechanisms involved are not well understood, particularly whether the physiology of taste-bud cells in the mouth is involved. Here we show that the electrical activity of type II taste-bud cells that sense sweet, bitter, and umami substances is strongly influenced by temperature. These results suggest a mechanism for the influence of temperature on the intensity of taste perception that resides in taste buds themselves.

CryoEM and CryoET Foskett

Efficient Formation of Single-copy Human Artificial Chromosomes

Gambogi CW, Mer E, Brown DM, Yankson G, Gavade JN, Logsdon GA, Heun P, Glass JI, Black BE. Efficient Formation of Single-copy Human Artificial Chromosomes. bioRxiv [Preprint]. 2023 Jun 30:2023.06.30.547284. doi: 10.1101/2023.06.30.547284. PMID: 37546784; PMCID: PMC10402137.

Abstract

Large DNA assembly methodologies underlie milestone achievements in synthetic prokaryotic and budding yeast chromosomes. While budding yeast control chromosome inheritance through ~125 bp DNA sequence-defined centromeres, mammals and many other eukaryotes use large, epigenetic centromeres. Harnessing centromere epigenetics permits human artificial chromosome (HAC) formation but is not sufficient to avoid rampant multimerization of the initial DNA molecule upon introduction to cells. Here, we describe an approach that efficiently forms single-copy HACs. It employs a ~750 kb construct that is sufficiently large to house the distinct chromatin types present at the inner and outer centromere, obviating the need to multimerize. Delivery to mammalian cells is streamlined by employing yeast spheroplast fusion. These developments permit faithful chromosome engineering in the context of metazoan cells.

Black Lab CryoEM and CryoET Mass Spectrometry

Design of Inhibitors That Target the Menin–Mixed-Lineage Leukemia Interaction

Moses N Arthur, Kristeen Bebla, Emmanuel Broni, Carolyn Ashley, Miriam Velazquez, Xianin Hua, Ravi Radhakrishnan, Samuel K Kwofie, Whelton A Miller

 

Abstract The prognosis of mixed-lineage leukemia (MLL) has remained a significant health concern, especially for infants. The minimal treatments available for this aggressive type of leukemia has been an ongoing problem. Chromosomal translocations of the KMT2A gene are known as MLL, which expresses MLL fusion proteins. A protein called menin is an important oncogenic cofactor for these MLL fusion proteins, thus providing a new avenue for treatments against this subset of acute leukemias. In this study, we report results using the structure-based drug design (SBDD) approach to discover potential novel MLL-mediated leukemia inhibitors from natural products against menin. The three-dimensional (3D) protein model was derived from Protein Databank (Protein ID: 4GQ4), and EasyModeller 4.0 and I-TASSER were used to fix missing residues during rebuilding. Out of the ten protein models generated (five from EasyModeller and I-TASSER each), one model was selected. The selected model demonstrated the most reasonable quality and had 75.5% of residues in the most favored regions, 18.3% of residues in additionally allowed regions, 3.3% of residues in generously allowed regions, and 2.9% of residues in disallowed regions. A ligand library containing 25,131 ligands from a Chinese database was virtually screened using AutoDock Vina, in addition to three known menin inhibitors. The top 10 compounds including ZINC000103526876, ZINC000095913861, ZINC000095912705, ZINC000085530497, ZINC000095912718, ZINC000070451048, ZINC000085530488, ZINC000095912706, ZINC000103580868, and ZINC000103584057 had binding energies of −11.0, −10.7, −10.6, −10.2, −10.2, −9.9, −9.9, −9.9, −9.9, and −9.9 kcal/mol, respectively. To confirm the stability of the menin–ligand complexes and the binding mechanisms, molecular dynamics simulations including molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) computations were performed. The amino acid residues that were found to be potentially crucial in ligand binding included Phe243,
Computational Biology Radhakrishnan Lab

Semi-Synthetic CoA-α-Synuclein Constructs Trap N-Terminal Acetyltransferase NatB for Binding Mechanism Studies

Pan B, Gardner SM, Schultz K, Perez RM, Deng S, Shimogawa M, Sato K, Rhoades E, Marmorstein R, Petersson EJ. Semi-Synthetic CoA-α-Synuclein Constructs Trap N-Terminal Acetyltransferase NatB for Binding Mechanism Studies. J Am Chem Soc. 2023 Jun 28;145(25):14019-14030. doi: 10.1021/jacs.3c03887. Epub 2023 Jun 15. PMID: 37319422; PMCID: PMC10728591.

Abstract

N-terminal acetylation is a chemical modification carried out by N-terminal acetyltransferases. A major member of this enzyme family, NatB, acts on much of the human proteome, including α-synuclein (αS), a synaptic protein that mediates vesicle trafficking. NatB acetylation of αS modulates its lipid vesicle binding properties and amyloid fibril formation, which underlies its role in the pathogenesis of Parkinson’s disease. Although the molecular details of the interaction between human NatB (hNatB) and the N-terminus of αS have been resolved, whether the remainder of the protein plays a role in interacting with the enzyme is unknown. Here, we execute the first synthesis, by native chemical ligation, of a bisubstrate inhibitor of NatB consisting of coenzyme A and full-length human αS, additionally incorporating two fluorescent probes for studies of conformational dynamics. We use cryo-electron microscopy (cryo-EM) to characterize the structural features of the hNatB/inhibitor complex and show that, beyond the first few residues, αS remains disordered when in complex with hNatB. We further probe changes in the αS conformation by single molecule Förster resonance energy transfer (smFRET) to reveal that the C-terminus expands when bound to hNatB. Computational models based on the cryo-EM and smFRET data help to explain the conformational changes as well as their implications for hNatB substrate recognition and specific inhibition of the interaction with αS. Beyond the study of αS and NatB, these experiments illustrate valuable strategies for the study of challenging structural biology targets through a combination of protein semi-synthesis, cryo-EM, smFRET, and computational modeling.

Chemical Biology CryoEM and CryoET Single Molecule Imaging X-ray Crystallography Marmorstein Lab Rhoades Lab

A Chicken Tapasin ortholog can chaperone empty HLA molecules independently of other peptide-loading components

Papadaki GF, Woodward CH, Young MC, Winters TJ, Burslem GM, Sgourakis NG. A Chicken Tapasin ortholog can chaperone empty HLA molecules independently of other peptide-loading components. bioRxiv [Preprint]. 2023 Jun 26:2023.06.23.546255. doi: 10.1101/2023.06.23.546255. Update in: J Biol Chem. 2023 Aug 3;:105136. PMID: 37425753; PMCID: PMC10326978.

Abstract

Human Tapasin (hTapasin) is the main chaperone of MHC-I molecules, enabling peptide loading and antigen repertoire optimization across HLA allotypes. However, it is restricted to the endoplasmic reticulum (ER) lumen as part of the protein loading complex (PLC) and therefore is highly unstable when expressed in recombinant form. Additional stabilizing co-factors such as ERp57 are required to catalyze peptide exchange in vitro , limiting uses for the generation of pMHC-I molecules of desired antigen specificities. Here, we show that the chicken Tapasin (chTapasin) ortholog can be expressed recombinantly at high yields in stable form, independently of co-chaperones. chTapasin can bind the human HLA-B * 37:01 with low micromolar-range affinity to form a stable tertiary complex. Biophysical characterization by methyl-based NMR methods reveals that chTapasin recognizes a conserved β 2 m epitope on HLA-B * 37:01, consistent with previously solved X-ray structures of hTapasin. Finally, we provide evidence that the B * 37:01/chTapasin complex is peptide-receptive and can be dissociated upon binding of high-affinity peptides. Our results highlight the use of chTapasin as a stable scaffold for future protein engineering applications aiming to expand the ligand exchange function on human MHC-I and MHC-like molecules.

Chemical Biology Burslem Lab Mass Spectrometry

BacPROTACs to basics: Targeted protein degradation in bacteria

Burslem GM. BacPROTACs to basics: Targeted protein degradation in bacteria. Cell. 2022 Jun 23;185(13):2203-2205. doi: 10.1016/j.cell.2022.05.026. PMID: 35750029.

Abstract

Targeted protein degradation has emerged as a powerful tool for therapeutic development and biological exploration. In this issue of Cell, Morreale et al. report the development of the BacPROTAC technology to enable targeted protein degradation in Gram-positive bacteria and mycobacteria via reprogramming of Clp proteases.

Chemical Biology Burslem Lab Mass Spectrometry

Structures of the free and capped ends of the actin filament

Carman PJ, Barrie KR, Rebowski G, Dominguez R. Structures of the free and capped ends of the actin filament. Science. 2023 Jun 23;380(6651):1287-1292. doi: 10.1126/science.adg6812. Epub 2023 May 25. PMID: 37228182.

Abstract

The barbed and pointed ends of the actin filament (F-actin) are the sites of growth and shrinkage and the targets of capping proteins that block subunit exchange, including CapZ at the barbed end and tropomodulin at the pointed end. We describe cryo-electron microscopy structures of the free and capped ends of F-actin. Terminal subunits at the free barbed end adopt a “flat” F-actin conformation. CapZ binds with minor changes to the barbed end but with major changes to itself. By contrast, subunits at the free pointed end adopt a “twisted” monomeric actin (G-actin) conformation. Tropomodulin binding forces the second subunit into an F-actin conformation. The structures reveal how the ends differ from the middle in F-actin and how these differences control subunit addition, dissociation, capping, and interactions with end-binding proteins.

CryoEM and CryoET Dominguez Lab X-ray Crystallography

To Isomerize or not to Isomerize? E/Z Isomers of Cyclic Azobenzene Derivatives and Their Reactivity Upon One-Electron Reduction

Glotz G, Knaipp K, Maier MS, Hüll K, Novak A, Kelterer AM, Griebenow T, Herges R, Trauner D, Gescheidt G. To Isomerize or not to Isomerize? E/Z Isomers of Cyclic Azobenzene Derivatives and Their Reactivity Upon One-Electron Reduction. Chemistry. 2023 Jun 22;29(35):e202300146. doi: 10.1002/chem.202300146. Epub 2023 May 8. PMID: 37040130.

Abstract

Azo compounds are efficient electron acceptors. Upon one-electron reduction they generally isomerize forming the thermodynamically most stable radical anion. Herein we show that the size of the central ring in 1,2-diazocines and diazonines has a ruling influence on the configuration of the one-electron reduced species. Markedly, diazonines, which bear a central nine membered heterocycle, show light-induced E/Z isomerization, but retain the configuration of the diazene N=N moiety upon one-electron reduction. Accordingly, E/Z isomerization is not induced by reduction.

Chemical Biology CryoEM and CryoET Trauner Group

Single particle cryo-EM analysis of Rickettsia conorii Sca2 reveals a formin-like core

Carman PJ, Rebowski G, Dominguez R, Alqassim SS. Single particle cryo-EM analysis of Rickettsia conorii Sca2 reveals a formin-like core. J Struct Biol. 2023 Jun;215(2):107960. doi: 10.1016/j.jsb.2023.107960. Epub 2023 Apr 5. PMID: 37028467; PMCID: PMC10200769.

Abstract

Spotted fever group Rickettsia undergo actin-based motility inside infected eukaryotic cells using Sca2 (surface cell antigen 2): an ∼ 1800 amino-acid monomeric autotransporter protein that is surface-attached to the bacterium and responsible for the assembly of long unbranched actin tails. Sca2 is the only known functional mimic of eukaryotic formins, yet it shares no sequence similarities to the latter. Using structural and biochemical approaches we have previously shown that Sca2 uses a novel actin assembly mechanism. The first ∼ 400 amino acids fold into helix-loop-helix repeats that form a crescent shape reminiscent of a formin FH2 monomer. Additionally, the N- and C- terminal halves of Sca2 display intramolecular interaction in an end-to-end manner and cooperate for actin assembly, mimicking a formin FH2 dimer. Towards a better structural understanding of this mechanism, we performed single-particle cryo-electron microscopy analysis of Sca2. While high-resolution structural details remain elusive, our model confirms the presence of a formin-like core: Sca2 indeed forms a doughnut shape, similar in diameter to a formin FH2 dimer and can accommodate two actin subunits. Extra electron density, thought to be contributed by the C-terminal repeat domain (CRD), covering one side is also observed. This structural analysis allows us to propose an updated model where nucleation proceeds by encircling two actin subunits, and elongation proceeds either by a formin-like mechanism that necessitates conformational changes in the observed Sca2 model, or via an insertional mechanism akin to that observed in the ParMRC system.

CryoEM and CryoET Dominguez Lab X-ray Crystallography

Universal open MHC-I molecules for rapid peptide loading and enhanced complex stability across HLA allotypes

Sun Y, Young MC, Woodward CH, Danon JN, Truong HV, Gupta S, Winters TJ, Font-Burgada J, Burslem GM, Sgourakis NG. Proc. Natl. Acad. Sci. U.S.A. 2023 Jun 20. doi: 10.1073/pnas.2304055120.

Abstract

The polymorphic nature and intrinsic instability of class I major histocompatibility complex (MHC-I) and MHC-like molecules loaded with suboptimal peptides, metabolites, or glycolipids presents a fundamental challenge for identifying disease-relevant antigens and antigen-specific T cell receptors (TCRs), hindering the development of autologous therapeutics. Here, we leverage the positive allosteric coupling between the peptide and light chain (β2 microglobulin, β2m) subunits for binding to the MHC-I heavy chain (HC) through an engineered disulfide bond bridging conserved epitopes across the HC/β2m interface, to generate conformationally stable, peptide-receptive molecules named “open MHC-I.” Biophysical characterization shows that open MHC-I molecules are properly folded protein complexes of enhanced thermal stability compared to the wild type when loaded with low- to moderate-affinity peptides. Using solution NMR, we characterize the effects of the disulfide bond on the conformation and dynamics of the MHC-I structure, ranging from local changes in β2m-interacting sites of the peptide-binding groove to long-range effects on the α2-1 helix and α3 domain. The interchain disulfide bond stabilizes MHC-I molecules in an open conformation to promote peptide exchange across multiple human leukocyte antigen (HLA) allotypes, covering representatives from five HLA-A supertypes, six HLA-B supertypes, and oligomorphic HLA-Ib molecules. Our structure-guided design, combined with conditional β-peptide ligands, provides a universal platform to generate ready-to-load MHC-I systems of enhanced stability, enabling a range of approaches to screen antigenic epitope libraries and probe polyclonal TCR repertoires covering highly polymorphic HLA-I allotypes, as well as oligomorphic nonclassical molecules.

