Judith Frydman

All Publications

• Brain malformations and seizures by impaired chaperonin function of TRiC. Science (New York, N.Y.) Kraft, F., Rodriguez-Aliaga, P., Yuan, W., Franken, L., Zajt, K., Hasan, D., Lee, T. T., Flex, E., Hentschel, A., Innes, A. M., Zheng, B., Julia Suh, D. S., Knopp, C., Lausberg, E., Krause, J., Zhang, X., Trapane, P., Carroll, R., McClatchey, M., Fry, A. E., Wang, L., Giesselmann, S., Hoang, H., Baldridge, D., Silverman, G. A., Radio, F. C., Bertini, E., Ciolfi, A., Blood, K. A., de Sainte Agathe, J. M., Charles, P., Bergant, G., Čuturilo, G., Peterlin, B., Diderich, K., Streff, H., Robak, L., Oegema, R., van Binsbergen, E., Herriges, J., Saunders, C. J., Maier, A., Wolking, S., Weber, Y., Lochmüller, H., Meyer, S., Aleman, A., Polavarapu, K., Nicolas, G., Goldenberg, A., Guyant, L., Pope, K., Hehmeyer, K. N., Monaghan, K. G., Quade, A., Smol, T., Caumes, R., Duerinckx, S., Depondt, C., Van Paesschen, W., Rieubland, C., Poloni, C., Guipponi, M., Arcioni, S., Meuwissen, M., Jansen, A. C., Rosenblum, J., Haack, T. B., Bertrand, M., Gerstner, L., Magg, J., Riess, O., Schulz, J. B., Wagner, N., Wiesmann, M., Weis, J., Eggermann, T., Begemann, M., Roos, A., Häusler, M., Schedl, T., Tartaglia, M., Bremer, J., Pak, S. C., Frydman, J., Elbracht, M., Kurth, I. 2024; 386 (6721): 516-525

  Abstract

  Malformations of the brain are common and vary in severity, from negligible to potentially fatal. Their causes have not been fully elucidated. Here, we report pathogenic variants in the core protein-folding machinery TRiC/CCT in individuals with brain malformations, intellectual disability, and seizures. The chaperonin TRiC is an obligate hetero-oligomer, and we identify variants in seven of its eight subunits, all of which impair function or assembly through different mechanisms. Transcriptome and proteome analyses of patient-derived fibroblasts demonstrate the various consequences of TRiC impairment. The results reveal an unexpected and potentially widespread role for protein folding in the development of the central nervous system and define a disease spectrum of "TRiCopathies."

  View details for DOI 10.1126/science.adp8721

  View details for PubMedID 39480921

• A machine learning approach uncovers principles and determinants of eukaryotic ribosome pausing. Science advances Aguilar Rangel, M., Stein, K., Frydman, J. 2024; 10 (42): eado0738

  Abstract

  Nonuniform local translation speed dictates diverse protein biogenesis outcomes. To unify known and uncover unknown principles governing eukaryotic elongation rate, we developed a machine learning pipeline to analyze RiboSeq datasets. We find that the chemical nature of the incoming amino acid determines how codon optimality influences elongation rate, with hydrophobic residues more dependent on transfer RNA (tRNA) levels than charged residues. Unexpectedly, we find that wobble interactions exert a widespread effect on elongation pausing, with wobble-mediated decoding being slower than Watson-Crick decoding, irrespective of tRNA levels. Applying our ribosome pausing principles to ribosome collisions reveals that disomes arise upon apposition of fast-decoding and slow-decoding signatures. We conclude that codon choice and tRNA pools are evolutionarily constrained to harmonize elongation rate with cotranslational folding while minimizing wobble pairing and deleterious stalling.

  View details for DOI 10.1126/sciadv.ado0738

  View details for PubMedID 39423268

• Polyglutamine-mediated ribotoxicity disrupts proteostasis and stress responses in Huntington's disease. Nature cell biology Aviner, R., Lee, T., Masto, V. B., Li, K. H., Andino, R., Frydman, J. 2024

  Abstract

  Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG trinucleotide repeat in the Huntingtin (HTT) gene, encoding a homopolymeric polyglutamine (polyQ) tract. Although mutant HTT (mHTT) protein is known to aggregate, the links between aggregation and neurotoxicity remain unclear. Here we show that both translation and aggregation of wild-type HTT and mHTT are regulated by a stress-responsive upstream open reading frame and that polyQ expansions cause abortive translation termination and release of truncated, aggregation-prone mHTT fragments. Notably, we find that mHTT depletes translation elongation factor eIF5A in brains of symptomatic HD mice and cultured HD cells, leading to pervasive ribosome pausing and collisions. Loss of eIF5A disrupts homeostatic controls and impairs recovery from acute stress. Importantly, drugs that inhibit translation initiation reduce premature termination and mitigate this escalating cascade of ribotoxic stress and dysfunction in HD.

  View details for DOI 10.1038/s41556-024-01414-x

  View details for PubMedID 38741019

• Impaired biogenesis of basic proteins impacts multiple hallmarks of the aging brain. bioRxiv : the preprint server for biology Di Fraia, D., Marino, A., Lee, J. H., Kelmer Sacramento, E., Baumgart, M., Bagnoli, S., Tomaz da Silva, P., Kumar Sahu, A., Siano, G., Tiessen, M., Terzibasi-Tozzini, E., Gagneur, J., Frydman, J., Cellerino, A., Ori, A. 2024

  Abstract

  Aging and neurodegeneration entail diverse cellular and molecular hallmarks. Here, we studied the effects of aging on the transcriptome, translatome, and multiple layers of the proteome in the brain of a short-lived killifish. We reveal that aging causes widespread reduction of proteins enriched in basic amino acids that is independent of mRNA regulation, and it is not due to impaired proteasome activity. Instead, we identify a cascade of events where aberrant translation pausing leads to reduced ribosome availability resulting in proteome remodeling independently of transcriptional regulation. Our research uncovers a vulnerable point in the aging brain's biology - the biogenesis of basic DNA/RNA binding proteins. This vulnerability may represent a unifying principle that connects various aging hallmarks, encompassing genome integrity and the biosynthesis of macromolecules.

  View details for DOI 10.1101/2023.07.20.549210

  View details for PubMedID 38260253

  View details for PubMedCentralID PMC10802395

• A hierarchical assembly pathway directs the unique subunit arrangement of TRiC/CCT. Molecular cell Betancourt Moreira, K., Collier, M. P., Leitner, A., Li, K. H., Lachapel, I. L., McCarthy, F., Opoku-Nsiah, K. A., Morales-Polanco, F., Barbosa, N., Gestaut, D., Samant, R. S., Roh, S., Frydman, J. 2023

  Abstract

  How the essential eukaryotic chaperonin TRiC/CCT assembles from eight distinct subunits into a unique double-ring architecture remains undefined. We show TRiC assembly involves a hierarchical pathway that segregates subunits with distinct functional properties until holocomplex (HC) completion. A stable, likely early intermediate arises from small oligomers containing CCT2, CCT4, CCT5, and CCT7, contiguous subunits that constitute the negatively charged hemisphere of the TRiC chamber, which has weak affinity for unfolded actin. The remaining subunits CCT8, CCT1, CCT3, and CCT6, which comprise the positively charged chamber hemisphere that binds unfolded actin more strongly, join the ring individually. Unincorporated late-assembling subunits are highly labile in cells, which prevents their accumulation and premature substrate binding. Recapitulation of assembly in a recombinant system demonstrates that the subunits in each hemisphere readily form stable, noncanonical TRiC-like HCs with aberrant functional properties. Thus, regulation of TRiC assembly along a biochemical axis disfavors the formation of stable alternative chaperonin complexes.