Chemical Biology Burslem Lab Computational Biology CryoEM and CryoET Mass Spectrometry X-ray Crystallography SgourakisLab

Purification of Recombinant Human Amphiphysin 1 and its N-BAR Domain

Mondal S, James HP, Milano F, Jin R, Baumgart T. Purification of Recombinant Human Amphiphysin 1 and its N-BAR Domain. Bio Protoc. 2023 Jun 20;13(12):e4699. doi: 10.21769/BioProtoc.4699. PMID: 37397795; PMCID: PMC10308189.

Abstract

Bin/Amphiphysin/Rvs (BAR) proteins are known as classical membrane curvature generators during endocytosis. Amphiphysin, a member of the N-BAR sub-family of proteins that contain a characteristic amphipathic sequence at the N-terminus of the BAR domain, is involved in clathrin-mediated endocytosis. Full-length amphiphysin contains a ~ 400 amino acid long disordered linker connecting the N-BAR domain and a C-terminal Src homology 3 (SH3) domain. We express and purify recombinant amphiphysin and its N-BAR domain along with an N-terminal glutathione-S-transferase (GST) tag. The GST tag allows extraction of the protein of interest using affinity chromatography and is removed in the subsequent protease treatment and ion-exchange chromatography steps. In the case of the N-BAR domain, cleavage of the GST tag was found to cause precipitation. This issue can be minimized by adding glycerol to the protein purification buffers. In the final step, size exclusion chromatography removes any potential oligomeric species. This protocol has also been successfully used to purify other N-BAR proteins, such as endophilin, Bin1, and their corresponding BAR domains.

Baumgart Lab CryoEM and CryoET Single Molecule Imaging

RAB27B controls palmitoylation-dependent NRAS trafficking and signaling in myeloid leukemi

Ren JG, Xing B, Lv K, O’Keefe RA, Wu M, Wang R, Bauer KM, Ghazaryan A, Burslem GM, Zhang J, O’Connell RM, Pillai V, Hexner EO, Philips MR, Tong W. RAB27B controls palmitoylation-dependent NRAS trafficking and signaling in myeloid leukemia. J Clin Invest. 2023 Jun 15;133(12):e165510. doi: 10.1172/JCI165510. PMID: 37317963; PMCID: PMC10266782.

Abstract

RAS mutations are among the most prevalent oncogenic drivers in cancers. RAS proteins propagate signals only when associated with cellular membranes as a consequence of lipid modifications that impact their trafficking. Here, we discovered that RAB27B, a RAB family small GTPase, controlled NRAS palmitoylation and trafficking to the plasma membrane, a localization required for activation. Our proteomic studies revealed RAB27B upregulation in CBL- or JAK2-mutated myeloid malignancies, and its expression correlated with poor prognosis in acute myeloid leukemias (AMLs). RAB27B depletion inhibited the growth of CBL-deficient or NRAS-mutant cell lines. Strikingly, Rab27b deficiency in mice abrogated mutant but not WT NRAS-mediated progenitor cell growth, ERK signaling, and NRAS palmitoylation. Further, Rab27b deficiency significantly reduced myelomonocytic leukemia development in vivo. Mechanistically, RAB27B interacted with ZDHHC9, a palmitoyl acyltransferase that modifies NRAS. By regulating palmitoylation, RAB27B controlled c-RAF/MEK/ERK signaling and affected leukemia development. Importantly, RAB27B depletion in primary human AMLs inhibited oncogenic NRAS signaling and leukemic growth. We further revealed a significant correlation between RAB27B expression and sensitivity to MEK inhibitors in AMLs. Thus, our studies presented a link between RAB proteins and fundamental aspects of RAS posttranslational modification and trafficking, highlighting future therapeutic strategies for RAS-driven cancers.

Chemical Biology Burslem Lab Mass Spectrometry

The mitochondrial Ca2+ channel MCU is critical for tumor growth by supporting cell cycle progression and proliferation

Fernandez Garcia E, Paudel U, Noji MC, Bowman CE, Rustgi AK, Pitarresi JR, Wellen KE, Arany Z, Weissenrieder JS, Foskett JK. The mitochondrial Ca2+ channel MCU is critical for tumor growth by supporting cell cycle progression and proliferation. Front Cell Dev Biol. 2023 Jun 8;11:1082213. doi: 10.3389/fcell.2023.1082213. PMID: 37363724; PMCID: PMC10285664.

Abstract

Introduction: The mitochondrial uniporter (MCU) Ca2+ ion channel represents the primary means for Ca2+ uptake by mitochondria. Mitochondrial matrix Ca2+ plays critical roles in mitochondrial bioenergetics by impinging upon respiration, energy production, and flux of biochemical intermediates through the TCA cycle. Inhibition of MCU in oncogenic cell lines results in an energetic crisis and reduced cell proliferation unless media is supplemented with nucleosides, pyruvate or α-KG. Nevertheless, the roles of MCU-mediated Ca2+ influx in cancer cells remain unclear, in part because of a lack of genetic models.

CryoEM and CryoET Foskett

Structural diversity of photoswitchable sphingolipids for optodynamic control of lipid microdomains

Hartrampf N, Leitao SM, Winter N, Toombs-Ruane H, Frank JA, Schwille P, Trauner D, Franquelim HG. Structural diversity of photoswitchable sphingolipids for optodynamic control of lipid microdomains. Biophys J. 2023 Jun 6;122(11):2325-2341. doi: 10.1016/j.bpj.2023.02.029. Epub 2023 Mar 3. PMID: 36869591; PMCID: PMC10257215.

Abstract

Sphingolipids are a structurally diverse class of lipids predominantly found in the plasma membrane of eukaryotic cells. These lipids can laterally segregate with other rigid lipids and cholesterol into liquid-ordered domains that act as organizing centers within biomembranes. Owing the vital role of sphingolipids for lipid segregation, controlling their lateral organization is of utmost significance. Hence, we made use of the light-induced trans-cis isomerization of azobenzene-modified acyl chains to develop a set of photoswitchable sphingolipids with different headgroups (hydroxyl, galactosyl, phosphocholine) and backbones (sphingosine, phytosphingosine, tetrahydropyran-blocked sphingosine) that are able to shuttle between liquid-ordered and liquid-disordered regions of model membranes upon irradiation with UV-A (λ = 365 nm) and blue (λ = 470 nm) light, respectively. Using combined high-speed atomic force microscopy, fluorescence microscopy, and force spectroscopy, we investigated how these active sphingolipids laterally remodel supported bilayers upon photoisomerization, notably in terms of domain area changes, height mismatch, line tension, and membrane piercing. Hereby, we show that the sphingosine-based (Azo-β-Gal-Cer, Azo-SM, Azo-Cer) and phytosphingosine-based (Azo-α-Gal-PhCer, Azo-PhCer) photoswitchable lipids promote a reduction in liquid-ordered microdomain area when in the UV-adapted cis-isoform. In contrast, azo-sphingolipids having tetrahydropyran groups that block H-bonding at the sphingosine backbone (lipids named Azo-THP-SM, Azo-THP-Cer) induce an increase in the liquid-ordered domain area when in cis, accompanied by a major rise in height mismatch and line tension. These changes were fully reversible upon blue light-triggered isomerization of the various lipids back to trans, pinpointing the role of interfacial interactions for the formation of stable liquid-ordered domains.

Chemical Biology CryoEM and CryoET Trauner Group

Structural basis of purine nucleotide inhibition of human uncoupling protein 1

Jones SA, Gogoi P, Ruprecht JJ, King MS, Lee Y, Zögg T, Pardon E, Chand D, Steimle S, Copeman DM, Cotrim CA, Steyaert J, Crichton PG, Moiseenkova-Bell V, Kunji ERS. Structural basis of purine nucleotide inhibition of human uncoupling protein 1. Sci Adv. 2023 Jun 2;9(22):eadh4251. doi: 10.1126/sciadv.adh4251. Epub 2023 May 31. PMID: 37256948; PMCID: PMC10413660.

Abstract Mitochondrial uncoupling protein

Mitochondrial uncoupling protein 1 (UCP1) gives brown adipose tissue of mammals its specialized ability to burn calories as heat for thermoregulation. When activated by fatty acids, UCP1 catalyzes the leak of protons across the mitochondrial inner membrane, short-circuiting the mitochondrion to generate heat, bypassing ATP synthesis. In contrast, purine nucleotides bind and inhibit UCP1, regulating proton leak by a molecular mechanism that is unclear. We present the cryo-electron microscopy structure of the GTP-inhibited state of UCP1, which is consistent with its nonconducting state. The purine nucleotide cross-links the transmembrane helices of UCP1 with an extensive interaction network. Our results provide a structural basis for understanding the specificity and pH dependency of the regulatory mechanism. UCP1 has retained all of the key functional and structural features required for a mitochondrial carrier-like transport mechanism. The analysis shows that inhibitor binding prevents the conformational changes that UCP1 uses to facilitate proton leak.

CryoEM and CryoET Mass Spectrometry Vera Moiseenkova-Bell Lab

Development of Light-Activated LXR Agonists

Mukhopadhyay TK, Willems S, Arp CJ, Morstein J, Haake CT, Merk D, Trauner D. Development of Light-Activated LXR Agonists. ChemMedChem. 2023 Jun 1;18(11):e202200647. doi: 10.1002/cmdc.202200647. Epub 2023 Jun 1. PMID: 36896647.

Abstract

Activation of the oxysterol-sensing transcription factor liver X receptor (LXR) has been studied as a therapeutic strategy in metabolic diseases and cancer but is compromised by the side effects of LXR agonists. Local LXR activation in cancer treatment may offer an opportunity to overcome this issue suggesting potential uses of photopharmacology. We report the computer-aided development of photoswitchable LXR agonists based on the T0901317 scaffold, which is a known LXR agonist. Azologization and structure-guided structure-activity relationship evaluation enabled the design of an LXR agonist, which activated LXR with low micromolar potency in its light-induced (Z)-state and was inactive as (E)-isomer. This tool sensitized human lung cancer cells to chemotherapeutic treatment in a light-dependent manner supporting potential of locally activated LXR agonists as adjuvant cancer treatment.

Chemical Biology CryoEM and CryoET Trauner Group

Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and ARF6 GTPase disrupts the axonal transport of autophagosomes

Dou D, Smith EM, Evans CS, Boecker CA, Holzbaur ELF. Regulatory imbalance between LRRK2 kinase, PPM1H phosphatase, and ARF6 GTPase disrupts the axonal transport of autophagosomes. Cell Rep. 2023 May 30;42(5):112448. doi: 10.1016/j.celrep.2023.112448. Epub 2023 May 1. PMID: 37133994; PMCID: PMC10304398.

Abstract

Gain-of-function mutations in the LRRK2 gene cause Parkinson’s disease (PD), increasing phosphorylation of RAB GTPases through hyperactive kinase activity. We find that LRRK2-hyperphosphorylated RABs disrupt the axonal transport of autophagosomes by perturbing the coordinated regulation of cytoplasmic dynein and kinesin. In iPSC-derived human neurons, knockin of the strongly hyperactive LRRK2-p.R1441H mutation causes striking impairments in autophagosome transport, inducing frequent directional reversals and pauses. Knockout of the opposing protein phosphatase 1H (PPM1H) phenocopies the effect of hyperactive LRRK2. Overexpression of ADP-ribosylation factor 6 (ARF6), a GTPase that acts as a switch for selective activation of dynein or kinesin, attenuates transport defects in both p.R1441H knockin and PPM1H knockout neurons. Together, these findings support a model where a regulatory imbalance between LRRK2-hyperphosphorylated RABs and ARF6 induces an unproductive “tug-of-war” between dynein and kinesin, disrupting processive autophagosome transport. This disruption may contribute to PD pathogenesis by impairing the essential homeostatic functions of axonal autophagy.

CryoEM and CryoET Holzbaur Lab Single Molecule Imaging

The structural basis of the multi-step allosteric activation of Aurora B kinase

Segura-Peña D, Hovet O, Gogoi H, Dawicki-McKenna J, Hansen Wøien SM, Carrer M, Black BE, Cascella M, Sekulic N. The structural basis of the multi-step allosteric activation of Aurora B kinase. Elife. 2023 May 25;12:e85328. doi: 10.7554/eLife.85328. PMID: 37227118; PMCID: PMC10259393.

Abstract

Aurora B, together with IN-box, the C-terminal part of INCENP, forms an enzymatic complex that ensures faithful cell division. The [Aurora B/IN-box] complex is activated by autophosphorylation in the Aurora B activation loop and in IN-box, but it is not clear how these phosphorylations activate the enzyme. We used a combination of experimental and computational studies to investigate the effects of phosphorylation on the molecular dynamics and structure of [Aurora B/IN-box]. In addition, we generated partially phosphorylated intermediates to analyze the contribution of each phosphorylation independently. We found that the dynamics of Aurora and IN-box are interconnected, and IN-box plays both positive and negative regulatory roles depending on the phosphorylation status of the enzyme complex. Phosphorylation in the activation loop of Aurora B occurs intramolecularly and prepares the enzyme complex for activation, but two phosphorylated sites are synergistically responsible for full enzyme activity.

Black Lab CryoEM and CryoET Mass Spectrometry

Centromere Innovations Within a Mouse Species

Gambogi CW, Pandey N, Dawicki-McKenna JM, Arora UP, Liskovykh MA, Ma J, Lamelza P, Larionov V, Lampson MA, Logsdon GA, Dumont BL, Black BE. Centromere Innovations Within a Mouse Species. bioRxiv [Preprint]. 2023 May 13:2023.05.11.540353. doi: 10.1101/2023.05.11.540353. Update in: Sci Adv. 2023 Nov 17;9(46):eadi5764. PMID: 37333154; PMCID: PMC10274901.

Abstract

Mammalian centromeres direct faithful genetic inheritance and are typically characterized by regions of highly repetitive and rapidly evolving DNA. We focused on a mouse species, Mus pahari, that we found has evolved to house centromere-specifying CENP-A nucleosomes at the nexus of a satellite repeat that we identified and term π-satellite (π-sat), a small number of recruitment sites for CENP-B, and short stretches of perfect telomere repeats. One M. pahari chromosome, however, houses a radically divergent centromere harboring ~6 Mbp of a homogenized π-sat-related repeat, π-satB, that contains >20,000 functional CENP-B boxes. There, CENP-B abundance drives accumulation of microtubule-binding components of the kinetochore, as well as a microtubule-destabilizing kinesin of the inner centromere. The balance of pro- and anti-microtubule-binding by the new centromere permits it to segregate during cell division with high fidelity alongside the older ones whose sequence creates a markedly different molecular composition.