  View details for DOI 10.1016/j.molcel.2023.07.031

  View details for PubMedID 37625406

• High-resolution mapping reveals the mechanism and contribution of genome insertions and deletions to RNA virus evolution. Proceedings of the National Academy of Sciences of the United States of America Aguilar Rangel, M., Dolan, P. T., Taguwa, S., Xiao, Y., Andino, R., Frydman, J. 2023; 120 (31): e2304667120

  Abstract

  RNA viruses rapidly adapt to selective conditions due to the high intrinsic mutation rates of their RNA-dependent RNA polymerases (RdRps). Insertions and deletions (indels) in viral genomes are major contributors to both deleterious mutational load and evolutionary novelty, but remain understudied. To characterize the mechanistic details of their formation and evolutionary dynamics during infection, we developed a hybrid experimental-bioinformatic approach. This approach, called MultiMatch, extracts insertions and deletions from ultradeep sequencing experiments, including those occurring at extremely low frequencies, allowing us to map their genomic distribution and quantify the rates at which they occur. Mapping indel mutations in adapting poliovirus and dengue virus populations, we determine the rates of indel generation and identify mechanistic and functional constraints shaping indel diversity. Using poliovirus RdRp variants of distinct fidelity and genome recombination rates, we demonstrate tradeoffs between fidelity and Indel generation. Additionally, we show that maintaining translation frame and viral RNA structures constrain the Indel landscape and that, due to these significant fitness effects, Indels exert a significant deleterious load on adapting viral populations. Conversely, we uncover positively selected Indels that modulate RNA structure, generate protein variants, and produce defective interfering genomes in viral populations. Together, our analyses establish the kinetic and mechanistic tradeoffs between misincorporation, recombination, and Indel rates and reveal functional principles defining the central role of Indels in virus evolution, emergence, and the regulation of viral infection.

  View details for DOI 10.1073/pnas.2304667120

  View details for PubMedID 37487061

• Novel Mode of nanoLuciferase Packaging in SARS-CoV-2 Virions and VLPs Provides Versatile Reporters for Virus Production. Viruses Gullberg, R. C., Frydman, J. 2023; 15 (6)

  Abstract

  SARS-CoV-2 is a positive-strand RNA virus in the Coronaviridae family that is responsible for morbidity and mortality worldwide. To better understand the molecular pathways leading to SARS-CoV-2 virus assembly, we examined a virus-like particle (VLP) system co-expressing all structural proteins together with an mRNA reporter encoding nanoLuciferase (herein nLuc). Surprisingly, the 19 kDa nLuc protein itself was encapsidated into VLPs, providing a better reporter than nLuc mRNA itself. Strikingly, infecting nLuc-expressing cells with the SARS-CoV-2, NL63 or OC43 coronaviruses yielded virions containing packaged nLuc that served to report viral production. In contrast, infection with the flaviviruses, dengue or Zika, did not lead to nLuc packaging and secretion. A panel of reporter protein variants revealed that the packaging is size-limited and requires cytoplasmic expression, indicating that the large virion of coronaviruses can encaspidate a small cytoplasmic reporter protein. Our findings open the way for powerful new approaches to measure coronavirus particle production, egress and viral entry mechanisms.

  View details for DOI 10.3390/v15061335

  View details for PubMedID 37376634

  View details for PubMedCentralID PMC10301038

• Nuclear and cytoplasmic spatial protein quality control is coordinated by nuclear-vacuolar junctions and perinuclear ESCRT. Nature cell biology Sontag, E. M., Morales-Polanco, F., Chen, J. H., McDermott, G., Dolan, P. T., Gestaut, D., Le Gros, M. A., Larabell, C., Frydman, J. 2023

  Abstract

  Effective protein quality control (PQC), essential for cellular health, relies on spatial sequestration of misfolded proteins into defined inclusions. Here we reveal the coordination of nuclear and cytoplasmic spatial PQC. Cytoplasmic misfolded proteins concentrate in a cytoplasmic juxtanuclear quality control compartment, while nuclear misfolded proteins sequester into an intranuclear quality control compartment (INQ). Particle tracking reveals that INQ and the juxtanuclear quality control compartment converge to face each other across the nuclear envelope at a site proximal to the nuclear-vacuolar junction marked by perinuclear ESCRT-II/III protein Chm7. Strikingly, convergence at nuclear-vacuolar junction contacts facilitates VPS4-dependent vacuolar clearance of misfolded cytoplasmic and nuclear proteins, the latter entailing extrusion of nuclear INQ into the vacuole. Finding that nuclear-vacuolar contact sites are cellular hubs of spatial PQC to facilitate vacuolar clearance of nuclear and cytoplasmic inclusions highlights the role of cellular architecture in proteostasis maintenance.

  View details for DOI 10.1038/s41556-023-01128-6

  View details for PubMedID 37081164

  View details for PubMedCentralID 6671943

• Structural visualization of the tubulin folding pathway directed by human chaperonin TRiC/CCT. Cell Gestaut, D., Zhao, Y., Park, J., Ma, B., Leitner, A., Collier, M., Pintilie, G., Roh, S. H., Chiu, W., Frydman, J. 2022; 185 (25): 4770-4787.e20

  Abstract

  The ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of β-tubulin using human prefoldin and TRiC. We find unstructured β-tubulin is delivered by prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. Cryo-EM resolves four near-atomic-resolution structures containing progressively folded β-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Intrinsically disordered CCT C termini within the chamber promote subsequent folding of tubulin's core and middle domains and GTP-binding. Thus, TRiC's chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.

  View details for DOI 10.1016/j.cell.2022.11.014

  View details for PubMedID 36493755

  View details for PubMedCentralID PMC9735246

• A campaign targeting a conserved Hsp70 binding site uncovers how subcellular localization is linked to distinct biological activities. Cell chemical biology Shao, H., Taguwa, S., Gilbert, L., Shkedi, A., Sannino, S., Guerriero, C. J., Gale-Day, Z. J., Young, Z. T., Brodsky, J. L., Weissman, J., Gestwicki, J. E., Frydman, J. 2022

  Abstract

  The potential of small molecules to localize within subcellular compartments is rarely explored. To probe this question, we measured the localization of Hsp70 inhibitors using fluorescence microscopy. We found that even closely related analogs had dramatically different distributions, with some residing predominantly in the mitochondria and others in the ER. CRISPRi screens supported this idea, showing that different compounds had distinct chemogenetic interactions with Hsp70s of the ER (HSPA5/BiP) and mitochondria (HSPA9/mortalin) and their co-chaperones. Moreover, localization seemed to determine function, even for molecules with conserved binding sites. Compounds with distinct partitioning have distinct anti-proliferative activity in breast cancer cells compared with anti-viral activity in cellular models of Dengue virus replication, likely because different sets of Hsp70s are required in these processes. These findings highlight the contributions of subcellular partitioning and chemogenetic interactions to small molecule activity, features that are rarely explored during medicinal chemistry campaigns.

  View details for DOI 10.1016/j.chembiol.2022.06.006

  View details for PubMedID 35830852

• Ageing exacerbates ribosome pausing to disrupt cotranslational proteostasis Nature Stein, K. C., Morales-Polanco, F., Leinden, J. v., Rainbolt, K. T., Frydman, J. 2022

  View details for DOI 10.1038/s41586-021-04295-4

• Cotranslational prolyl hydroxylation is essential for flavivirus biogenesis NATURE Aviner, R., Li, K. H., Frydman, J., Andino, R. 2021

  Abstract

  Viral pathogens are an ongoing threat to public health worldwide. Analysing their dependence on host biosynthetic pathways could lead to effective antiviral therapies1. Here we integrate proteomic analyses of polysomes with functional genomics and pharmacological interventions to define how enteroviruses and flaviviruses remodel host polysomes to synthesize viral proteins and disable host protein production. We find that infection with polio, dengue or Zika virus markedly modifies polysome composition, without major changes to core ribosome stoichiometry. These viruses use different strategies to evict a common set of translation initiation and RNA surveillance factors from polysomes while recruiting host machineries that are specifically required for viral biogenesis. Targeting these specialized viral polysomes could provide a new approach for antiviral interventions. For example, we find that both Zika and dengue use the collagen proline hydroxylation machinery to mediate cotranslational modification of conserved proline residues in the viral polyprotein. Genetic or pharmacological inhibition of proline hydroxylation impairs nascent viral polyprotein folding and induces its aggregation and degradation. Notably, such interventions prevent viral polysome remodelling and lower virus production. Our findings delineate the modular nature of polysome specialization at the virus-host interface and establish a powerful strategy to identify targets for selective antiviral interventions.

  View details for DOI 10.1038/s41586-021-03851-2

  View details for Web of Science ID 000686074400001

  View details for PubMedID 34408324

• CryoEM reveals the stochastic nature of individual ATP binding events in a group II chaperonin. Nature communications Zhao, Y., Schmid, M. F., Frydman, J., Chiu, W. 2021; 12 (1): 4754

  Abstract

  Chaperonins are homo- or hetero-oligomeric complexes that use ATP binding and hydrolysis to facilitate protein folding. ATP hydrolysis exhibits both positive and negative cooperativity. The mechanism by which chaperonins coordinate ATP utilization in their multiple subunits remains unclear. Here we use cryoEM to study ATP binding in the homo-oligomeric archaeal chaperonin from Methanococcus maripaludis (MmCpn), consisting of two stacked rings composed of eight identical subunits each. Using a series of image classification steps, we obtained different structural snapshots of individual chaperonins undergoing the nucleotide binding process. We identified nucleotide-bound and free states of individual subunits in each chaperonin, allowing us to determine the ATP occupancy state of each MmCpn particle. We observe distinctive tertiary and quaternary structures reflecting variations in nucleotide occupancy and subunit conformations in each chaperonin complex. Detailed analysis of the nucleotide distribution in each MmCpn complex indicates that individual ATP binding events occur in a statistically random manner for MmCpn, both within and across the rings. Our findings illustrate the power of cryoEM to characterize a biochemical property of multi-subunit ligand binding cooperativity at the individual particle level.

  View details for DOI 10.1038/s41467-021-25099-0

  View details for PubMedID 34362932

• Cryo-electron tomography provides topological insights into mutant huntingtin exon 1 and polyQ aggregates. Communications biology Galaz-Montoya, J. G., Shahmoradian, S. H., Shen, K., Frydman, J., Chiu, W. 2021; 4 (1): 849

  Abstract

  Huntington disease (HD) is a neurodegenerative trinucleotide repeat disorder caused by an expanded poly-glutamine (polyQ) tract in the mutant huntingtin (mHTT) protein. The formation and topology of filamentous mHTT inclusions in the brain (hallmarks of HD implicated in neurotoxicity) remain elusive. Using cryo-electron tomography and subtomogram averaging, here we show that mHTT exon 1 and polyQ-only aggregates in vitro are structurally heterogenous and filamentous, similar to prior observations with other methods. Yet, we find filaments in both types of aggregates under ~2nm in width, thinner than previously reported, and regions forming large sheets. In addition, our data show a prevalent subpopulation of filaments exhibiting a lumpy slab morphology in both aggregates, supportive of the polyQ core model. This provides a basis for future cryoET studies of various aggregated mHTT and polyQ constructs to improve their structure-based modeling as well as their identification in cells without fusion tags.

  View details for DOI 10.1038/s42003-021-02360-2

  View details for PubMedID 34239038

• Structural and functional dissection of reovirus capsid folding and assembly by the prefoldin-TRiC/CCT chaperone network PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Knowlton, J. J., Gestaut, D., Ma, B., Taylor, G., Seven, A., Leitner, A., Wilson, G. J., Shanker, S., Yates, N. A., Prasad, B., Aebersold, R., Chiu, W., Frydman, J., Dermody, T. S. 2021; 118 (11)

  View details for DOI 10.1073/pnas.2018127118

  View details for Web of Science ID 000629635100046

• Principles of dengue virus evolvability derived from genotype-fitness maps in human and mosquito cells. eLife Dolan, P. T., Taguwa, S., Rangel, M. A., Acevedo, A., Hagai, T., Andino, R., Frydman, J. 2021; 10

  Abstract

  Dengue virus (DENV) cycles between mosquito and mammalian hosts. To examine how DENV populations adapt to these different host environments we used serial passage in human and mosquito cell lines and estimated fitness effects for all single-nucleotide variants in these populations using ultra-deep sequencing. This allowed us to determine the contributions of beneficial and deleterious mutations to the collective fitness of the population. Our analysis revealed that the continuous influx of a large burden of deleterious mutations counterbalances the effect of rare, host-specific beneficial mutations to shape the path of adaptation. Beneficial mutations preferentially map to intrinsically disordered domains in the viral proteome and cluster to defined regions in the genome. These phenotypically redundant adaptive alleles may facilitate host-specific DENV adaptation. Importantly, the evolutionary constraints described in our simple system mirror trends observed across DENV and Zika strains, indicating it recapitulates key biophysical and biological constraints shaping long-term viral evolution.

  View details for DOI 10.7554/eLife.61921

  View details for PubMedID 33491648

• Proteostasis in Viral Infection: Unfolding the Complex Virus-Chaperone Interplay COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Aviner, R., Frydman, J. 2020; 12 (3)

  View details for DOI 10.1101/cshperspect.a034090

  View details for Web of Science ID 000518450100007

• Differentiation Drives Widespread Rewiring of the Neural Stem Cell Chaperone Network. Molecular cell Vonk, W. I., Rainbolt, T. K., Dolan, P. T., Webb, A. E., Brunet, A. n., Frydman, J. n. 2020

  Abstract

  Neural stem and progenitor cells (NSPCs) are critical for continued cellular replacement in the adult brain. Lifelong maintenance of a functional NSPC pool necessitates stringent mechanisms to preserve a pristine proteome. We find that the NSPC chaperone network robustly maintains misfolded protein solubility and stress resilience through high levels of the ATP-dependent chaperonin TRiC/CCT. Strikingly, NSPC differentiation rewires the cellular chaperone network, reducing TRiC/CCT levels and inducing those of the ATP-independent small heat shock proteins (sHSPs). This switches the proteostasis strategy in neural progeny cells to promote sequestration of misfolded proteins into protective inclusions. The chaperone network of NSPCs is more effective than that of differentiated cells, leading to improved management of proteotoxic stress and amyloidogenic proteins. However, NSPC proteostasis is impaired by brain aging. The less efficient chaperone network of differentiated neural progeny may contribute to their enhanced susceptibility to neurodegenerative diseases characterized by aberrant protein misfolding and aggregation.

  View details for DOI 10.1016/j.molcel.2020.03.009

  View details for PubMedID 32268122

• THE CHAPERONIN TRIC/CCT ASSOCIATES WITH PREFOLDIN THROUGH A CONSERVED ELECTROSTATIC INTERFACE ESSENTIAL FOR CELLULAR PROTEOSTASIS Gestaut, D., Roh, S., Ma, B., Pintile, G., Joachimiak, L., Leitner, A., Walzotheini, T., Aebersold, R., Chiu, W., Frydman, J. WILEY. 2019: 44

  View details for Web of Science ID 000488134600059

• Dual Role of Ribosome-Binding Domain of NAC as a Potent Suppressor of Protein Aggregation and Aging-Related Proteinopathies MOLECULAR CELL Shen, K., Gamerdinger, M., Chan, R., Gense, K., Martin, E. M., Sachs, N., Knight, P. D., Schloemer, R., Calabrese, A. N., Stewart, K. L., Leiendecker, L., Baghel, A., Radford, S. E., Frydman, J., Deuerling, E. 2019; 74 (4): 729-+

  View details for DOI 10.1016/j.molcel.2019.03.012

  View details for Web of Science ID 000468094300010

• The Chaperonin TRiC/CCT Associates with Prefoldin through a Conserved Electrostatic Interface Essential for Cellular Proteostasis CELL Gestaut, D., Roh, S., Ma, B., Pintilie, G., Joachimiak, L. A., Leitner, A., Walzthoeni, T., Aebersold, R., Chiu, W., Frydman, J. 2019; 177 (3): 751-+

  View details for DOI 10.1016/j.cell.2019.03.012

  View details for Web of Science ID 000464947700026

• The ATP-powered gymnastics of TRiC/CCT: an asymmetric protein folding machine with a symmetric origin story. Current opinion in structural biology Gestaut, D., Limatola, A., Joachimiak, L., Frydman, J. 2019; 55: 50–58

  Abstract

  The eukaryotic chaperonin TRiC/CCT is a large hetero-oligomeric complex that plays an essential role assisting cellular protein folding and suppressing protein aggregation. It consists of two rings, and each composed of eight different subunits; non-native polypeptides bind and fold in an ATP-dependent manner within their central chamber. Here, we review recent advances in our understanding of TRiC structure and mechanism enabled by application of hybrid structural methods including the integration of cryo-electron microscopy with distance constraints from crosslinking mass spectrometry. These new insights are revealing how the different TRiC/CCT subunits create asymmetry in its ATP-driven conformational cycle and its interaction with non-native polypeptides, which ultimately underlie its unique ability to fold proteins that cannot be folded by other chaperones.