Black Lab CryoEM and CryoET Mass Spectrometry

Metformin exerts antileukemic effects by modulating lactate metabolism and overcomes imatinib resistance in chronic myelogenous leukemia

Gayatri MB, Kancha RK, Patchva D, Velugonda N, Gundeti S, Reddy ABM. FEBS J. 2023 Sep;290(18):4480-4495. doi: 10.1111/febs.16818. Epub 2023 May 23.

Abstract

Imatinib is the frontline treatment option in treating chronic myelogenous leukemia (CML). Hitherto, some patients relapse following treatment. Biochemical analysis of a panel of clonally derived imatinib-resistant cells revealed enhanced glucose uptake and ATP production, suggesting increased rates of glycolysis. Interestingly, increased lactate export was also observed in imatinib-resistant cell lines. Here, we show that metformin inhibits the growth of imatinib-resistant cell lines as well as peripheral blood mononuclear cells isolated from patients who relapsed following imatinib treatment. Metformin exerted these antiproliferative effects by inhibiting MCT1 and MCT4, leading to the inhibition of lactate export. Furthermore, glucose uptake and ATP production were also inhibited following metformin treatment due to the inhibition of GLUT1 and HK-II in an AMPK-dependent manner. Our results also confirmed that metformin-mediated inhibition of lactate export and glucose uptake occurs through the regulation of mTORC1 and HIF-1α. These results delineate the molecular mechanisms underlying metabolic reprogramming leading to secondary imatinib resistance and the potential of metformin as a therapeutic option in CML.

 

CryoEM and CryoET Mass Spectrometry Reddy Lab

ECLiPSE: A Versatile Classification Technique for Structural and Morphological Analysis of Super-Resolution Microscopy Data

Hugelier S, Kim H, Gyparaki MT, Bond C, Tang Q, Santiago-Ruiz AN, Porta S, Lakadamyali M. ECLiPSE: A Versatile Classification Technique for Structural and Morphological Analysis of Super-Resolution Microscopy Data. bioRxiv [Preprint]. 2023 May 10:2023.05.10.540077. doi: 10.1101/2023.05.10.540077. PMID: 37215010; PMCID: PMC10197633.

Abstract

We introduce a new automated machine learning analysis pipeline to precisely classify cellular structures captured through single molecule localization microscopy, which we call ECLiPSE (Enhanced Classification of Localized Pointclouds by Shape Extraction). ECLiPSE leverages 67 comprehensive shape descriptors encompassing geometric, boundary, skeleton and other properties, the majority of which are directly extracted from the localizations to accurately characterize individual structures. We validate ECLiPSE through unsupervised and supervised classification on a dataset featuring five distinct cellular structures, achieving exceptionally high classification accuracies nearing 100%. Moreover, we demonstrate the versatility of our approach by applying it to two novel biological applications: quantifying the clearance of tau protein aggregates, a critical marker for neurodegenerative diseases, and differentiating between two distinct morphological features (morphotypes) of TAR DNA-binding protein 43 proteinopathy, potentially associated to different TDP-43 strains, each exhibiting unique seeding and spreading properties. We anticipate that this versatile approach will significantly enhance the way we study cellular structures across var

Single Molecule Imaging Lakadamyali Lab

Quantitative Single-Molecule Localization Microscopy

Hugelier S, Colosi PL, Lakadamyali M. Quantitative Single-Molecule Localization Microscopy. Annu Rev Biophys. 2023 May 9;52:139-160. doi: 10.1146/annurev-biophys-111622-091212. PMID: 37159293.

Abstract

Super-resolution fluorescence microscopy allows the investigation of cellular structures at nanoscale resolution using light. Current developments in super-resolution microscopy have focused on reliable quantification of the underlying biological data. In this review, we first describe the basic principles of super-resolution microscopy techniques such as stimulated emission depletion (STED) microscopy and single-molecule localization microscopy (SMLM), and then give a broad overview of methodological developments to quantify super-resolution data, particularly those geared toward SMLM data. We cover commonly used techniques such as spatial point pattern analysis, colocalization, and protein copy number quantification but also describe more advanced techniques such as structural modeling, single-particle tracking, and biosensing. Finally, we provide an outlook on exciting new research directions to which quantitative super-resolution microscopy might be applied.

Single Molecule Imaging Lakadamyali Lab

Circulating tumor DNA reveals mechanisms of lorlatinib resistance in patients with relapsed/refractory ALK-driven neuroblastoma

Berko ER, Witek GM, Matkar S, Petrova ZO, Wu MA, Smith CM, Daniels A, Kalna J, Kennedy A, Gostuski I, Casey C, Krytska K, Gerelus M, Pavlick D, Ghazarian S, Park JR, Marachelian A, Maris JM, Goldsmith KC, Radhakrishnan R, Lemmon MA, Mossé YP. Circulating tumor DNA reveals mechanisms of lorlatinib resistance in patients with relapsed/refractory ALK-driven neuroblastoma. Nat Commun. 2023 May 5;14(1):2601. doi: 10.1038/s41467-023-38195-0. PMID: 37147298; PMCID: PMC10163008.

Abstract

Activating point mutations in Anaplastic Lymphoma Kinase (ALK) have positioned ALK as the only mutated oncogene tractable for targeted therapy in neuroblastoma. Cells with these mutations respond to lorlatinib in pre-clinical studies, providing the rationale for a first-in-child Phase 1 trial (NCT03107988) in patients with ALK-driven neuroblastoma. To track evolutionary dynamics and heterogeneity of tumors, and to detect early emergence of lorlatinib resistance, we collected serial circulating tumor DNA samples from patients enrolled on this trial. Here we report the discovery of off-target resistance mutations in 11 patients (27%), predominantly in the RAS-MAPK pathway. We also identify newly acquired secondary compound ALK mutations in 6 (15%) patients, all acquired at disease progression. Functional cellular and biochemical assays and computational studies elucidate lorlatinib resistance mechanisms. Our results establish the clinical utility of serial circulating tumor DNA sampling to track response and progression and to discover acquired resistance mechanisms that can be leveraged to develop therapeutic strategies to overcome lorlatinib resistance.

Computational Biology Radhakrishnan Lab

FRETing about the details: Case studies in the use of a genetically encoded fluorescent amino acid for distance-dependent energy transfer

Cory MB, Jones CM, Shaffer KD, Venkatesh Y, Giannakoulias S, Perez RM, Lougee MG, Hummingbird E, Pagar VV, Hurley CM, Li A, Mach RH, Kohli RM, Petersson EJ. FRETing about the details: Case studies in the use of a genetically encoded fluorescent amino acid for distance-dependent energy transfer. Protein Sci. 2023 May;32(5):e4633. doi: 10.1002/pro.4633. PMID: 36974585; PMCID: PMC10108435.

Abstract

Förster resonance energy transfer (FRET) is a valuable method for monitoring protein conformation and biomolecular interactions. Intrinsically fluorescent amino acids that can be genetically encoded, such as acridonylalanine (Acd), are particularly useful for FRET studies. However, quantitative interpretation of FRET data to derive distance information requires careful use of controls and consideration of photophysical effects. Here we present two case studies illustrating how Acd can be used in FRET experiments to study small molecule induced conformational changes and multicomponent biomolecular complexes.

 

Chemical Biology CryoEM and CryoET Kohli Lab

Deubiquitinase CYLD regulates excitatory synaptic transmission and short-term plasticity in the hippocampus

Chen SY, Liu KF, Tan SY, Chen XS, Li HD, Li JJ, Zhou JW, Yang L, Long C. Deubiquitinase CYLD regulates excitatory synaptic transmission and short-term plasticity in the hippocampus. Brain Res. 2023 May 1;1806:148313. doi: 10.1016/j.brainres.2023.148313. Epub 2023 Mar 4. PMID: 36878342.

Abstract

The fate of proteins is determined by the addition of various forms of polyubiquitin during ubiquitin-mediated proteasomal degradation. Cylindromatosis (CYLD), a K63-specific deubiquitinase, is enriched in postsynaptic density fractions of the rodent central nervous system (CNS), but the synaptic role of CYLD in the CNS is poorly understand. Here we show that CYLD deficiency (Cyld-/-) results in reduced intrinsic hippocampal neuronal firing, a decrease in the frequency of spontaneous excitatory postsynaptic currents and a decrease in the amplitude of field excitatory postsynaptic potentials. Moreover, Cyld-/- hippocampus shows downregulated levels of presynaptic vesicular glutamate transporter 1 (vGlut1) and upregulated levels of postsynaptic GluA1, a subunit of the AMPA receptor, together with an altered paired-pulse ratio (PPR). We also found increased activation of astrocytes and microglia in the hippocampus of Cyld-/- mice. The present study suggests a critical role for CYLD in mediating hippocampal neuronal and synaptic activity.

Mass Spectrometry X-ray Crystallography Liu Lab

Computational Methods Toward Unbiased Pattern Mining and Structure Determination in Cryo-Electron Tomography Data

Kim HH, Uddin MR, Xu M, Chang YW. Computational Methods Toward Unbiased Pattern Mining and Structure Determination in Cryo-Electron Tomography Data. J Mol Biol. 2023 May 1;435(9):168068. doi: 10.1016/j.jmb.2023.168068. Epub 2023 Mar 31. PMID: 37003470; PMCID: PMC10164694.

Abstract

Cryo-electron tomography can uniquely probe the native cellular environment for macromolecular structures. Tomograms feature complex data with densities of diverse, densely crowded macromolecular complexes, low signal-to-noise, and artifacts such as the missing wedge effect. Post-processing of this data generally involves isolating regions or particles of interest from tomograms, organizing them into related groups, and rendering final structures through subtomogram averaging. Template-matching and reference-based structure determination are popular analysis methods but are vulnerable to biases and can often require significant user input. Most importantly, these approaches cannot identify novel complexes that reside within the imaged cellular environment. To reliably extract and resolve structures of interest, efficient and unbiased approaches are therefore of great value. This review highlights notable computational software and discusses how they contribute to making automated structural pattern discovery a possibility. Perspectives emphasizing the importance of features for user-friendliness and accessibility are also presented.

CryoEM and CryoET Mass Spectrometry Yi-Wei Chang Lab

The 5’UTR of HCoV-OC43 adopts a topologically constrained structure to intrinsically repress translation

Mackeown M, Kung YA, Davila-Calderon J, Ford WP, Luo L, Henry B, Li ML, Brewer G, Shih SR, Tolbert BS. The 5’UTR of HCoV-OC43 adopts a topologically constrained structure to intrinsically repress translation. J Biol Chem. 2023 Apr;299(4):103028. doi: 10.1016/j.jbc.2023.103028. Epub 2023 Feb 15. PMID: 36805339; PMCID: PMC9930382.

Abstract

The emergence of SARS-CoV-2, which is responsible for the COVID-19 pandemic, has highlighted the need for rapid characterization of viral mechanisms associated with cellular pathogenesis. Viral UTRs represent conserved genomic elements that contribute to such mechanisms. Structural details of most CoV UTRs are not available, however. Experimental approaches are needed to allow for the facile generation of high-quality viral RNA tertiary structural models, which can facilitate comparative mechanistic efforts. By integrating experimental and computational techniques, we herein report the efficient characterization of conserved RNA structures within the 5’UTR of the HCoV-OC43 genome, a lab-tractable model coronavirus. We provide evidence that the 5’UTR folds into a structure with well-defined stem-loops (SLs) as determined by chemical probing and direct detection of hydrogen bonds by NMR. We combine experimental base-pair restraints with global structural information from SAXS to generate a 3D model that reveals that SL1-4 adopts a topologically constrained structure wherein SLs 3 and 4 coaxially stack. Coaxial stacking is mediated by short linker nucleotides and allows SLs 1 to 2 to sample different cojoint orientations by pivoting about the SL3,4 helical axis. To evaluate the functional relevance of the SL3,4 coaxial helix, we engineered luciferase reporter constructs harboring the HCoV-OC43 5’UTR with mutations designed to abrogate coaxial stacking. Our results reveal that the SL3,4 helix intrinsically represses translation efficiency since the destabilizing mutations correlate with increased luciferase expression relative to wildtype without affecting reporter mRNA levels, thus highlighting how the 5’UTR structure contributes to the viral mechanism.

CryoEM and CryoET NMR Tolbert Lab

Remembering the Work of Phillip L. Geissler: A Coda to His Scientific Trajectory

Bowman GR, Cox SJ, Dellago C, DuBay KH, Eaves JD, Fletcher DA, Frechette LB, Grünwald M, Klymko K, Ku J, Omar AK, Rabani E, Reichman DR, Rogers JR, Rosnik AM, Rotskoff GM, Schneider AR, Schwierz N, Sivak DA, Vaikuntanathan S, Whitelam S, Widmer-Cooper A. Remembering the Work of Phillip L. Geissler: A Coda to His Scientific Trajectory. Annu Rev Phys Chem. 2023 Apr 24;74:1-27. doi: 10.1146/annurev-physchem-101422-030127. Epub 2023 Jan 31. PMID: 36719975.

Abstract

Phillip L. Geissler made important contributions to the statistical mechanics of biological polymers, heterogeneous materials, and chemical dynamics in aqueous environments. He devised analytical and computational methods that revealed the underlying organization of complex systems at the frontiers of biology, chemistry, and materials science. In this retrospective we celebrate his work at these frontiers.

Bowman Lab Computational Biology

Macrocyclic Octapeptide Binding and Inferences on Protein Substrate Binding to Histone Deacetylase 6

Watson PR, Gupta S, Hosseinzadeh P, Brown BP, Baker D, Christianson DW. Macrocyclic Octapeptide Binding and Inferences on Protein Substrate Binding to Histone Deacetylase 6. ACS Chem Biol. 2023 Apr 21;18(4):959-968. doi: 10.1021/acschembio.3c00113. Epub 2023 Apr 7. PMID: 37027789; PMCID: PMC10130746.