  View details for PubMedID 30978594

• The ATP-powered gymnastics of TRiC/CCT: an asymmetric protein folding machine with a symmetric origin story CURRENT OPINION IN STRUCTURAL BIOLOGY Gestaut, D., Limatola, A., Joachimiak, L., Frydman, J. 2019; 55: 50–58

  View details for DOI 10.1016/j.sbi.2019.03.002

  View details for Web of Science ID 000482514400008

• Proteostasis in Viral Infection: Unfolding the Complex Virus-Chaperone Interplay. Cold Spring Harbor perspectives in biology Aviner, R., Frydman, J. 2019

  Abstract

  Viruses are obligate intracellular parasites that rely on their hosts for protein synthesis, genome replication, and viral particle production. As such, they have evolved mechanisms to divert host resources, including molecular chaperones, facilitate folding and assembly of viral proteins, stabilize complex structures under constant mutational pressure, and modulate signaling pathways to dampen antiviral responses and prevent premature host death. Biogenesis of viral proteins often presents unique challenges to the proteostasis network, as it requires the rapid and orchestrated production of high levels of a limited number of multifunctional, multidomain, and aggregation-prone proteins. To overcome such challenges, viruses interact with the folding machinery not only as clients but also as regulators of chaperone expression, function, and subcellular localization. In this review, we summarize the main types of interactions between viral proteins and chaperones during infection, examine evolutionary aspects of this relationship, and discuss the potential of using chaperone inhibitors as broad-spectrum antivirals.

  View details for PubMedID 30858229

• Zika Virus Dependence on Host Hsp70 Provides a Protective Strategy against Infection and Disease. Cell reports Taguwa, S., Yeh, M., Rainbolt, T. K., Nayak, A., Shao, H., Gestwicki, J. E., Andino, R., Frydman, J. 2019; 26 (4): 906

  Abstract

  The spread of mosquito-borne Zika virus (ZIKV), which causes neurological disorders and microcephaly, highlights the need for countermeasures against sudden viral epidemics. Here, we tested the concept that drugs targeting host proteostasis provide effective antivirals. We show that different cytosolic Hsp70 isoforms are recruited to ZIKV-induced compartments and are required for virus replication at pre- and post-entry steps. Drugs targeting Hsp70 significantly reduce replication of different ZIKV strains in human and mosquito cells, including human neural stem cells and a placental trophoblast cell line, at doses without appreciable toxicity to the host cell. By targeting several ZIKV functions, including entry, establishment of active replication complexes, and capsid assembly, Hsp70 inhibitors are refractory to the emergence of drug-resistant virus. Importantly, these drugs protected mouse models from ZIKV infection, reducing viremia, mortality, and disease symptoms. Hsp70 inhibitors are thus attractive candidates for ZIKV therapeutics with the added benefit of a broad spectrum of action.

  View details for PubMedID 30673613

• Nascent Polypeptide Domain Topology and Elongation Rate Direct the Cotranslational Hierarchy of Hsp70 and TRiC/CCT. Molecular cell Stein, K. C., Kriel, A. n., Frydman, J. n. 2019

  Abstract

  Cotranslational protein folding requires assistance from elaborate ribosome-associated chaperone networks. It remains unclear how the changing information in a growing nascent polypeptide dictates the recruitment of functionally distinct chaperones. Here, we used ribosome profiling to define the principles governing the cotranslational action of the chaperones TRiC/CCT and Hsp70/Ssb. We show that these chaperones are sequentially recruited to specific sites within domain-encoding regions of select nascent polypeptides. Hsp70 associates first, binding select sites throughout domains, whereas TRiC associates later, upon the emergence of nearly complete domains that expose an unprotected hydrophobic surface. This suggests that transient topological properties of nascent folding intermediates drive sequential chaperone association. Moreover, cotranslational recruitment of both TRiC and Hsp70 correlated with translation elongation slowdowns. We propose that the temporal modulation of the nascent chain structural landscape is coordinated with local elongation rates to regulate the hierarchical action of Hsp70 and TRiC for cotranslational folding.

  View details for DOI 10.1016/j.molcel.2019.06.036

  View details for PubMedID 31400849

• Distinct proteostasis circuits cooperate in nuclear and cytoplasmic protein quality control. Nature Samant, R. S., Livingston, C. M., Sontag, E. M., Frydman, J. 2018; 563 (7731): 407-411

  Abstract

  Protein misfolding is linked to a wide array of human disorders, including Alzheimer's disease, Parkinson's disease and type II diabetes1,2. Protective cellular protein quality control (PQC) mechanisms have evolved to selectively recognize misfolded proteins and limit their toxic effects3-9, thus contributing to the maintenance of the proteome (proteostasis). Here we examine how molecular chaperones and the ubiquitin-proteasome system cooperate to recognize and promote the clearance of soluble misfolded proteins. Using a panel of PQC substrates with distinct characteristics and localizations, we define distinct chaperone and ubiquitination circuitries that execute quality control in the cytoplasm and nucleus. In the cytoplasm, proteasomal degradation of misfolded proteins requires tagging with mixed lysine 48 (K48)- and lysine 11 (K11)-linked ubiquitin chains. A distinct combination of E3 ubiquitin ligases and specific chaperones is required to achieve each type of linkage-specific ubiquitination. In the nucleus, however, proteasomal degradation of misfolded proteins requires only K48-linked ubiquitin chains, and is thus independent of K11-specific ligases and chaperones. The distinct ubiquitin codes for nuclear and cytoplasmic PQC appear to be linked to the function of the ubiquilin protein Dsk2, which is specifically required to clear nuclear misfolded proteins. Our work defines the principles of cytoplasmic and nuclear PQC as distinct, involving combinatorial recognition by defined sets of cooperating chaperones and E3 ligases. A better understanding of how these organelle-specific PQC requirements implement proteome integrity has implications for our understanding of diseases linked to impaired protein clearance and proteostasis dysfunction.

  View details for DOI 10.1038/s41586-018-0678-x

  View details for PubMedID 30429547

• Hsp90 shapes protein and RNA evolution to balance trade-offs between protein stability and aggregation NATURE COMMUNICATIONS Geller, R., Pechmann, S., Acevedo, A., Andino, R., Frydman, J. 2018; 9: 1781

  Abstract

  Acquisition of mutations is central to evolution; however, the detrimental effects of most mutations on protein folding and stability limit protein evolvability. Molecular chaperones, which suppress aggregation and facilitate polypeptide folding, may alleviate the effects of destabilizing mutations thus promoting sequence diversification. To illuminate how chaperones can influence protein evolution, we examined the effect of reduced activity of the chaperone Hsp90 on poliovirus evolution. We find that Hsp90 offsets evolutionary trade-offs between protein stability and aggregation. Lower chaperone levels favor variants of reduced hydrophobicity and protein aggregation propensity but at a cost to protein stability. Notably, reducing Hsp90 activity also promotes clusters of codon-deoptimized synonymous mutations at inter-domain boundaries, likely to facilitate cotranslational domain folding. Our results reveal how a chaperone can shape the sequence landscape at both the protein and RNA levels to harmonize competing constraints posed by protein stability, aggregation propensity, and translation rate on successful protein biogenesis.

  View details for PubMedID 29725062

• Lysosome activation clears aggregates and enhances quiescent neural stem cell activation during aging SCIENCE Leeman, D. S., Hebestreit, K., Ruetz, T., Webb, A. E., McKay, A., Pollina, E. A., Dulken, B. W., Zhao, X., Yeo, R. W., Ho, T. T., Mahmoudi, S., Devarajan, K., Passegue, E., Rando, T. A., Frydman, J., Brunet, A. 2018; 359 (6381): 1277–82

  Abstract

  In the adult brain, the neural stem cell (NSC) pool comprises quiescent and activated populations with distinct roles. Transcriptomic analysis revealed that quiescent and activated NSCs exhibited differences in their protein homeostasis network. Whereas activated NSCs had active proteasomes, quiescent NSCs contained large lysosomes. Quiescent NSCs from young mice accumulated protein aggregates, and many of these aggregates were stored in large lysosomes. Perturbation of lysosomal activity in quiescent NSCs affected protein-aggregate accumulation and the ability of quiescent NSCs to activate. During aging, quiescent NSCs displayed defects in their lysosomes, increased accumulation of protein aggregates, and reduced ability to activate. Enhancement of the lysosome pathway in old quiescent NSCs cleared protein aggregates and ameliorated the ability of quiescent NSCs to activate, allowing them to regain a more youthful state.

  View details for PubMedID 29590078

  View details for PubMedCentralID PMC5915358

• An information theoretic framework reveals a tunable allosteric network in group II chaperonins. Nature structural & molecular biology Lopez, T., Dalton, K., Tomlinson, A., Pande, V., Frydman, J. 2017; 24 (9): 726-733

  Abstract

  ATP-dependent allosteric regulation of the ring-shaped group II chaperonins remains ill defined, in part because their complex oligomeric topology has limited the success of structural techniques in suggesting allosteric determinants. Further, their high sequence conservation has hindered the prediction of allosteric networks using mathematical covariation approaches. Here, we develop an information theoretic strategy that is robust to residue conservation and apply it to group II chaperonins. We identify a contiguous network of covarying residues that connects all nucleotide-binding pockets within each chaperonin ring. An interfacial residue between the networks of neighboring subunits controls positive cooperativity by communicating nucleotide occupancy within each ring. Strikingly, chaperonin allostery is tunable through single mutations at this position. Naturally occurring variants at this position that double the extent of positive cooperativity are less prevalent in nature. We propose that being less cooperative than attainable allows chaperonins to support robust folding over a wider range of metabolic conditions.

  View details for DOI 10.1038/nsmb.3440

  View details for PubMedID 28741612

  View details for PubMedCentralID PMC5986071

• Structure and Dynamics of the Huntingtin Exon-1 N-Terminus: A Solution NMR Perspective. Journal of the American Chemical Society Baias, M., Smith, P. E., Shen, K., Joachimiak, L. A., Zerko, S., Kozminski, W., Frydman, J., Frydman, L. 2017; 139 (3): 1168-1176

  Abstract

  Many neurodegenerative diseases are characterized by misfolding and aggregation of an expanded polyglutamine tract (polyQ). Huntington's Disease, caused by expansion of the polyQ tract in exon 1 of the Huntingtin protein (Htt), is associated with aggregation and neuronal toxicity. Despite recent structural progress in understanding the structures of amyloid fibrils, little is known about the solution states of Htt in general, and about molecular details of their transition from soluble to aggregation-prone conformations in particular. This is an important question, given the increasing realization that toxicity may reside in soluble conformers. This study presents an approach that combines NMR with computational methods to elucidate the structural conformations of Htt Exon 1 in solution. Of particular focus was Htt's N17 domain sited N-terminal to the polyQ tract, which is key to enhancing aggregation and modulate Htt toxicity. Such in-depth structural study of Htt presents a number of unique challenges: the long homopolymeric polyQ tract contains nearly identical residues, exon 1 displays a high degree of conformational flexibility leading to a scaling of the NMR chemical shift dispersion, and a large portion of the backbone amide groups are solvent-exposed leading to fast hydrogen exchange and causing extensive line broadening. To deal with these problems, NMR assignment was achieved on a minimal Htt exon 1, comprising the N17 domain, a polyQ tract of 17 glutamines, and a short hexameric polyProline region that does not contribute to the spectrum. A pH titration method enhanced this polypeptide's solubility and, with the aid of ≤5D NMR, permitted the full assignment of N17 and the entire polyQ tract. Structural predictions were then derived using the experimental chemical shifts of the Htt peptide at low and neutral pH, together with various different computational approaches. All these methods concurred in indicating that low-pH protonation stabilizes a soluble conformation where a helical region of N17 propagates into the polyQ region, while at neutral pH both N17 and the polyQ become largely unstructured-thereby suggesting a mechanism for how N17 regulates Htt aggregation.

  View details for DOI 10.1021/jacs.6b10893

  View details for PubMedID 28085263

• Mechanisms and Functions of Spatial Protein Quality Control. Annual review of biochemistry Sontag, E. M., Samant, R. S., Frydman, J. 2017

  Abstract

  A healthy proteome is essential for cell survival. Protein misfolding is linked to a rapidly expanding list of human diseases, ranging from neurodegenerative diseases to aging and cancer. Many of these diseases are characterized by the accumulation of misfolded proteins in intra- and extracellular inclusions, such as amyloid plaques. The clear link between protein misfolding and disease highlights the need to better understand the elaborate machinery that manages proteome homeostasis, or proteostasis, in the cell. Proteostasis depends on a network of molecular chaperones and clearance pathways involved in the recognition, refolding, and/or clearance of aberrant proteins. Recent studies reveal that an integral part of the cellular management of misfolded proteins is their spatial sequestration into several defined compartments. Here, we review the properties, function, and formation of these compartments. Spatial sequestration plays a central role in protein quality control and cellular fitness and represents a critical link to the pathogenesis of protein aggregation-linked diseases.

  View details for DOI 10.1146/annurev-biochem-060815-014616

  View details for PubMedID 28489421

• Control of the structural landscape and neuronal proteotoxicity of mutant Huntingtin by domains flanking the polyQ tract ELIFE Shen, K., Calamini, B., Fauerbach, J. A., Ma, B., Shahmoradian, S. H., Lachapei, I. L., Chiu, W., Lo, D. C., Frydman, J. 2016; 5

  Abstract

  Many neurodegenerative diseases are linked to amyloid aggregation. In Huntington's disease (HD), neurotoxicity correlates with an increased aggregation propensity of a polyglutamine (polyQ) expansion in exon 1 of mutant huntingtin protein (mHtt). Here we establish how the domains flanking the polyQ tract shape the mHtt conformational landscape in vitro and in neurons. In vitro, the flanking domains have opposing effects on the conformation and stabilities of oligomers and amyloid fibrils. The N-terminal N17 promotes amyloid fibril formation, while the C-terminal Proline Rich Domain destabilizes fibrils and enhances oligomer formation. However, in neurons both domains act synergistically to engage protective chaperone and degradation pathways promoting mHtt proteostasis. Surprisingly, when proteotoxicity was assessed in rat corticostriatal brain slices, either flanking region alone sufficed to generate a neurotoxic conformation, while the polyQ tract alone exhibited minimal toxicity. Linking mHtt structural properties to its neuronal proteostasis should inform new strategies for neuroprotection in polyQ-expansion diseases.

  View details for DOI 10.7554/eLife.18065

  View details for Web of Science ID 000390025200001

  View details for PubMedCentralID PMC5135392

• Cotranslational signal-independent SRP preloading during membrane targeting NATURE Chartron, J. W., Hunt, K. C., Frydman, J. 2016; 536 (7615): 224-?

  Abstract

  Ribosome-associated factors must properly decode the limited information available in nascent polypeptides to direct them to their correct cellular fate. It is unclear how the low complexity information exposed by the nascent chain suffices for accurate recognition by the many factors competing for the limited surface near the ribosomal exit site. Questions remain even for the well-studied cotranslational targeting cycle to the endoplasmic reticulum, involving recognition of linear hydrophobic signal sequences or transmembrane domains by the signal recognition particle (SRP). Notably, the SRP has low abundance relative to the large number of ribosome-nascent-chain complexes (RNCs), yet it accurately selects those destined for the endoplasmic reticulum. Despite their overlapping specificities, the SRP and the cotranslationally acting Hsp70 display precise mutually exclusive selectivity in vivo for their cognate RNCs. To understand cotranslational nascent chain recognition in vivo, here we investigate the cotranslational membrane-targeting cycle using ribosome profiling in yeast cells coupled with biochemical fractionation of ribosome populations. We show that the SRP preferentially binds secretory RNCs before their targeting signals are translated. Non-coding mRNA elements can promote this signal-independent pre-recruitment of SRP. Our study defines the complex kinetic interaction between elongation in the cytosol and determinants in the polypeptide and mRNA that modulate SRP–substrate selection and membrane targeting.