Abstract

Histone deacetylases (HDACs) are essential for the regulation of myriad biological processes, and their aberrant function is implicated in cancer, neurodegeneration, and other diseases. The cytosolic isozyme HDAC6 is unique among the greater family of deacetylases in that it contains two catalytic domains, CD1 and CD2. HDAC6 CD2 is responsible for tubulin deacetylase and tau deacetylase activities, inhibition of which is a key goal as new therapeutic approaches are explored. Of particular interest as HDAC inhibitors are naturally occurring cyclic tetrapeptides such as Trapoxin A or HC Toxin, or the cyclic depsipeptides Largazole and Romidepsin. Even more intriguing are larger, computationally designed macrocyclic peptide inhibitors. Here, we report the 2.0 Å resolution crystal structure of HDAC6 CD2 complexed with macrocyclic octapeptide 1. Comparison with the previously reported structure of the complex with macrocyclic octapeptide 2 reveals that a potent thiolate-zinc interaction made by the unnatural amino acid (S)-2-amino-7-sulfanylheptanoic acid contributes to nanomolar inhibitory potency for each inhibitor. Apart from this zinc-binding residue, octapeptides adopt strikingly different overall conformations and make few direct hydrogen bonds with the protein. Intermolecular interactions are dominated by water-mediated hydrogen bonds; in essence, water molecules appear to cushion the enzyme-octapeptide interface. In view of the broad specificity observed for protein substrates of HDAC6 CD2, we suggest that the binding of macrocyclic octapeptides may mimic certain features of the binding of macromolecular protein substrates.

CryoEM and CryoET Christianson Group X-ray Crystallography

Influenza virus mRNAs encode determinants for nuclear export via the cellular TREX-2 complex

Bhat P, Aksenova V, Gazzara M, Rex EA, Aslam S, Haddad C, Gao S, Esparza M, Cagatay T, Batten K, El Zahed SS, Arnaoutov A, Zhong H, Shay JW, Tolbert BS, Dasso M, Lynch KW, García-Sastre A, Fontoura BMA. Influenza virus mRNAs encode determinants for nuclear export via the cellular TREX-2 complex. Nat Commun. 2023 Apr 21;14(1):2304. doi: 10.1038/s41467-023-37911-0. PMID: 37085480; PMCID: PMC10121598.

Abstract

Nuclear export of influenza A virus (IAV) mRNAs occurs through the nuclear pore complex (NPC). Using the Auxin-Induced Degron (AID) system to rapidly degrade proteins, we show that among the nucleoporins localized at the nucleoplasmic side of the NPC, TPR is the key nucleoporin required for nuclear export of influenza virus mRNAs. TPR recruits the TRanscription and EXport complex (TREX)-2 to the NPC for exporting a subset of cellular mRNAs. By degrading components of the TREX-2 complex (GANP, Germinal-center Associated Nuclear Protein; PCID2, PCI domain containing 2), we show that influenza mRNAs require the TREX-2 complex for nuclear export and replication. Furthermore, we found that cellular mRNAs whose export is dependent on GANP have a small number of exons, a high mean exon length, long 3′ UTR, and low GC content. Some of these features are shared by influenza virus mRNAs. Additionally, we identified a 45 nucleotide RNA signal from influenza virus HA mRNA that is sufficient to mediate GANP-dependent mRNA export. Thus, we report a role for the TREX-2 complex in nuclear export of influenza mRNAs and identified RNA determinants associated with the TREX-2-dependent mRNA export.

CryoEM and CryoET NMR Tolbert Lab

Allosteric role of the citrate synthase homology domain of ATP citrate lyase

Wei X, Schultz K, Pepper HL, Megill E, Vogt A, Snyder NW, Marmorstein R. Allosteric role of the citrate synthase homology domain of ATP citrate lyase. Nat Commun. 2023 Apr 19;14(1):2247. doi: 10.1038/s41467-023-37986-9. PMID: 37076498; PMCID: PMC10115795.

Abstract

ATP citrate lyase (ACLY) is the predominant nucleocytosolic source of acetyl-CoA and is aberrantly regulated in many diseases making it an attractive therapeutic target. Structural studies of ACLY reveal a central homotetrameric core citrate synthase homology (CSH) module flanked by acyl-CoA synthetase homology (ASH) domains, with ATP and citrate binding the ASH domain and CoA binding the ASH-CSH interface to produce acetyl-CoA and oxaloacetate products. The specific catalytic role of the CSH module and an essential D1026A residue contained within it has been a matter of debate. Here, we report biochemical and structural analysis of an ACLY-D1026A mutant demonstrating that this mutant traps a (3S)-citryl-CoA intermediate in the ASH domain in a configuration that is incompatible with the formation of acetyl-CoA, is able to convert acetyl-CoA and OAA to (3S)-citryl-CoA in the ASH domain, and can load CoA and unload acetyl-CoA in the CSH module. Together, this data support an allosteric role for the CSH module in ACLY catalysis.

Chemical Biology CryoEM and CryoET X-ray Crystallography Marmorstein Lab

Discovery of a cryptic pocket in the AI-predicted structure of PPM1D phosphatase explains the binding site and potency of its allosteric inhibitors

Meller A, De Oliveira S, Davtyan A, Abramyan T, Bowman GR, van den Bedem H. Discovery of a cryptic pocket in the AI-predicted structure of PPM1D phosphatase explains the binding site and potency of its allosteric inhibitors. Front Mol Biosci. 2023 Apr 18;10:1171143. doi: 10.3389/fmolb.2023.1171143. PMID: 37143823; PMCID: PMC10151774.

Abstract

Virtual screening is a widely used tool for drug discovery, but its predictive power can vary dramatically depending on how much structural data is available. In the best case, crystal structures of a ligand-bound protein can help find more potent ligands. However, virtual screens tend to be less predictive when only ligand-free crystal structures are available, and even less predictive if a homology model or other predicted structure must be used. Here, we explore the possibility that this situation can be improved by better accounting for protein dynamics, as simulations started from a single structure have a reasonable chance of sampling nearby structures that are more compatible with ligand binding. As a specific example, we consider the cancer drug target PPM1D/Wip1 phosphatase, a protein that lacks crystal structures. High-throughput screens have led to the discovery of several allosteric inhibitors of PPM1D, but their binding mode remains unknown. To enable further drug discovery efforts, we assessed the predictive power of an AlphaFold-predicted structure of PPM1D and a Markov state model (MSM) built from molecular dynamics simulations initiated from that structure. Our simulations reveal a cryptic pocket at the interface between two important structural elements, the flap and hinge regions. Using deep learning to predict the pose quality of each docked compound for the active site and cryptic pocket suggests that the inhibitors strongly prefer binding to the cryptic pocket, consistent with their allosteric effect. The predicted affinities for the dynamically uncovered cryptic pocket also recapitulate the relative potencies of the compounds (τb = 0.70) better than the predicted affinities for the static AlphaFold-predicted structure (τb = 0.42).

Bowman Lab Computational Biology

High-throughput cryo-ET structural pattern mining by unsupervised deep iterative subtomogram clustering

Zeng X, Kahng A, Xue L, Mahamid J, Chang YW, Xu M. High-throughput cryo-ET structural pattern mining by unsupervised deep iterative subtomogram clustering. Proc Natl Acad Sci U S A. 2023 Apr 11;120(15):e2213149120. doi: 10.1073/pnas.2213149120. Epub 2023 Apr 7. PMID: 37027429; PMCID: PMC10104553.

Abstract

Cryoelectron tomography directly visualizes heterogeneous macromolecular structures in their native and complex cellular environments. However, existing computer-assisted structure sorting approaches are low throughput or inherently limited due to their dependency on available templates and manual labels. Here, we introduce a high-throughput template-and-label-free deep learning approach, Deep Iterative Subtomogram Clustering Approach (DISCA), that automatically detects subsets of homogeneous structures by learning and modeling 3D structural features and their distributions. Evaluation on five experimental cryo-ET datasets shows that an unsupervised deep learning based method can detect diverse structures with a wide range of molecular sizes. This unsupervised detection paves the way for systematic unbiased recognition of macromolecular complexes in situ.

CryoEM and CryoET Mass Spectrometry Yi-Wei Chang Lab

Photoswitchable Molecular Glues Enable Optical Control of Transcription Factor Degradation

Arp CJ, Reynders M, Sreekanth V, Kokkonda P, Pagano M, Choudhary A, Trauner D. Photoswitchable Molecular Glues Enable Optical Control of Transcription Factor Degradation. bioRxiv [Preprint]. 2023 Apr 9:2023.04.09.536172. doi: 10.1101/2023.04.09.536172. PMID: 37066279; PMCID: PMC10104231.

Abstract

Immunomodulatory drugs (IMiDs), which include thalidomide and its derivatives, have emerged as the standard of care against multiple myeloma. They function as molecular glues that bind to the E3 ligase cereblon (CRBN) and induce protein interactions with neosubstrates, including the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3). The subsequent ubiquitylation and degradation of these transcription factors underlies the antiproliferative activity of IMiDs. Here, we introduce photoswitchable immunomodulatory drugs (PHOIMiDs) that can be used to degrade Ikaros and Aiolos in a light-dependent fashion. Our lead compound shows minimal activity in the dark and becomes an active degrader upon irradiation with violet light. It shows high selectivity over other transcription factors, regardless of its state, and could therefore be used to control the levels of Ikaros and Aiolos with high spatiotemporal precision.

Chemical Biology CryoEM and CryoET Trauner Group

Transitions in the framework of condensate biology

Seydoux G, Zhang M, Forman-Kay JD, McStay B, Liu KF, Li P. Transitions in the framework of condensate biology. Mol Cell. 2023 Apr 6;83(7):1016-1021. doi: 10.1016/j.molcel.2023.03.014. PMID: 37028411; PMCID: PMC10627508.

Abstract

As phase separation is found in an increasing variety of biological contexts, additional challenges have arisen in understanding the underlying principles of condensate formation and function. We spoke with researchers across disciplines about their views on the ever-changing landscape of biomolecular condensates.

Mass Spectrometry X-ray Crystallography Liu Lab

Functional and Phylogenetic Diversity of Cas10 Proteins

Wiegand T, Wilkinson R, Santiago-Frangos A, Lynes M, Hatzenpichler R, Wiedenheft B. Functional and Phylogenetic Diversity of Cas10 Proteins. CRISPR J. 2023 Apr;6(2):152-162. doi: 10.1089/crispr.2022.0085. Epub 2023 Mar 13. PMID: 36912817; PMCID: PMC10123807.

Abstract

Cas10 proteins are large subunits of type III CRISPR RNA (crRNA)-guided surveillance complexes, many of which have nuclease and cyclase activities. Here, we use computational and phylogenetic methods to identify and analyze 2014 Cas10 sequences from genomic and metagenomic databases. Cas10 proteins cluster into five distinct clades that mirror previously established CRISPR-Cas subtypes. Most Cas10 proteins (85.0%) have conserved polymerase active-site motifs, while HD-nuclease domains are less well conserved (36.0%). We identify Cas10 variants that are split over multiple genes or genetically fused to nucleases activated by cyclic nucleotides (i.e., NucC) or components of toxin-antitoxin systems (i.e., AbiEii). To clarify the functional diversification of Cas10 proteins, we cloned, expressed, and purified five representatives from three phylogenetically distinct clades. None of the Cas10s are functional cyclases in isolation, and activity assays performed with polymerase domain active site mutants indicate that previously reported Cas10 DNA-polymerase activity may be a result of contamination. Collectively, this work helps clarify the phylogenetic and functional diversity of Cas10 proteins in type III CRISPR systems.

Computational Biology CryoEM and CryoET Santiago-Frangos Lab

Transferase-Mediated Labeling of Protein N-Termini with Click Chemistry Handles

Wagner AM, Warner JB, Garrett HE, Walters CR, Petersson EJ. Transferase-Mediated Labeling of Protein N-Termini with Click Chemistry Handles. Methods Mol Biol. 2023;2620:157-175. doi: 10.1007/978-1-0716-2942-0_21. PMID: 37010762.

Abstract

The E. coli aminoacyl transferase (AaT) can be used to transfer a variety of unnatural amino acids, including those with azide or alkyne groups, to the α-amine of a protein with an N-terminal Lys or Arg. Subsequent functionalization through either copper-catalyzed or strain-promoted click reactions can be used to label the protein with fluorophores or biotin. This can be used to directly detect AaT substrates or in a two-step protocol to detect substrates of the mammalian ATE1 transferase.

CryoEM and CryoET NMR Petersson Lab

Synthesis of Peptides and Proteins with Site-Specific Glutamate Arginylation

Shimogawa M, Huang Y, Pan B, Petersson EJ. Synthesis of Peptides and Proteins with Site-Specific Glutamate Arginylation. Methods Mol Biol. 2023;2620:177-207. doi: 10.1007/978-1-0716-2942-0_22. PMID: 37010763; PMCID: PMC10752357.

Abstract

Solid-phase peptide synthesis and protein semi-synthesis are powerful methods for site-specific modification of peptides and proteins. We describe protocols using these techniques for the syntheses of peptides and proteins bearing glutamate arginylation (EArg) at specific sites. These methods overcome challenges posed by enzymatic arginylation methods and allow for a comprehensive study of the effects of EArg on protein folding and interactions. Potential applications include biophysical analyses, cell-based microscopic studies, and profiling of EArg levels and interactomes in human tissue samples.

CryoEM and CryoET NMR Petersson Lab

Spurious intragenic transcription is a feature of mammalian cellular senescence and tissue aging

Sen P, Donahue G, Li C, Egervari G, Yang N, Lan Y, Robertson N, Shah PP, Kerkhoven E, Schultz DC, Adams PD, Berger SL. Spurious intragenic transcription is a feature of mammalian cellular senescence and tissue aging. Nat Aging. 2023 Apr;3(4):402-417. doi: 10.1038/s43587-023-00384-3. Epub 2023 Mar 30. PMID: 37117791; PMCID: PMC10165726.

Abstract

Mammalian aging is characterized by the progressive loss of tissue function and increased risk for disease. Accumulation of senescent cells in aging tissues partly contributes to this decline, and targeted depletion of senescent cells in vivo ameliorates many age-related phenotypes. The fundamental molecular mechanisms responsible for the decline of cellular health and fitness during senescence and aging are largely unknown. In this study, we investigated whether chromatin-mediated loss of transcriptional fidelity, known to contribute to fitness and survival in yeast and worms, also occurs during human cellular senescence and mouse aging. Our findings reveal aberrant transcription initiation inside genes during senescence and aging that co-occurs with changes in the chromatin landscape. Interventions that alter these spurious transcripts have profound consequences on cellular health, primarily affecting intracellular signal transduction pathways. We propose that age-related spurious transcription promotes a noisy transcriptome and degradation of coherent transcriptional networks.

Chemical Biology Protein Production Services Schultz Lab

Noncatalytic regulation of 18 S rRNA methyltransferase DIMT1 in acute myeloid leukemia

Gonskikh Y, Stoute J, Shen H, Budinich K, Pingul B, Schultz K, Elashal H, Marmorstein R, Shi J, Liu KF. Noncatalytic regulation of 18S rRNA methyltransferase DIMT1 in acute myeloid leukemia. Genes Dev. 2023 Apr 1;37(7-8):321-335. doi: 10.1101/gad.350298.122. Epub 2023 Apr 6. PMID: 37024283; PMCID: PMC10153457.

Abstract

Several rRNA-modifying enzymes install rRNA modifications while participating in ribosome assembly. Here, we show that 18S rRNA methyltransferase DIMT1 is essential for acute myeloid leukemia (AML) proliferation through a noncatalytic function. We reveal that targeting a positively charged cleft of DIMT1, remote from the catalytic site, weakens the binding of DIMT1 to rRNA and mislocalizes DIMT1 to the nucleoplasm, in contrast to the primarily nucleolar localization of wild-type DIMT1. Mechanistically, rRNA binding is required for DIMT1 to undergo liquid-liquid phase separation, which explains the distinct nucleoplasm localization of the rRNA binding-deficient DIMT1. Re-expression of wild-type or a catalytically inactive mutant E85A, but not the rRNA binding-deficient DIMT1, supports AML cell proliferation. This study provides a new strategy to target DIMT1-regulated AML proliferation via targeting this essential noncatalytic region.

Chemical Biology CryoEM and CryoET Mass Spectrometry X-ray Crystallography Liu Lab Marmorstein Lab

Mpox Evolution: Has the Current Outbreak Revealed a Pox on “U”?

Kohli RM, Isaacs SN. Mpox Evolution: Has the Current Outbreak Revealed a Pox on “U”? J Infect Dis. 2023 Mar 28;227(6):828-830. doi: 10.1093/infdis/jiac471. PMID: 36481811.

Abstract

The recent mpox (monkeypox) outbreak has prompted genomic studies to track global spread of the disease. These studies have also revealed unexpected patterns of mutations that implicate the action of the immune defense APOBEC3 family of enzymes, which catalyze conversion of cytosine (C) to uracil (U) in DNA, in viral evolution. As poxviruses have conventionally been regarded as slow-evolving viruses, the rapid emergence of APOBEC3 mutational signatures begs a series of important and open questions regarding how host-pathogen interactions may have changed and whether these mutations are bystanders or have roles in pathogenesis.

Chemical Biology CryoEM and CryoET Kohli Lab

Optopharmacological tools for precise spatiotemporal control of oxytocin signaling in the central nervous system and periphery

Ahmed IA, Liu JJ, Gieniec KA, Bair-Marshall CJ, Adewakun AB, Hetzler BE, Arp CJ, Khatri L, Vanwalleghem GC, Seidenberg AT, Cowin P, Trauner D, Chao MV, Davis FM, Tsien RW, Froemke RC. Optopharmacological tools for precise spatiotemporal control of oxytocin signaling in the central nervous system and periphery. Res Sq [Preprint]. 2023 Mar 27:rs.3.rs-2715993. doi: 10.21203/rs.3.rs-2715993/v1. PMID: 37034806; PMCID: PMC10081362.

Abstract

Oxytocin is a neuropeptide critical for maternal physiology and social behavior, and is thought to be dysregulated in several neuropsychiatric disorders. Despite the biological and neurocognitive importance of oxytocin signaling, methods are lacking to activate oxytocin receptors with high spatiotemporal precision in the brain and peripheral mammalian tissues. Here we developed and validated caged analogs of oxytocin which are functionally inert until cage release is triggered by ultraviolet light. We examined how focal versus global oxytocin application affected oxytocin-driven Ca2+ wave propagation in mouse mammary tissue. We also validated the application of caged oxytocin in the hippocampus and auditory cortex with electrophysiological recordings in vitro, and demonstrated that oxytocin uncaging can accelerate the onset of mouse maternal behavior in vivo. Together, these results demonstrate that optopharmacological control of caged peptides is a robust tool with spatiotemporal precision for modulating neuropeptide signaling throughout the brain and body.

Chemical Biology CryoEM and CryoET Trauner Group

Stiff matrix induces exosome secretion to promote tumour growth

Wu B, Liu DA, Guan L, Myint PK, Chin L, Dang H, Xu Y, Ren J, Li T, Yu Z, Jabban S, Mills GB, Nukpezah J, Chen YH, Furth EE, Gimotty PA, Wells RG, Weaver VM, Radhakrishnan R, Wang XW, Guo W. Stiff matrix induces exosome secretion to promote tumour growth. Nat Cell Biol. 2023 Mar;25(3):415-424. doi: 10.1038/s41556-023-01092-1. Epub 2023 Feb 16. Erratum in: Nat Cell Biol. 2024 Feb 12;: PMID: 36797475; PMCID: PMC10351222.

Abstract

Tissue fibrosis and extracellular matrix (ECM) stiffening promote tumour progression. The mechanisms by which ECM regulates its contacting cells have been extensively studied. However, how stiffness influences intercellular communications in the microenvironment for tumour progression remains unknown. Here we report that stiff ECM stimulates the release of exosomes from cancer cells. We delineate a molecular pathway that links stiff ECM to activation of Akt, which in turn promotes GTP loading to Rab8 that drives exosome secretion. We further show that exosomes generated from cells grown on stiff ECM effectively promote tumour growth. Proteomic analysis revealed that the Notch signalling pathway is activated in cells treated with exosomes derived from tumour cells grown on stiff ECM, consistent with our gene expression analysis of liver tissues from patients. Our study reveals a molecular mechanism that regulates exosome secretion and provides insight into how mechanical properties of the ECM control the tumour microenvironment for tumour growth.

Computational Biology Radhakrishnan Lab

Discovery of a cryptic pocket in the AI-predicted structure of PPM1D phosphatase explains the binding site and potency of its allosteric inhibitors

Meller A, de Oliveira S, Davtyan A, Abramyan T, Bowman GR, van den Bedem H. Discovery of a cryptic pocket in the AI-predicted structure of PPM1D phosphatase explains the binding site and potency of its allosteric inhibitors. bioRxiv [Preprint]. 2023 Mar 24:2023.03.22.533829. doi: 10.1101/2023.03.22.533829. Update in: Front Mol Biosci. 2023 Apr 18;10:1171143. PMID: 36993233; PMCID: PMC10055338.

Abstract

Virtual screening is a widely used tool for drug discovery, but its predictive power can vary dramatically depending on how much structural data is available. In the best case, crystal structures of a ligand-bound protein can help find more potent ligands. However, virtual screens tend to be less predictive when only ligand-free crystal structures are available, and even less predictive if a homology model or other predicted structure must be used. Here, we explore the possibility that this situation can be improved by better accounting for protein dynamics, as simulations started from a single structure have a reasonable chance of sampling nearby structures that are more compatible with ligand binding. As a specific example, we consider the cancer drug target PPM1D/Wip1 phosphatase, a protein that lacks crystal structures. High-throughput screens have led to the discovery of several allosteric inhibitors of PPM1D, but their binding mode remains unknown. To enable further drug discovery efforts, we assessed the predictive power of an AlphaFold-predicted structure of PPM1D and a Markov state model (MSM) built from molecular dynamics simulations initiated from that structure. Our simulations reveal a cryptic pocket at the interface between two important structural elements, the flap and hinge regions. Using deep learning to predict the pose quality of each docked compound for the active site and cryptic pocket suggests that the inhibitors strongly prefer binding to the cryptic pocket, consistent with their allosteric effect. The predicted affinities for the dynamically uncovered cryptic pocket also recapitulate the relative potencies of the compounds (τ b =0.70) better than the predicted affinities for the static AlphaFold-predicted structure (τ b =0.42).

Bowman Lab Computational Biology

Accelerating Cryptic Pocket Discovery Using AlphaFold

Meller A, Bhakat S, Solieva S, Bowman GR. Accelerating Cryptic Pocket Discovery Using AlphaFold. J Chem Theory Comput. 2023 Jul 25;19(14):4355-4363. doi: 10.1021/acs.jctc.2c01189. Epub 2023 Mar 22. PMID: 36948209; PMCID: PMC10373493.

Abstract Cryptic pockets, or

Cryptic pockets, or pockets absent in ligand-free, experimentally determined structures, hold great potential as drug targets. However, cryptic pocket openings are often beyond the reach of conventional biomolecular simulations because certain cryptic pocket openings involve slow motions. Here, we investigate whether AlphaFold can be used to accelerate cryptic pocket discovery either by generating structures with open pockets directly or generating structures with partially open pockets that can be used as starting points for simulations. We use AlphaFold to generate ensembles for 10 known cryptic pocket examples, including five that were deposited after AlphaFold’s training data were extracted from the PDB. We find that in 6 out of 10 cases AlphaFold samples the open state. For plasmepsin II, an aspartic protease from the causative agent of malaria, AlphaFold only captures a partial pocket opening. As a result, we ran simulations from an ensemble of AlphaFold-generated structures and show that this strategy samples cryptic pocket opening, even though an equivalent amount of simulations launched from a ligand-free experimental structure fails to do so. Markov state models (MSMs) constructed from the AlphaFold-seeded simulations quickly yield a free energy landscape of cryptic pocket opening that is in good agreement with the same landscape generated with well-tempered metadynamics. Taken together, our results demonstrate that AlphaFold has a useful role to play in cryptic pocket discovery but that many cryptic pockets may remain difficult to sample using AlphaFold alone.

Bowman Lab Computational Biology

Folding@home: achievements from over twenty years of citizen science herald the exascale era

Voelz VA, Pande VS, Bowman GR. Folding@home: achievements from over twenty years of citizen science herald the exascale era. ArXiv [Preprint]. 2023 Mar 15:arXiv:2303.08993v1. Update in: Biophys J. 2023 Mar 21;: PMID: 36994157; PMCID: PMC10055475.

Abstract

Simulations of biomolecules have enormous potential to inform our understanding of biology but require extremely demanding calculations. For over twenty years, the Folding@home distributed computing project has pioneered a massively parallel approach to biomolecular simulation, harnessing the resources of citizen scientists across the globe. Here, we summarize the scientific and technical advances this perspective has enabled. As the project’s name implies, the early years of Folding@home focused on driving advances in our understanding of protein folding by developing statistical methods for capturing long-timescale processes and facilitating insight into complex dynamical processes. Success laid a foundation for broadening the scope of Folding@home to address other functionally relevant conformational changes, such as receptor signaling, enzyme dynamics, and ligand binding. Continued algorithmic advances, hardware developments such as GPU-based computing, and the growing scale of Folding@home have enabled the project to focus on new areas where massively parallel sampling can be impactful. While previous work sought to expand toward larger proteins with slower conformational changes, new work focuses on large-scale comparative studies of different protein sequences and chemical compounds to better understand biology and inform the development of small molecule drugs. Progress on these fronts enabled the community to pivot quickly in response to the COVID-19 pandemic, expanding to become the world’s first exascale computer and deploying this massive resource to provide insight into the inner workings of the SARS-CoV-2 virus and aid the development of new antivirals. This success provides a glimpse of what’s to come as exascale supercomputers come online, and Folding@home continues its work.

Bowman Lab Computational Biology

Drosophila Tropomodulin is required for multiple actin-dependent processes within developing myofibers

Zapater I Morales C, Carman PJ, Soffar DB, Windner SE, Dominguez R, Baylies MK. Drosophila Tropomodulin is required for multiple actin-dependent processes within developing myofibers. Development. 2023 Mar 15;150(6):dev201194. doi: 10.1242/dev.201194. Epub 2023 Mar 24. PMID: 36806912; PMCID: PMC10112908.

Abstract

Proper muscle contraction requires the assembly and maintenance of sarcomeres and myofibrils. Although the protein components of myofibrils are generally known, less is known about the mechanisms by which they individually function and together synergize for myofibril assembly and maintenance. For example, it is unclear how the disruption of actin filament (F-actin) regulatory proteins leads to the muscle weakness observed in myopathies. Here, we show that knockdown of Drosophila Tropomodulin (Tmod), results in several myopathy-related phenotypes, including reduction of muscle cell (myofiber) size, increased sarcomere length, disorganization and misorientation of myofibrils, ectopic F-actin accumulation, loss of tension-mediating proteins at the myotendinous junction, and misshaped and internalized nuclei. Our findings support and extend the tension-driven self-organizing myofibrillogenesis model. We show that, like its mammalian counterpart, Drosophila Tmod caps F-actin pointed-ends, and we propose that this activity is crucial for cellular processes in different locations within the myofiber that directly and indirectly contribute to the maintenance of muscle function. Our findings provide significant insights to the role of Tmod in muscle development, maintenance and disease.

CryoEM and CryoET Dominguez Lab X-ray Crystallography

Predicting locations of cryptic pockets from single protein structures using the PocketMiner graph neural network

Meller A, Ward M, Borowsky J, Kshirsagar M, Lotthammer JM, Oviedo F, Ferres JL, Bowman GR. Predicting locations of cryptic pockets from single protein structures using the PocketMiner graph neural network. Nat Commun. 2023 Mar 1;14(1):1177. doi: 10.1038/s41467-023-36699-3. PMID: 36859488; PMCID: PMC9977097.

Abstract

Cryptic pockets expand the scope of drug discovery by enabling targeting of proteins currently considered undruggable because they lack pockets in their ground state structures. However, identifying cryptic pockets is labor-intensive and slow. The ability to accurately and rapidly predict if and where cryptic pockets are likely to form from a structure would greatly accelerate the search for druggable pockets. Here, we present PocketMiner, a graph neural network trained to predict where pockets are likely to open in molecular dynamics simulations. Applying PocketMiner to single structures from a newly curated dataset of 39 experimentally confirmed cryptic pockets demonstrates that it accurately identifies cryptic pockets (ROC-AUC: 0.87) >1,000-fold faster than existing methods. We apply PocketMiner across the human proteome and show that predicted pockets open in simulations, suggesting that over half of proteins thought to lack pockets based on available structures likely contain cryptic pockets, vastly expanding the potentially druggable proteome.

Bowman Lab Computational Biology

Rational design of a genetically encoded NMR zinc sensor

Zhao Z, Zhou M, Zemerov SD, Marmorstein R, Dmochowski IJ. Rational design of a genetically encoded NMR zinc sensor. Chem Sci. 2023 Mar 13;14(14):3809-3815. doi: 10.1039/d3sc00437f. PMID: 37035699; PMCID: PMC10074429.