  View details for DOI 10.1038/nature19309

  View details for Web of Science ID 000381472100039

  View details for PubMedID 27487213

  View details for PubMedCentralID PMC5120976

• Delayed emergence of subdiffraction-sized mutant huntingtin fibrils following inclusion body formation. Quarterly reviews of biophysics Sahl, S. J., Lau, L., Vonk, W. I., Weiss, L. E., Frydman, J., Moerner, W. E. 2016; 49

  Abstract

  Aberrant aggregation of improperly folded proteins is the hallmark of several human neurodegenerative disorders, including Huntington's Disease (HD) with autosomal-dominant inheritance. In HD, expansion of the CAG-repeat-encoded polyglutamine (polyQ) stretch beyond ~40 glutamines in huntingtin (Htt) and its N-terminal fragments leads to the formation of large (up to several μm) globular neuronal inclusion bodies (IBs) over time. We report direct observations of aggregating Htt exon 1 in living and fixed cells at enhanced spatial resolution by stimulated emission depletion (STED) microscopy and single-molecule super-resolution optical imaging. Fibrils of Htt exon 1 arise abundantly across the cytosolic compartment and also in neuritic processes only after nucleation and aggregation into a fairly advanced stage of growth of the prominent IB have taken place. Structural characterizations of fibrils by STED show a distinct length cutoff at ~1·5 µm and reveal subsequent coalescence (bundling/piling). Cytosolic fibrils are observed even at late stages in the process, side-by-side with the mature IB. Htt sequestration into the IB, which in neurons has been argued to be a cell-protective phenomenon, thus appears to saturate and over-power the cellular degradation systems and leaves cells vulnerable to further aggregation producing much smaller, potentially toxic, conformational protein species of which the fibrils may be comprised. We further found that exogenous delivery of the apical domain of the chaperonin subunit CCT1 to the cells via the cell medium reduced the aggregation propensity of mutant Htt exon 1 in general, and strongly reduced the occurrence of such late-stage fibrils in particular.

  View details for PubMedID 26350150

• Defining Hsp70 Subnetworks in Dengue Virus Replication Reveals Key Vulnerability in Flavivirus Infection. Cell Taguwa, S., Maringer, K., Li, X., Bernal-Rubio, D., Rauch, J. N., Gestwicki, J. E., Andino, R., Fernandez-Sesma, A., Frydman, J. 2015; 163 (5): 1108-1123

  View details for DOI 10.1016/j.cell.2015.10.046

  View details for PubMedID 26582131

• Local slowdown of translation by nonoptimal codons promotes nascent-chain recognition by SRP in vivo. Nature structural & molecular biology Pechmann, S., Chartron, J. W., Frydman, J. 2014; 21 (12): 1100-1105

  Abstract

  The genetic code allows most amino acids a choice of optimal and nonoptimal codons. We report that synonymous codon choice is tuned to promote interaction of nascent polypeptides with the signal recognition particle (SRP), which assists in protein translocation across membranes. Cotranslational recognition by the SRP in vivo is enhanced when mRNAs contain nonoptimal codon clusters 35-40 codons downstream of the SRP-binding site, the distance that spans the ribosomal polypeptide exit tunnel. A local translation slowdown upon ribosomal exit of SRP-binding elements in mRNAs containing these nonoptimal codon clusters is supported experimentally by ribosome profiling analyses in yeast. Modulation of local elongation rates through codon choice appears to kinetically enhance recognition by ribosome-associated factors. We propose that cotranslational regulation of nascent-chain fate may be a general constraint shaping codon usage in the genome.

  View details for DOI 10.1038/nsmb.2919

  View details for PubMedID 25420103

• Local slowdown of translation by nonoptimal codons promotes nascent-chain recognition by SRP in vivo NATURE STRUCTURAL & MOLECULAR BIOLOGY Pechmann, S., Chartron, J. W., Frydman, J. 2014; 21 (12): 1100-1105

  View details for DOI 10.1038/nsmb.2919

  View details for Web of Science ID 000345893300015

  View details for PubMedID 25420103

• The Structural Basis of Substrate Recognition by the Eukaryotic Chaperonin TRiC/CCT CELL Joachimiak, L. A., Walzthoeni, T., Liu, C. W., Aebersold, R., Frydman, J. 2014; 159 (5): 1042-1055

  View details for DOI 10.1016/j.cell.2014.10.042

  View details for Web of Science ID 000345506300017

• The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT. Cell Joachimiak, L. A., Walzthoeni, T., Liu, C. W., Aebersold, R., Frydman, J. 2014; 159 (5): 1042-1055

  Abstract

  The eukaryotic chaperonin TRiC (also called CCT) is the obligate chaperone for many essential proteins. TRiC is hetero-oligomeric, comprising two stacked rings of eight different subunits each. Subunit diversification from simpler archaeal chaperonins appears linked to proteome expansion. Here, we integrate structural, biophysical, and modeling approaches to identify the hitherto unknown substrate-binding site in TRiC and uncover the basis of substrate recognition. NMR and modeling provided a structural model of a chaperonin-substrate complex. Mutagenesis and crosslinking-mass spectrometry validated the identified substrate-binding interface and demonstrate that TRiC contacts full-length substrates combinatorially in a subunit-specific manner. The binding site of each subunit has a distinct, evolutionarily conserved pattern of polar and hydrophobic residues specifying recognition of discrete substrate motifs. The combinatorial recognition of polypeptides broadens the specificity of TRiC and may direct the topology of bound polypeptides along a productive folding trajectory, contributing to TRiC's unique ability to fold obligate substrates.

  View details for DOI 10.1016/j.cell.2014.10.042

  View details for PubMedID 25416944

  View details for PubMedCentralID PMC4298165

• Interplay between chaperones and protein disorder promotes the evolution of protein networks. PLoS computational biology Pechmann, S., Frydman, J. 2014; 10 (6)

  Abstract

  Evolution is driven by mutations, which lead to new protein functions but come at a cost to protein stability. Non-conservative substitutions are of interest in this regard because they may most profoundly affect both function and stability. Accordingly, organisms must balance the benefit of accepting advantageous substitutions with the possible cost of deleterious effects on protein folding and stability. We here examine factors that systematically promote non-conservative mutations at the proteome level. Intrinsically disordered regions in proteins play pivotal roles in protein interactions, but many questions regarding their evolution remain unanswered. Similarly, whether and how molecular chaperones, which have been shown to buffer destabilizing mutations in individual proteins, generally provide robustness during proteome evolution remains unclear. To this end, we introduce an evolutionary parameter λ that directly estimates the rate of non-conservative substitutions. Our analysis of λ in Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens sequences reveals how co- and post-translationally acting chaperones differentially promote non-conservative substitutions in their substrates, likely through buffering of their destabilizing effects. We further find that λ serves well to quantify the evolution of intrinsically disordered proteins even though the unstructured, thus generally variable regions in proteins are often flanked by very conserved sequences. Crucially, we show that both intrinsically disordered proteins and highly re-wired proteins in protein interaction networks, which have evolved new interactions and functions, exhibit a higher λ at the expense of enhanced chaperone assistance. Our findings thus highlight an intricate interplay of molecular chaperones and protein disorder in the evolvability of protein networks. Our results illuminate the role of chaperones in enabling protein evolution, and underline the importance of the cellular context and integrated approaches for understanding proteome evolution. We feel that the development of λ may be a valuable addition to the toolbox applied to understand the molecular basis of evolution.

  View details for DOI 10.1371/journal.pcbi.1003674

  View details for PubMedID 24968255

  View details for PubMedCentralID PMC4072544

• Sorting out the trash: the spatial nature of eukaryotic protein quality control CURRENT OPINION IN CELL BIOLOGY Sontag, E. M., Vonk, W. I., Frydman, J. 2014; 26: 139-146

  Abstract

  Failure to maintain protein homeostasis is associated with aggregation and cell death, and underies a growing list of pathologies including neurodegenerative diseases, aging, and cancer. Misfolded proteins can be toxic and interfere with normal cellular functions, particularly during proteotoxic stress. Accordingly, molecular chaperones, the ubiquitin-proteasome system (UPS) and autophagy together promote refolding or clearance of misfolded proteins. Here we discuss emerging evidence that the pathways of protein quality control (PQC) are intimately linked to cell architecture, and sequester proteins into spatially and functionally distinct PQC compartments. This sequestration serves a number of functions, including enhancing the efficiency of quality control; clearing the cellular milieu of potentially toxic species and facilitating asymmetric inheritance of damaged proteins to promote rejuvenation of daughter cells.