Abstract

Elucidating the biochemical roles of the essential metal ion, Zn2+, motivates detection strategies that are sensitive, selective, quantitative, and minimally invasive in living systems. Fluorescent probes have identified Zn2+ in cells but complementary approaches employing nuclear magnetic resonance (NMR) are lacking. Recent studies of maltose binding protein (MBP) using ultrasensitive 129Xe NMR spectroscopy identified a switchable salt bridge which causes slow xenon exchange and elicits strong hyperpolarized 129Xe chemical exchange saturation transfer (hyper-CEST) NMR contrast. To engineer the first genetically encoded, NMR-active sensor for Zn2+, we converted the MBP salt bridge into a Zn2+ binding site, while preserving the specific xenon binding cavity. The zinc sensor (ZS) at only 1 μM achieved ‘turn-on’ detection of Zn2+ with pronounced hyper-CEST contrast. This made it possible to determine different Zn2+ levels in a biological fluid via hyper-CEST. ZS was responsive to low-micromolar Zn2+, only modestly responsive to Cu2+, and nonresponsive to other biologically important metal ions, according to hyper-CEST NMR spectroscopy and isothermal titration calorimetry (ITC). Protein X-ray crystallography confirmed the identity of the bound Zn2+ ion using anomalous scattering: Zn2+ was coordinated with two histidine side chains and three water molecules. Penta-coordinate Zn2+ forms a hydrogen-bond-mediated gate that controls the Xe exchange rate. Metal ion binding affinity, 129Xe NMR chemical shift, and exchange rate are tunable parameters via protein engineering, which highlights the potential to develop proteins as selective metal ion sensors for NMR spectroscopy and imaging.

Chemical Biology CryoEM and CryoET X-ray Crystallography Marmorstein Lab

Mapping light distribution in tissue by using MRI-detectable photosensitive liposomes

Simon J, Schwalm M, Morstein J, Trauner D, Jasanoff A. Mapping light distribution in tissue by using MRI-detectable photosensitive liposomes. Nat Biomed Eng. 2023 Mar;7(3):313-322. doi: 10.1038/s41551-022-00982-3. Epub 2022 Dec 22. PMID: 36550300.

Abstract

Characterizing sources and targets of illumination in living tissue is challenging. Here we show that spatial distributions of light in tissue can be mapped by using magnetic resonance imaging (MRI) in the presence of photosensitive nanoparticle probes. Each probe consists of a reservoir of paramagnetic molecules enclosed by a liposomal membrane incorporating photosensitive lipids. Incident light causes the photoisomerization of the lipids and alters hydrodynamic exchange across the membrane, thereby affecting longitudinal relaxation-weighted contrast in MRI. We injected the nanoparticles into the brains of live rats and used MRI to map responses to illumination profiles characteristic of widely used applications of photostimulation, photometry and phototherapy. The responses deviated from simple photon propagation models and revealed signatures of light scattering and nonlinear responsiveness. Paramagnetic liposomal nanoparticles may enable MRI to map a broad range of optical phenomena in deep tissue and other opaque environments.

Chemical Biology CryoEM and CryoET Trauner Group

Structure of a HIV-1 IN-Allosteric inhibitor complex at 2.93 Å resolution: Routes to inhibitor optimization

Eilers G, Gupta K, Allen A, Montermoso S, Murali H, Sharp R, Hwang Y, Bushman FD, Van Duyne G. Structure of a HIV-1 IN-Allosteric inhibitor complex at 2.93 Å resolution: Routes to inhibitor optimization. PLoS Pathog. 2023 Mar 3;19(3):e1011097. doi: 10.1371/journal.ppat.1011097. PMID: 36867659; PMCID: PMC10016701.

Abstract

HIV integrase (IN) inserts viral DNA into the host genome and is the target of the strand transfer inhibitors (STIs), a class of small molecules currently in clinical use. Another potent class of antivirals is the allosteric inhibitors of integrase, or ALLINIs. ALLINIs promote IN aggregation by stabilizing an interaction between the catalytic core domain (CCD) and carboxy-terminal domain (CTD) that undermines viral particle formation in late replication. Ongoing challenges with inhibitor potency, toxicity, and viral resistance motivate research to understand their mechanism. Here, we report a 2.93 Å X-ray crystal structure of the minimal ternary complex between CCD, CTD, and the ALLINI BI-224436. This structure reveals an asymmetric ternary complex with a prominent network of π-mediated interactions that suggest specific avenues for future ALLINI development and optimization

CryoEM and CryoET Gupta Lab X-ray Crystallography

The ion channel CALHM6 controls bacterial infection-induced cellular cross-talk at the immunological synapse

Danielli S, Ma Z, Pantazi E, Kumar A, Demarco B, Fischer FA, Paudel U, Weissenrieder J, Lee RJ, Joyce S, Foskett JK, Bezbradica JS. The ion channel CALHM6 controls bacterial infection-induced cellular cross-talk at the immunological synapse. EMBO J. 2023 Apr 3;42(7):e111450. doi: 10.15252/embj.2022111450. Epub 2023 Mar 2. PMID: 36861806; PMCID: PMC10068325.

Abstract

Membrane ion channels of the calcium homeostasis modulator (CALHM) family promote cell-cell crosstalk at neuronal synapses via ATP release, where ATP acts as a neurotransmitter. CALHM6, the only CALHM highly expressed in immune cells, has been linked to the induction of natural killer (NK) cell anti-tumour activity. However, its mechanism of action and broader functions in the immune system remain unclear. Here, we generated Calhm6-/- mice and report that CALHM6 is important for the regulation of the early innate control of Listeria monocytogenes infection in vivo. We find that CALHM6 is upregulated in macrophages by pathogen-derived signals and that it relocates from the intracellular compartment to the macrophage-NK cell synapse, facilitating ATP release and controlling the kinetics of NK cell activation. Anti-inflammatory cytokines terminate CALHM6 expression. CALHM6 forms an ion channel when expressed in the plasma membrane of Xenopus oocytes, where channel opening is controlled by a conserved acidic residue, E119. In mammalian cells, CALHM6 is localised to intracellular compartments. Our results contribute to the understanding of neurotransmitter-like signal exchange between immune cells that fine-tunes the timing of innate immune responses.

CryoEM and CryoET Foskett

Myo19 tethers mitochondria to endoplasmic reticulum-associated actin to promote mitochondrial fission

Coscia SM, Thompson CP, Tang Q, Baltrusaitis EE, Rhodenhiser JA, Quintero-Carmona OA, Ostap EM, Lakadamyali M, Holzbaur ELF. Myo19 tethers mitochondria to endoplasmic reticulum-associated actin to promote mitochondrial fission. J Cell Sci. 2023 Mar 1;136(5):jcs260612. doi: 10.1242/jcs.260612. Epub 2023 Mar 2. PMID: 36744380; PMCID: PMC10022680.

Abstract

Mitochondrial homeostasis requires a dynamic balance of fission and fusion. The actin cytoskeleton promotes fission, and we found that the mitochondrially localized myosin, myosin 19 (Myo19), is integral to this process. Myo19 knockdown induced mitochondrial elongation, whereas Myo19 overexpression induced fragmentation. This mitochondrial fragmentation was blocked by a Myo19 mutation predicted to inhibit ATPase activity and strong actin binding but not by mutations predicted to affect the working stroke of the motor that preserve ATPase activity. Super-resolution imaging indicated a dispersed localization of Myo19 on mitochondria, which we found to be dependent on metaxins. These observations suggest that Myo19 acts as a dynamic actin-binding tether that facilitates mitochondrial fragmentation. Myo19-driven fragmentation was blocked by depletion of either the CAAX splice variant of the endoplasmic reticulum (ER)-anchored formin INF2 or the mitochondrially localized F-actin nucleator Spire1C (a splice variant of Spire1), which together polymerize actin at sites of mitochondria-ER contact for fission. These observations imply that Myo19 promotes fission by stabilizing mitochondria-ER contacts; we used a split-luciferase system to demonstrate a reduction in these contacts following Myo19 depletion. Our data support a model in which Myo19 tethers mitochondria to ER-associated actin to promote mitochondrial fission.

CryoEM and CryoET Holzbaur Lab Single Molecule Imaging Lakadamyali Lab Ostap Lab

The selective autophagy adaptor p62/SQSTM1 forms phase condensates regulated by HSP27 that facilitate the clearance of damaged lysosomes via lysophagy

Gallagher ER, Holzbaur ELF. The selective autophagy adaptor p62/SQSTM1 forms phase condensates regulated by HSP27 that facilitate the clearance of damaged lysosomes via lysophagy. Cell Rep. 2023 Feb 28;42(2):112037. doi: 10.1016/j.celrep.2023.112037. Epub 2023 Jan 25. PMID: 36701233; PMCID: PMC10366342.

Abstract

In response to lysosomal damage, cells engage several quality-control mechanisms, including the selective isolation and degradation of damaged lysosomes by lysophagy. Here, we report that the selective autophagy adaptor SQSTM1/p62 is recruited to damaged lysosomes in both HeLa cells and neurons and is required for lysophagic flux. The Phox and Bem1p (PB1) domain of p62 mediates oligomerization and is specifically required for lysophagy. Consistent with this observation, we find that p62 forms condensates on damaged lysosomes. These condensates are precisely tuned by the small heat shock protein HSP27, which is phosphorylated in response to lysosomal injury and maintains the liquidity of p62 condensates, facilitating autophagosome formation. Mutations in p62 have been identified in patients with amyotrophic lateral sclerosis (ALS); ALS-associated mutations in p62 impair lysophagy, suggesting that deficits in this pathway may contribute to neurodegeneration. Thus, p62 condensates regulated by HSP27 promote lysophagy by forming platforms for autophagosome biogenesis at damaged lysosomes.

 

CryoEM and CryoET Holzbaur Lab Single Molecule Imaging

In Situ Structure Determination of Bacterial Surface Nanomachines Using Cryo-Electron Tomography

Lai L, Cheung YW, Martinez M, Kixmoeller K, Palao L 3rd, Steimle S, Ho MC, Black BE, Lai EM, Chang YW. In Situ Structure Determination of Bacterial Surface Nanomachines Using Cryo-Electron Tomography. Methods Mol Biol. 2023;2646:211-248. doi: 10.1007/978-1-0716-3060-0_18. PMID: 36842118.

Abstract

Bacterial surface nanomachines are often refractory to structural determination in their intact form due to their extensive association with the cell envelope preventing them from being properly purified for traditional structural biology methods. Cryo-electron tomography (cryo-ET) is an emerging branch of cryo-electron microscopy that can visualize supramolecular complexes directly inside frozen-hydrated cells in 3D at nanometer resolution, therefore posing a unique capability to study the intact structures of bacterial surface nanomachines in situ and reveal their molecular association with other cellular components. Furthermore, the resolution of cryo-ET is continually improving alongside methodological advancement. Here, using the type IV pilus machine in Myxococcus xanthus as an example, we describe a step-by-step workflow for in situ structure determination including sample preparation and screening, microscope and camera tuning, tilt series acquisition, data processing and tomogram reconstruction, subtomogram averaging, and structural analysis.

CryoEM and CryoET Mass Spectrometry Yi-Wei Chang Lab

In Situ Structure Determination of Bacterial Surface Nanomachines Using Cryo-Electron Tomography

Lai L, Cheung YW, Martinez M, Kixmoeller K, Palao L 3rd, Steimle S, Ho MC, Black BE, Lai EM, Chang YW. In Situ Structure Determination of Bacterial Surface Nanomachines Using Cryo-Electron Tomography. Methods Mol Biol. 2023;2646:211-248. doi: 10.1007/978-1-0716-3060-0_18. PMID: 36842118.

Abstract

Bacterial surface nanomachines are often refractory to structural determination in their intact form due to their extensive association with the cell envelope preventing them from being properly purified for traditional structural biology methods. Cryo-electron tomography (cryo-ET) is an emerging branch of cryo-electron microscopy that can visualize supramolecular complexes directly inside frozen-hydrated cells in 3D at nanometer resolution, therefore posing a unique capability to study the intact structures of bacterial surface nanomachines in situ and reveal their molecular association with other cellular components. Furthermore, the resolution of cryo-ET is continually improving alongside methodological advancement. Here, using the type IV pilus machine in Myxococcus xanthus as an example, we describe a step-by-step workflow for in situ structure determination including sample preparation and screening, microscope and camera tuning, tilt series acquisition, data processing and tomogram reconstruction, subtomogram averaging, and structural analysis.

Black Lab CryoEM and CryoET Mass Spectrometry

Decoupling peptide binding from T cell receptor recognition with engineered chimeric MHC-I molecules

Papadaki GF, Ani O, Florio TJ, Young MC, Danon JN, Sun Y, Dersh D, Sgourakis NG. Front. Immunol. 2023 Jan 25. doi: 10.3389/fimmu.2023.1116906.

Major Histocompatibility Complex class I (MHC-I) molecules display self, viral or aberrant epitopic peptides to T cell receptors (TCRs), which employ interactions between complementarity-determining regions with both peptide and MHC-I heavy chain ‘framework’ residues to recognize specific Human Leucocyte Antigens (HLAs). The highly polymorphic nature of the HLA peptide-binding groove suggests a malleability of interactions within a common structural scaffold. Here, using structural data from peptide:MHC-I and pMHC:TCR structures, we first identify residues important for peptide and/or TCR binding. We then outline a fixed-backbone computational design approach for engineering synthetic molecules that combine peptide binding and TCR recognition surfaces from existing HLA allotypes. X-ray crystallography demonstrates that chimeric molecules bridging divergent HLA alleles can bind selected peptide antigens in a specified backbone conformation. Finally, in vitro tetramer staining and biophysical binding experiments using chimeric pMHC-I molecules presenting established antigens further demonstrate the requirement of TCR recognition on interactions with HLA framework residues, as opposed to interactions with peptide-centric Chimeric Antigen Receptors (CARs). Our results underscore a novel, structure-guided platform for developing synthetic HLA molecules with desired properties as screening probes for peptide-centric interactions with TCRs and other therapeutic modalities.

Computational Biology NMR X-ray Crystallography SgourakisLab

Xeno interactions between MHC-I proteins and molecular chaperones enable ligand exchange on a broad repertoire of HLA allotypes

Sun Y, Papadaki GF, Devlin CA, Danon JN, Young MC, Winters TJ, Burslem GM, Procko E, Sgourakis NG, Sci. Adv. 2023 Feb 24. doi: 10.1126/sciadv.ade7151.