  View details for DOI 10.1016/j.ceb.2013.12.006

  View details for Web of Science ID 000331860400018

  View details for PubMedID 24463332

• Spatial sequestration of misfolded proteins by a dynamic chaperone pathway enhances cellular fitness during stress. Nature cell biology Escusa-Toret, S., Vonk, W. I., Frydman, J. 2013; 15 (10): 1231-1243

  Abstract

  The extensive links between proteotoxic stress, protein aggregation and pathologies ranging from ageing to neurodegeneration underscore the importance of understanding how cells manage protein misfolding. Using live-cell imaging, we determine the fate of stress-induced misfolded proteins from their initial appearance until their elimination. Upon denaturation, misfolded proteins are sequestered from the bulk cytoplasm into dynamic endoplasmic reticulum (ER)-associated puncta that move and coalesce into larger structures in an energy-dependent but cytoskeleton-independent manner. These puncta, which we name Q-bodies, concentrate different misfolded and stress-denatured proteins en route to degradation, but do not contain amyloid aggregates, which localize instead to the insoluble protein deposit compartment. Q-body formation and clearance depends on an intact cortical ER and a complex chaperone network that is affected by rapamycin and impaired during chronological ageing. Importantly, Q-body formation enhances cellular fitness during stress. We conclude that spatial sequestration of misfolded proteins in Q-bodies is an early quality control strategy occurring synchronously with degradation to clear the cytoplasm of potentially toxic species.

  View details for DOI 10.1038/ncb2838

  View details for PubMedID 24036477

• Spatial sequestration of misfolded proteins by a dynamic chaperone pathway enhances cellular fitness during stress NATURE CELL BIOLOGY Escusa-Toret, S., Vonk, W. I., Frydman, J. 2013; 15 (10): 1231-U253

  View details for DOI 10.1038/ncb2838

  View details for Web of Science ID 000325200300014

  View details for PubMedID 24036477

• Principles of cotranslational ubiquitination and quality control at the ribosome. Molecular cell Duttler, S., Pechmann, S., Frydman, J. 2013; 50 (3): 379-393

  Abstract

  Achieving efficient cotranslational folding of complex proteomes poses a challenge for eukaryotic cells. Nascent polypeptides that emerge vectorially from the ribosome often cannot fold stably and may be susceptible to misfolding and degradation. The extent to which nascent chains are subject to cotranslational quality control and degradation remains unclear. Here, we directly and quantitatively assess cotranslational ubiquitination and identify, at a systems level, the determinants and factors governing this process. Cotranslational ubiquitination occurs at very low levels and is carried out by a complex network of E3 ubiquitin ligases. Ribosome-associated chaperones and cotranslational folding protect the majority of nascent chains from premature quality control. Nonetheless, a number of nascent chains whose intrinsic properties hinder efficient cotranslational folding remain susceptible for cotranslational ubiquitination. We find that quality control at the ribosome is achieved through a tiered system wherein nascent polypeptides have a chance to fold before becoming accessible to ubiquitination.

  View details for DOI 10.1016/j.molcel.2013.03.010

  View details for PubMedID 23583075

• The Ribosome as a Hub for Protein Quality Control MOLECULAR CELL Pechmann, S., Willmund, F., Frydman, J. 2013; 49 (3): 411-421

  Abstract

  Cells face a constant challenge as they produce new proteins. The newly synthesized polypeptides must be folded properly to avoid aggregation. If proteins do misfold, they must be cleared to maintain a functional and healthy proteome. Recent work is revealing the complex mechanisms that work cotranslationally to ensure protein quality control during biogenesis at the ribosome. Indeed, the ribosome is emerging as a central hub in coordinating these processes, particularly in sensing the nature of the nascent protein chain, recruiting protein folding and translocation components, and integrating mRNA and nascent chain quality control. The tiered and complementary nature of these decision-making processes confers robustness and fidelity to protein homeostasis during protein synthesis.

  View details for DOI 10.1016/j.molcel.2013.01.020

  View details for Web of Science ID 000314792500004

  View details for PubMedID 23395271

  View details for PubMedCentralID PMC3593112

• Evolutionary conservation of codon optimality reveals hidden signatures of cotranslational folding. Nature structural & molecular biology Pechmann, S., Frydman, J. 2013; 20 (2): 237-243

  Abstract

  The choice of codons can influence local translation kinetics during protein synthesis. Whether codon preference is linked to cotranslational regulation of polypeptide folding remains unclear. Here, we derive a revised translational efficiency scale that incorporates the competition between tRNA supply and demand. Applying this scale to ten closely related yeast species, we uncover the evolutionary conservation of codon optimality in eukaryotes. This analysis reveals universal patterns of conserved optimal and nonoptimal codons, often in clusters, which associate with the secondary structure of the translated polypeptides independent of the levels of expression. Our analysis suggests an evolved function for codon optimality in regulating the rhythm of elongation to facilitate cotranslational polypeptide folding, beyond its previously proposed role of adapting to the cost of expression. These findings establish how mRNA sequences are generally under selection to optimize the cotranslational folding of corresponding polypeptides.

  View details for DOI 10.1038/nsmb.2466

  View details for PubMedID 23262490

  View details for PubMedCentralID PMC3565066

• Evolutionary conservation of codon optimality reveals hidden signatures of cotranslational folding NATURE STRUCTURAL & MOLECULAR BIOLOGY Pechmann, S., Frydman, J. 2013; 20 (2): 237-243

  View details for DOI 10.1038/nsmb.2466

  View details for Web of Science ID 000314623400017

  View details for PubMedID 23262490

• The Cotranslational Function of Ribosome-Associated Hsp70 in Eukaryotic Protein Homeostasis CELL Willmund, F., del Alamo, M., Pechmann, S., Chen, T., Albanese, V., Dammer, E. B., Peng, J., Frydman, J. 2013; 152 (1-2): 196-209

  Abstract

  In eukaryotic cells a molecular chaperone network associates with translating ribosomes, assisting the maturation of emerging nascent polypeptides. Hsp70 is perhaps the major eukaryotic ribosome-associated chaperone and the first reported to bind cotranslationally to nascent chains. However, little is known about the underlying principles and function of this interaction. Here, we use a sensitive and global approach to define the cotranslational substrate specificity of the yeast Hsp70 SSB. We find that SSB binds to a subset of nascent polypeptides whose intrinsic properties and slow translation rates hinder efficient cotranslational folding. The SSB-ribosome cycle and substrate recognition is modulated by its ribosome-bound cochaperone, RAC. Deletion of SSB leads to widespread aggregation of newly synthesized polypeptides. Thus, cotranslationally acting Hsp70 meets the challenge of folding the eukaryotic proteome by stabilizing its longer, more slowly translated, and aggregation-prone nascent polypeptides.

  View details for DOI 10.1016/j.cell.2012.12.001

  View details for Web of Science ID 000313719800017

  View details for PubMedID 23332755

  View details for PubMedCentralID PMC3553497

• TRiC's tricks inhibit huntingtin aggregation. eLife Shahmoradian, S. H., Galaz-Montoya, J. G., Schmid, M. F., Cong, Y., Ma, B., Spiess, C., Frydman, J., Ludtke, S. J., Chiu, W. 2013; 2

  Abstract

  In Huntington's disease, a mutated version of the huntingtin protein leads to cell death. Mutant huntingtin is known to aggregate, a process that can be inhibited by the eukaryotic chaperonin TRiC (TCP1-ring complex) in vitro and in vivo. A structural understanding of the genesis of aggregates and their modulation by cellular chaperones could facilitate the development of therapies but has been hindered by the heterogeneity of amyloid aggregates. Using cryo-electron microscopy (cryoEM) and single particle cryo-electron tomography (SPT) we characterize the growth of fibrillar aggregates of mutant huntingtin exon 1 containing an expanded polyglutamine tract with 51 residues (mhttQ51), and resolve 3-D structures of the chaperonin TRiC interacting with mhttQ51. We find that TRiC caps mhttQ51 fibril tips via the apical domains of its subunits, and also encapsulates smaller mhtt oligomers within its chamber. These two complementary mechanisms provide a structural description for TRiC's inhibition of mhttQ51 aggregation in vitro. DOI:http://dx.doi.org/10.7554/eLife.00710.001.