Abstract Immunological chaperones tapasin and TAP binding protein, related (TAPBPR) play key roles in antigenic peptide optimization and quality control of nascent class I major histocompatibility complex (MHC-I) molecules. The polymorphic nature of MHC-I proteins leads to a range of allelic dependencies on chaperones for assembly and cell-surface expression, limiting chaperone-mediated peptide exchange to a restricted set of human leukocyte antigen (HLA) allotypes. Here, we demonstrate and characterize xeno interactions between a chicken TAPBPR ortholog and a complementary repertoire of HLA allotypes, relative to its human counterpart. We find that TAPBPR orthologs recognize empty MHC-I with broader allele specificity and facilitate peptide exchange by maintaining a reservoir of receptive molecules. Deep mutational scanning of human TAPBPR further identifies gain-of-function mutants, resembling the chicken sequence, which can enhance HLA-A*01:01 expression in situ and promote peptide exchange in vitro. These results highlight that polymorphic sites on MHC-I and chaperone surfaces can be engineered to manipulate their interactions, enabling chaperone-mediated peptide exchange on disease-relevant HLA alleles.
Computational Biology NMR X-ray Crystallography SgourakisLab

Xeno interactions between MHC-I proteins and molecular chaperones enable ligand exchange on a broad repertoire of HLA allotypes

Sun Y, Papadaki GF, Devlin CA, Danon JN, Young MC, Winters TJ, Burslem GM, Procko E, Sgourakis NG. Xeno interactions between MHC-I proteins and molecular chaperones enable ligand exchange on a broad repertoire of HLA allotypes. Sci Adv. 2023 Feb 24;9(8):eade7151. doi: 10.1126/sciadv.ade7151. Epub 2023 Feb 24. PMID: 36827371; PMCID: PMC9956121.

Abstract

Immunological chaperones tapasin and TAP binding protein, related (TAPBPR) play key roles in antigenic peptide optimization and quality control of nascent class I major histocompatibility complex (MHC-I) molecules. The polymorphic nature of MHC-I proteins leads to a range of allelic dependencies on chaperones for assembly and cell-surface expression, limiting chaperone-mediated peptide exchange to a restricted set of human leukocyte antigen (HLA) allotypes. Here, we demonstrate and characterize xeno interactions between a chicken TAPBPR ortholog and a complementary repertoire of HLA allotypes, relative to its human counterpart. We find that TAPBPR orthologs recognize empty MHC-I with broader allele specificity and facilitate peptide exchange by maintaining a reservoir of receptive molecules. Deep mutational scanning of human TAPBPR further identifies gain-of-function mutants, resembling the chicken sequence, which can enhance HLA-A*01:01 expression in situ and promote peptide exchange in vitro. These results highlight that polymorphic sites on MHC-I and chaperone surfaces can be engineered to manipulate their interactions, enabling chaperone-mediated peptide exchange on disease-relevant HLA alleles.

Chemical Biology Burslem Lab Mass Spectrometry

Engineered antibody cytokine chimera synergizes with DNA-launched nanoparticle vaccines to potentiate melanoma suppression in vivo

Tursi NJ, Xu Z, Helble M, Walker S, Liaw K, Chokkalingam N, Kannan T, Wu Y, Tello-Ruiz E, Park DH, Zhu X, Wise MC, Smith TRF, Majumdar S, Kossenkov A, Kulp DW, Weiner DB. Engineered antibody cytokine chimera synergizes with DNA-launched nanoparticle vaccines to potentiate melanoma suppression in vivo. Front Immunol. 2023 Feb 23;14:1072810. doi: 10.3389/fimmu.2023.1072810. PMID: 36911698; PMCID: PMC9997082.

Abstract

Cancer immunotherapy has demonstrated great promise with several checkpoint inhibitors being approved as the first-line therapy for some types of cancer, and new engineered cytokines such as Neo2/15 now being evaluated in many studies. In this work, we designed antibody-cytokine chimera (ACC) scaffolding cytokine mimetics on a full-length tumor-specific antibody. We characterized the pharmacokinetic (PK) and pharmacodynamic (PD) properties of first-generation ACC TA99-Neo2/15, which synergized with DLnano-vaccines to suppress in vivo melanoma proliferation and induced significant systemic cytokine activation. A novel second-generation ACC TA99-HL2-KOA1, with retained IL-2Rβ/γ binding and attenuated but preserved IL-2Rα binding, induced lower systemic cytokine activation with non-inferior protection in murine tumor studies. Transcriptomic analyses demonstrated an upregulation of Type I interferon responsive genes, particularly ISG15, in dendritic cells, macrophages and monocytes following TA99-HL2-KOA1 treatment. Characterization of additional ACCs in combination with cancer vaccines will likely be an important area of research for treating melanoma and other types of cancer.

Computational Biology CryoEM and CryoET Kulp Lab

Chemical probe mediated visualization of protein S-palmitoylation in patient tissue samples

Schek N, Lee JY, Burslem GM, Witze E. Chemical probe mediated visualization of protein S-palmitoylation in patient tissue samples. Front Physiol. 2023 Feb 21;14:1063247. doi: 10.3389/fphys.2023.1063247. Erratum in: Front Physiol. 2023 May 03;14:1208618. PMID: 36895631; PMCID: PMC9988892.

Abstract

While protein palmitoylation has been studied for decades, our understanding of its clinical importance is minimal compared to other post translational modifications. As a result of the inherent challenges preventing the production of antibodies to palmitoylated epitopes we are unable to correlate levels of protein palmitoylation in biopsied tissues at a meaningful resolution. The most common method for detecting palmitoylated proteins without metabolic labelling is through chemical labeling of palmitoylated cysteines with the acyl-biotinyl exchange (ABE) assay. We have adapted the ABE assay to detect protein palmitoylation in formalin fixed paraffin embedded (FFPE) tissue sections. The assay is sufficient to detect subcellular regions of cells with increased labeling which indicates areas enriched in palmitoylated proteins. To visualize specific palmitoylated proteins in both cultured cells and in FFPE preserved tissue arrays we have integrated the ABE assay with a proximity ligation assay (ABE-PLA). Our findings demonstrate for the first time that FFPE preserved tissues can be labelled with unique chemical probes to detect either areas enriched in palmitoylated proteins or the localization of specific palmitoylated proteins using our ABE-PLA methodology.

Chemical Biology Burslem Lab Mass Spectrometry

TET1 Catalytic Activity is Required for Reprogramming of Imprinting Control Regions and Patterning of Sperm-Specific Hypomethylated Regions

Prasasya RD, Caldwell BA, Liu Z, Wu S, Leu NA, Fowler JM, Cincotta SA, Laird DJ, Kohli RM, Bartolomei MS. TET1 Catalytic Activity is Required for Reprogramming of Imprinting Control Regions and Patterning of Sperm-Specific Hypomethylated Regions. bioRxiv [Preprint]. 2023 Feb 21:2023.02.21.529426. doi: 10.1101/2023.02.21.529426. PMID: 36865267; PMCID: PMC9980038.

Abstract

DNA methylation erasure is required for mammalian primordial germ cell reprogramming. TET enzymes iteratively oxidize 5-methylcytosine to generate 5-hyroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxycytosine to facilitate active genome demethylation. Whether these bases are required to promote replication-coupled dilution or activate base excision repair during germline reprogramming remains unresolved due to the lack of genetic models that decouple TET activities. Here, we generated two mouse lines expressing catalytically inactive TET1 ( Tet1-HxD ) and TET1 that stalls oxidation at 5hmC ( Tet1-V ). Tet1 -/- , Tet1 V/V , and Tet1 HxD/HxD sperm methylomes show that TET1 V and TET1 HxD rescue most Tet1 -/- hypermethylated regions, demonstrating the importance of TET1’s extra-catalytic functions. Imprinted regions, in contrast, require iterative oxidation. We further reveal a broader class of hypermethylated regions in sperm of Tet1 mutant mice that are excluded from de novo methylation during male germline development and depend on TET oxidation for reprogramming. Our study underscores the link between TET1-mediated demethylation during reprogramming and sperm methylome patterning.

Chemical Biology CryoEM and CryoET Kohli Lab

Structural basis of a transcription pre-initiation complex on a divergent promoter

Gorbea Colón JJ, Palao L 3rd, Chen SF, Kim HJ, Snyder L, Chang YW, Tsai KL, Murakami K. Structural basis of a transcription pre-initiation complex on a divergent promoter. Mol Cell. 2023 Feb 16;83(4):574-588.e11. doi: 10.1016/j.molcel.2023.01.011. Epub 2023 Feb 1. PMID: 36731470; PMCID: PMC10162435.

Abstract

Most eukaryotic promoter regions are divergently transcribed. As the RNA polymerase II pre-initiation complex (PIC) is intrinsically asymmetric and responsible for transcription in a single direction, it is unknown how divergent transcription arises. Here, the Saccharomyces cerevisiae Mediator complexed with a PIC (Med-PIC) was assembled on a divergent promoter and analyzed by cryoelectron microscopy. The structure reveals two distinct Med-PICs forming a dimer through the Mediator tail module, induced by a homodimeric activator protein localized near the dimerization interface. The tail dimer is associated with ∼80-bp upstream DNA, such that two flanking core promoter regions are positioned and oriented in a suitable form for PIC assembly in opposite directions. Also, cryoelectron tomography visualized the progress of the PIC assembly on the two core promoter regions, providing direct evidence for the role of the Med-PIC dimer in divergent transcription.

CryoEM and CryoET Mass Spectrometry Murakami Lab Yi-Wei Chang Lab

Apolipoprotein E4 has extensive conformational heterogeneity in lipid-free and lipid-bound forms

Stuchell-Brereton MD, Zimmerman MI, Miller JJ, Mallimadugula UL, Incicco JJ, Roy D, Smith LG, Cubuk J, Baban B, DeKoster GT, Frieden C, Bowman GR, Soranno A. Apolipoprotein E4 has extensive conformational heterogeneity in lipid-free and lipid-bound forms. Proc Natl Acad Sci U S A. 2023 Feb 14;120(7):e2215371120. doi: 10.1073/pnas.2215371120. Epub 2023 Feb 7. PMID: 36749730; PMCID: PMC9963066.

Abstract

The ε4-allele variant of apolipoprotein E (ApoE4) is the strongest genetic risk factor for Alzheimer’s disease, although it only differs from its neutral counterpart ApoE3 by a single amino acid substitution. While ApoE4 influences the formation of plaques and neurofibrillary tangles, the structural determinants of pathogenicity remain undetermined due to limited structural information. Previous studies have led to conflicting models of the C-terminal region positioning with respect to the N-terminal domain across isoforms largely because the data are potentially confounded by the presence of heterogeneous oligomers. Here, we apply a combination of single-molecule spectroscopy and molecular dynamics simulations to construct an atomically detailed model of monomeric ApoE4 and probe the effect of lipid association. Importantly, our approach overcomes previous limitations by allowing us to work at picomolar concentrations where only the monomer is present. Our data reveal that ApoE4 is far more disordered and extended than previously thought and retains significant conformational heterogeneity after binding lipids. Comparing the proximity of the N- and C-terminal domains across the three major isoforms (ApoE4, ApoE3, and ApoE2) suggests that all maintain heterogeneous conformations in their monomeric form, with ApoE2 adopting a slightly more compact ensemble. Overall, these data provide a foundation for understanding how ApoE4 differs from nonpathogenic and protective variants of the protein.

Bowman Lab Computational Biology

Mechanosensitive (de) regulation of exosome production in tumors

Kshitiz Parihar, Ravi Radhakrishnan

Computational Biology Radhakrishnan Lab

Aberrant chromatin reorganization in cells from diseased fibrous connective tissue in response to altered chemomechanical cues

Heo SJ, Thakur S, Chen X, Loebel C, Xia B, McBeath R, Burdick JA, Shenoy VB, Mauck RL, Lakadamyali M. Aberrant chromatin reorganization in cells from diseased fibrous connective tissue in response to altered chemomechanical cues. Nat Biomed Eng. 2023 Feb;7(2):177-191. doi: 10.1038/s41551-022-00910-5. Epub 2022 Aug 22. PMID: 35996026; PMCID: PMC10053755.

Abstract

Changes in the micro-environment of fibrous connective tissue can lead to alterations in the phenotypes of tissue-resident cells, yet the underlying mechanisms are poorly understood. Here, by visualizing the dynamics of histone spatial reorganization in tenocytes and mesenchymal stromal cells from fibrous tissue of human donors via super-resolution microscopy, we show that physiological and pathological chemomechanical cues can directly regulate the spatial nanoscale organization and density of chromatin in these tissue-resident cell populations. Specifically, changes in substrate stiffness, altered oxygen tension and the presence of inflammatory signals drive chromatin relocalization and compaction into the nuclear boundary, mediated by the activity of the histone methyltransferase EZH2 and an intact cytoskeleton. In healthy cells, chemomechanically triggered changes in the spatial organization and density of chromatin are reversible and can be attenuated by dynamically stiffening the substrate. In diseased human cells, however, the link between mechanical or chemical inputs and chromatin remodelling is abrogated. Our findings suggest that aberrant chromatin organization in fibrous connective tissue may be a hallmark of disease progression that could be leveraged for therapeutic intervention.

Single Molecule Imaging Lakadamyali Lab

Efficient engineering of human and mouse primary cells using peptide-assisted genome editing

Zhang Z, Baxter AE, Ren D, Qin K, Chen Z, Collins SM, Huang H, Komar CA, Bailer PF, Parker JB, Blobel GA, Kohli RM, Wherry EJ, Berger SL, Shi J. Efficient engineering of human and mouse primary cells using peptide-assisted genome editing. Nat Biotechnol. 2024 Feb;42(2):305-315. doi: 10.1038/s41587-023-01756-1. Epub 2023 Apr 24. PMID: 37095348.

Abstract

Simple, efficient and well-tolerated delivery of CRISPR genome editing systems into primary cells remains a major challenge. Here we describe an engineered Peptide-Assisted Genome Editing (PAGE) CRISPR-Cas system for rapid and robust editing of primary cells with minimal toxicity. The PAGE system requires only a 30-min incubation with a cell-penetrating Cas9 or Cas12a and a cell-penetrating endosomal escape peptide to achieve robust single and multiplex genome editing. Unlike electroporation-based methods, PAGE gene editing has low cellular toxicity and shows no significant transcriptional perturbation. We demonstrate rapid and efficient editing of primary cells, including human and mouse T cells, as well as human hematopoietic progenitor cells, with editing efficiencies upwards of 98%. PAGE provides a broadly generalizable platform for next-generation genome engineering in primary cells.

Chemical Biology CryoEM and CryoET Kohli Lab

Ensembles of human myosin-19 bound to calmodulin and regulatory light chain RLC12B drive multimicron transport

Pollard LW, Coscia SM, Rebowski G, Palmer NJ, Holzbaur ELF, Dominguez R, Ostap EM. Ensembles of human myosin-19 bound to calmodulin and regulatory light chain RLC12B drive multimicron transport. J Biol Chem. 2023 Feb;299(2):102906. doi: 10.1016/j.jbc.2023.102906. Epub 2023 Jan 13. PMID: 36642185; PMCID: PMC9929473.

Abstract

Myosin-19 (Myo19) controls the size, morphology, and distribution of mitochondria, but the underlying role of Myo19 motor activity is unknown. Complicating mechanistic in vitro studies, the identity of the light chains (LCs) of Myo19 remains unsettled. Here, we show by coimmunoprecipitation, reconstitution, and proteomics that the three IQ motifs of human Myo19 expressed in Expi293 human cells bind regulatory light chain (RLC12B) and calmodulin (CaM). We demonstrate that overexpression of Myo19 in HeLa cells enhances the recruitment of both Myo19 and RLC12B to mitochondria, suggesting cellular association of RLC12B with the motor. Further experiments revealed that RLC12B binds IQ2 and is flanked by two CaM molecules. In vitro, we observed that the maximal speed (∼350 nm/s) occurs when Myo19 is supplemented with CaM, but not RLC12B, suggesting maximal motility requires binding of CaM to IQ-1 and IQ-3. The addition of calcium slowed actin gliding (∼200 nm/s) without an apparent effect on CaM affinity. Furthermore, we show that small ensembles of Myo19 motors attached to quantum dots can undergo processive runs over several microns, and that calcium reduces the attachment frequency and run length of Myo19. Together, our data are consistent with a model where a few single-headed Myo19 molecules attached to a mitochondrion can sustain prolonged motile associations with actin in a CaM- and calcium-dependent manner. Based on these properties, we propose that Myo19 can function in mitochondria transport along actin filaments, tension generation on multiple randomly oriented filaments, and/or pushing against branched actin networks assembled near the membrane surface.

CryoEM and CryoET Dominguez Lab Holzbaur Lab Single Molecule Imaging X-ray Crystallography Ostap Lab

Molecular role of NAA38 in thermostability and catalytic activity of the human NatC N-terminal acetyltransferase

Deng S, Gardner SM, Gottlieb L, Pan B, Petersson EJ, Marmorstein R. Molecular role of NAA38 in thermostability and catalytic activity of the human NatC N-terminal acetyltransferase. Structure. 2023 Feb 2;31(2):166-173.e4. doi: 10.1016/j.str.2022.12.008. Epub 2023 Jan 12. PMID: 36638802; PMCID: PMC9898148.

Abstract

N-terminal acetylation occurs on over 80% of human proteins and is catalyzed by a family of N-terminal acetyltransferases (NATs). All NATs contain a small catalytic subunit, while some also contain a large auxiliary subunit that facilitates catalysis and ribosome targeting for co-translational acetylation. NatC is one of the major NATs containing an NAA30 catalytic subunit, but uniquely contains two auxiliary subunits, large NAA35 and small NAA38. Here, we report the cryo-EM structures of human NatC (hNatC) complexes with and without NAA38, together with biochemical studies, to reveal that NAA38 increases the thermostability and broadens the substrate-specificity profile of NatC by ordering an N-terminal segment of NAA35 and reorienting an NAA30 N-terminal peptide binding loop for optimal catalysis, respectively. We also note important differences in engagement with a stabilizing inositol hexaphosphate molecule between human and yeast NatC. These studies provide new insights for the function and evolution of the NatC complex.

 

Chemical Biology CryoEM and CryoET X-ray Crystallography Marmorstein Lab

Drug specificity and affinity are encoded in the probability of cryptic pocket opening in myosin motor domains

Meller A, Lotthammer JM, Smith LG, Novak B, Lee LA, Kuhn CC, Greenberg L, Leinwand LA, Greenberg MJ, Bowman GR. Drug specificity and affinity are encoded in the probability of cryptic pocket opening in myosin motor domains. Elife. 2023 Jan 27;12:e83602. doi: 10.7554/eLife.83602. PMID: 36705568; PMCID: PMC9995120.

Abstract

The design of compounds that can discriminate between closely related target proteins remains a central challenge in drug discovery. Specific therapeutics targeting the highly conserved myosin motor family are urgently needed as mutations in at least six of its members cause numerous diseases. Allosteric modulators, like the myosin-II inhibitor blebbistatin, are a promising means to achieve specificity. However, it remains unclear why blebbistatin inhibits myosin-II motors with different potencies given that it binds at a highly conserved pocket that is always closed in blebbistatin-free experimental structures. We hypothesized that the probability of pocket opening is an important determinant of the potency of compounds like blebbistatin. To test this hypothesis, we used Markov state models (MSMs) built from over 2 ms of aggregate molecular dynamics simulations with explicit solvent. We find that blebbistatin’s binding pocket readily opens in simulations of blebbistatin-sensitive myosin isoforms. Comparing these conformational ensembles reveals that the probability of pocket opening correctly identifies which isoforms are most sensitive to blebbistatin inhibition and that docking against MSMs quantitatively predicts blebbistatin binding affinities (R2=0.82). In a blind prediction for an isoform (Myh7b) whose blebbistatin sensitivity was unknown, we find good agreement between predicted and measured IC50s (0.67 μM vs. 0.36 μM). Therefore, we expect this framework to be useful for the development of novel specific drugs across numerous protein targets.

 

Bowman Lab Computational Biology

Viewing Ca2+-binding sites in the inositol trisphosphate receptor

Lunko O, Foskett JK. Viewing Ca2+-binding sites in the inositol trisphosphate receptor. Cell Calcium. 2023 Mar;110:102697. doi: 10.1016/j.ceca.2023.102697. Epub 2023 Jan 27. PMID: 36736164; PMCID: PMC10173365.

Abstract

Ca2+ is a major ligand of the inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+-release channel. Fan et al. [1] recently solved additional cryo-electron microscopy (cryo-EM) structures of the IP3R in different ligand-binding states, revealing new Ca2+ binding sites.

CryoEM and CryoET Foskett

Structure-function correlates of fibrinogen binding by Acinetobacter adhesins critical in catheter-associated urinary tract infections

Tamadonfar KO, Di Venanzio G, Pinkner JS, Dodson KW, Kalas V, Zimmerman MI, Bazan Villicana J, Bowman GR, Feldman MF, Hultgren SJ. Structure-function correlates of fibrinogen binding by Acinetobacter adhesins critical in catheter-associated urinary tract infections. Proc Natl Acad Sci U S A. 2023 Jan 24;120(4):e2212694120. doi: 10.1073/pnas.2212694120. Epub 2023 Jan 18. PMID: 36652481; PMCID: PMC9942807.

Abstract Multidrug-resistant

Multidrug-resistant Acinetobacter baumannii infections are an urgent clinical problem and can cause difficult-to-treat nosocomial infections. During such infections, like catheter-associated urinary tract infections (CAUTI), A. baumannii rely on adhesive, extracellular fibers, called chaperone-usher pathway (CUP) pili for critical binding interactions. The A. baumannii uropathogenic strain, UPAB1, and the pan-European subclone II isolate, ACICU, use the CUP pili Abp1 and Abp2 (previously termed Cup and Prp, respectively) in tandem to establish CAUTIs, specifically to facilitate bacterial adherence and biofilm formation on the implanted catheter. Abp1 and Abp2 pili are tipped with two domain tip adhesins, Abp1D and Abp2D, respectively. We discovered that both adhesins bind fibrinogen, a critical host wound response protein that is released into the bladder upon catheterization and is subsequently deposited on the catheter. The crystal structures of the Abp1D and Abp2D receptor-binding domains were determined and revealed that they both contain a large, distally oriented pocket, which mediates binding to fibrinogen and other glycoproteins. Genetic, biochemical, and biophysical studies revealed that interactions with host proteins are governed by several critical residues in and along the edge of the binding pocket, one of which regulates the structural stability of an anterior loop motif. K34, located outside of the pocket but interacting with the anterior loop, also regulates the binding affinity of the protein. This study illuminates the mechanistic basis of the critical fibrinogen-coated catheter colonization step in A.

Bowman Lab Computational Biology

Fluorescent azobenzene-confined coiled-coil mesofibers

Punia K, Britton D, Hüll K, Yin L, Wang Y, Renfrew PD, Gilchrist ML, Bonneau R, Trauner D, Montclare JK. Fluorescent azobenzene-confined coiled-coil mesofibers. Soft Matter. 2023 Jan 18;19(3):497-501. doi: 10.1039/d2sm01578a. PMID: 36538008.

Abstract

Fluorescent protein biomaterials have important applications such as bioimaging in pharmacological studies. Self-assembly of proteins, especially into fibrils, is known to produce fluorescence in the blue band. Capable of self-assembly into nanofibers, we have shown we can modulate its aggregation into mesofibers by encapsulation of a small hydrophobic molecule. Conversely, azobenzenes are hydrophobic small molecules that are virtually non-fluorescent in solution due to their highly efficient photoisomerization. However, they demonstrate fluorogenic properties upon confinement in nanoscale assemblies by reducing the non-radiative photoisomerization. Here, we report the fluorescence of a hybrid protein-small molecule system in which azobenzene is confined in our protein assembly leading to fiber thickening and increased fluorescence. We show our engineered protein Q encapsulates AzoCholine, bearing a photoswitchable azobenzene moiety, in the hydrophobic pore to produce fluorescent mesofibers. This study further investigates the photocontrol of protein conformation as well as fluorescence of an azobenze-containing biomaterial.

Chemical Biology CryoEM and CryoET Trauner Group

Optical control of neuronal activities with photoswitchable nanovesicles

Xiong H, Alberto KA, Youn J, Taura J, Morstein J, Li X, Wang Y, Trauner D, Slesinger PA, Nielsen SO, Qin Z. Optical control of neuronal activities with photoswitchable nanovesicles. Nano Res. 2023 Jan;16(1):1033-1041. doi: 10.1007/s12274-022-4853-x. Epub 2022 Sep 2. PMID: 37063114; PMCID: PMC10103898.

Abstract

Precise modulation of neuronal activity by neuroactive molecules is essential for understanding brain circuits and behavior. However, tools for highly controllable molecular release are lacking. Here, we developed a photoswitchable nanovesicle with azobenzene-containing phosphatidylcholine (azo-PC), coined ‘azosome’, for neuromodulation. Irradiation with 365 nm light triggers the trans-to-cis isomerization of azo-PC, resulting in a disordered lipid bilayer with decreased thickness and cargo release. Irradiation with 455 nm light induces reverse isomerization and switches the release off. Real-time fluorescence imaging shows controllable and repeatable cargo release within seconds (< 3 s). Importantly, we demonstrate that SKF-81297, a dopamine D1-receptor agonist, can be repeatedly released from the azosome to activate cultures of primary striatal neurons. Azosome shows promise for precise optical control over the molecular release and can be a valuable tool for molecular neuroscience studies.

Chemical Biology CryoEM and CryoET Trauner Group

KIF1A is kinetically tuned to be a superengaging motor under hindering loads

Pyrpassopoulos S, Gicking AM, Zaniewski TM, Hancock WO, Ostap EM. KIF1A is kinetically tuned to be a superengaging motor under hindering loads. Proc Natl Acad Sci U S A. 2023 Jan 10;120(2):e2216903120. doi: 10.1073/pnas.2216903120. Epub 2023 Jan 4. PMID: 36598948; PMCID: PMC9926277.

Abstract

KIF1A is a highly processive vesicle transport motor in the kinesin-3 family. Mutations in KIF1A lead to neurodegenerative diseases including hereditary spastic paraplegia. We applied optical tweezers to study the ability of KIF1A to generate and sustain force against hindering loads. We used both the three-bead assay, where force is oriented parallel to the microtubule, and the traditional single-bead assay, where force is directed along the radius of the bead, resulting in a vertical force component. The average force and attachment duration of KIF1A in the three-bead assay were substantially greater than those observed in the single-bead assay. Thus, vertical forces accelerate termination of force ramps of KIF1A. Average KIF1A termination forces were slightly lower than the kinesin-1 KIF5B, and the median attachment duration of KIF1A was >10-fold shorter than KIF5B under hindering loads. KIF1A rapidly reengages with microtubules after detachment, as observed previously. Strikingly, quantification enabled by the three-bead assay shows that reengagement largely occurs within 2 ms of detachment, indicating that KIF1A has a nearly 10-fold faster reengagement rate than KIF5B. We found that rapid microtubule reengagement is not due to KIF1A’s positively charged loop-12; however, removal of charge from this loop diminished the unloaded run length at near physiological ionic strength. Both loop-12 and the microtubule nucleotide state have modulatory effects on reengagement under load, suggesting a role for the microtubule lattice in KIF1A reengagement. Our results reveal adaptations of KIF1A that lead to a model of superengaging transport under load.

CryoEM and CryoET Single Molecule Imaging Ostap Lab

Involvement of Target of Rapamycin (TOR) Signaling in the Regulation of Crosstalk between Ribosomal Protein Small Subunit 6 Kinase-1 (RPS6K-1) and Ribosomal Proteins

Bakshi A, Moin M, Gayatri MB, Reddy ABM, Datla R, Madhav MS, Kirti PB. Plants (Basel). 2023 Jan 1;12(1):176. doi: 10.3390/plants12010176

Abstract

The target of rapamycin (TOR) protein phosphorylates its downstream effector p70kDa ribosomal protein S6 kinases (S6K1) for ribosome biogenesis and translation initiation in eukaryotes. However, the molecular mechanism of TOR-S6K1-ribosomal protein (RP) signaling is not well understood in plants. In the present study, we report the transcriptional upregulation of ribosomal protein large and small subunit (RPL and RPS) genes in the previously established TOR overexpressing transgenic lines of rice (in Oryza sativa ssp. indica, variety BPT-5204, TR-2.24 and TR-15.1) and of Arabidopsis thaliana (in Col 0 ecotype, ATR-1.4.27 and ATR-3.7.32). The mRNA levels of RP genes from this study were compared with those previously available in transcriptomic datasets on the expression of RPs in relation to TOR inhibitor and in the TOR-RNAi lines of Arabidopsis thaliana. We further analyzed TOR activity, i.e., S6K1 phosphorylation in SALK lines of Arabidopsis with mutation in rpl6, rpl18, rpl23, rpl24 and rps28C, where the rpl18 mutant showed inactivation of S6K1 phosphorylation. We also predicted similar putative Ser/Thr phosphorylation sites for ribosomal S6 kinases (RSKs) in the RPs of Oryza sativa ssp. indica and Arabidopsis thaliana. The findings of this study indicate that the TOR pathway is possibly interlinked in a cyclic manner via the phosphorylation of S6K1 as a modulatory step for the regulation of RP function to switch ‘on’/‘off’ the translational regulation for balanced plant growth.
CryoEM and CryoET Mass Spectrometry Reddy Lab