  View details for DOI 10.7554/eLife.00710

  View details for PubMedID 23853712

  View details for PubMedCentralID PMC3707056

• A Gradient of ATP Affinities Generates an Asymmetric Power Stroke Driving the Chaperonin TRIC/CCT Folding Cycle CELL REPORTS Reissmann, S., Joachimiak, L. A., Chen, B., Meyer, A. S., Nguyen, A., Frydman, J. 2012; 2 (4): 866-877

  Abstract

  The eukaryotic chaperonin TRiC/CCT uses ATP cycling to fold many essential proteins that other chaperones cannot fold. This 1 MDa hetero-oligomer consists of two identical stacked rings assembled from eight paralogous subunits, each containing a conserved ATP-binding domain. Here, we report a dramatic asymmetry in the ATP utilization cycle of this ring-shaped chaperonin, despite its apparently symmetric architecture. Only four of the eight different subunits bind ATP at physiological concentrations. ATP binding and hydrolysis by the low-affinity subunits is fully dispensable for TRiC function in vivo. The conserved nucleotide-binding hierarchy among TRiC subunits is evolutionarily modulated through differential nucleoside contacts. Strikingly, high- and low-affinity subunits are spatially segregated within two contiguous hemispheres in the ring, generating an asymmetric power stroke that drives the folding cycle. This unusual mode of ATP utilization likely serves to orchestrate a directional mechanism underlying TRiC/CCT's unique ability to fold complex eukaryotic proteins.

  View details for DOI 10.1016/j.celrep.2012.08.036

  View details for Web of Science ID 000314455600018

  View details for PubMedID 23041314

  View details for PubMedCentralID PMC3543868

• Systematic Functional Prioritization of Protein Posttranslational Modifications CELL Beltrao, P., Albanese, V., Kenner, L. R., Swaney, D. L., Burlingame, A., Villen, J., Lim, W. A., Fraser, J. S., Frydman, J., Krogan, N. J. 2012; 150 (2): 413-425

  Abstract

  Protein function is often regulated by posttranslational modifications (PTMs), and recent advances in mass spectrometry have resulted in an exponential increase in PTM identification. However, the functional significance of the vast majority of these modifications remains unknown. To address this problem, we compiled nearly 200,000 phosphorylation, acetylation, and ubiquitination sites from 11 eukaryotic species, including 2,500 newly identified ubiquitylation sites for Saccharomyces cerevisiae. We developed methods to prioritize the functional relevance of these PTMs by predicting those that likely participate in cross-regulatory events, regulate domain activity, or mediate protein-protein interactions. PTM conservation within domain families identifies regulatory "hot spots" that overlap with functionally important regions, a concept that we experimentally validated on the HSP70 domain family. Finally, our analysis of the evolution of PTM regulation highlights potential routes for neutral drift in regulatory interactions and suggests that only a fraction of modification sites are likely to have a significant biological role.

  View details for DOI 10.1016/j.cell.2012.05.036

  View details for Web of Science ID 000306595700019

  View details for PubMedID 22817900

• The Molecular Architecture of the Eukaryotic Chaperonin TRiC/CCT STRUCTURE Leitner, A., Joachimiak, L. A., Bracher, A., Moenkemeyer, L., Walzthoeni, T., Chen, B., Pechmann, S., Holmes, S., Cong, Y., Ma, B., Ludtke, S., Chiu, W., Hartl, F. U., Aebersold, R., Frydman, J. 2012; 20 (5): 814-825

  Abstract

  TRiC/CCT is a highly conserved and essential chaperonin that uses ATP cycling to facilitate folding of approximately 10% of the eukaryotic proteome. This 1 MDa hetero-oligomeric complex consists of two stacked rings of eight paralogous subunits each. Previously proposed TRiC models differ substantially in their subunit arrangements and ring register. Here, we integrate chemical crosslinking, mass spectrometry, and combinatorial modeling to reveal the definitive subunit arrangement of TRiC. In vivo disulfide mapping provided additional validation for the crosslinking-derived arrangement as the definitive TRiC topology. This subunit arrangement allowed the refinement of a structural model using existing X-ray diffraction data. The structure described here explains all available crosslink experiments, provides a rationale for previously unexplained structural features, and reveals a surprising asymmetry of charges within the chaperonin folding chamber.

  View details for DOI 10.1016/j.str.2012.03.007

  View details for Web of Science ID 000304214400008

  View details for PubMedID 22503819

  View details for PubMedCentralID PMC3350567

• Cellular Strategies of Protein Quality Control COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Chen, B., Retzlaff, M., Roos, T., Frydman, J. 2011; 3 (8)

  Abstract

  Eukaryotic cells must contend with a continuous stream of misfolded proteins that compromise the cellular protein homeostasis balance and jeopardize cell viability. An elaborate network of molecular chaperones and protein degradation factors continually monitor and maintain the integrity of the proteome. Cellular protein quality control relies on three distinct yet interconnected strategies whereby misfolded proteins can either be refolded, degraded, or delivered to distinct quality control compartments that sequester potentially harmful misfolded species. Molecular chaperones play a critical role in determining the fate of misfolded proteins in the cell. Here, we discuss the spatial and temporal organization of cellular quality control strategies and their implications for human diseases linked to protein misfolding and aggregation.

  View details for DOI 10.1101/cshperspect.a004374

  View details for Web of Science ID 000294124200004

  View details for PubMedID 21746797

• Defining the Specificity of Cotranslationally Acting Chaperones by Systematic Analysis of mRNAs Associated with Ribosome-Nascent Chain Complexes PLOS BIOLOGY del Alamo, M., Hogan, D. J., Pechmann, S., Albanese, V., Brown, P. O., Frydman, J. 2011; 9 (7)

  Abstract

  Polypeptides exiting the ribosome must fold and assemble in the crowded environment of the cell. Chaperones and other protein homeostasis factors interact with newly translated polypeptides to facilitate their folding and correct localization. Despite the extensive efforts, little is known about the specificity of the chaperones and other factors that bind nascent polypeptides. To address this question we present an approach that systematically identifies cotranslational chaperone substrates through the mRNAs associated with ribosome-nascent chain-chaperone complexes. We here focused on two Saccharomyces cerevisiae chaperones: the Signal Recognition Particle (SRP), which acts cotranslationally to target proteins to the ER, and the Nascent chain Associated Complex (NAC), whose function has been elusive. Our results provide new insights into SRP selectivity and reveal that NAC is a general cotranslational chaperone. We found surprising differential substrate specificity for the three subunits of NAC, which appear to recognize distinct features within nascent chains. Our results also revealed a partial overlap between the sets of nascent polypeptides that interact with NAC and SRP, respectively, and showed that NAC modulates SRP specificity and fidelity in vivo. These findings give us new insight into the dynamic interplay of chaperones acting on nascent chains. The strategy we used should be generally applicable to mapping the specificity, interplay, and dynamics of the cotranslational protein homeostasis network.

  View details for DOI 10.1371/journal.pbio.1001100

  View details for Web of Science ID 000293219800007

  View details for PubMedID 21765803

  View details for PubMedCentralID PMC3134442

• Dual Action of ATP Hydrolysis Couples Lid Closure to Substrate Release into the Group II Chaperonin Chamber CELL Douglas, N. R., Reissmann, S., Zhang, J., Chen, B., Jakana, J., Kumar, R., Chiu, W., Frydman, J. 2011; 144 (2): 240-252

  Abstract

  Group II chaperonins are ATP-dependent ring-shaped complexes that bind nonnative polypeptides and facilitate protein folding in archaea and eukaryotes. A built-in lid encapsulates substrate proteins within the central chaperonin chamber. Here, we describe the fate of the substrate during the nucleotide cycle of group II chaperonins. The chaperonin substrate-binding sites are exposed, and the lid is open in both the ATP-free and ATP-bound prehydrolysis states. ATP hydrolysis has a dual function in the folding cycle, triggering both lid closure and substrate release into the central chamber. Notably, substrate release can occur in the absence of a lid, and lid closure can occur without substrate release. However, productive folding requires both events, so that the polypeptide is released into the confined space of the closed chamber where it folds. Our results show that ATP hydrolysis coordinates the structural and functional determinants that trigger productive folding.

  View details for DOI 10.1016/j.cell.2010.12.017

  View details for Web of Science ID 000286459900009

  View details for PubMedID 21241893

  View details for PubMedCentralID PMC3055171

• A ribosome-anchored chaperone network that facilitates eukaryotic ribosome biogenesis JOURNAL OF CELL BIOLOGY Albanese, V., Reissmann, S., Frydman, J. 2010; 189 (1): 69-U105

  Abstract

  Molecular chaperones assist cellular protein folding as well as oligomeric complex assembly. In eukaryotic cells, several chaperones termed chaperones linked to protein synthesis (CLIPS) are transcriptionally and physically linked to ribosomes and are implicated in protein biosynthesis. In this study, we show that a CLIPS network comprising two ribosome-anchored J-proteins, Jjj1 and Zuo1, function together with their partner Hsp70 proteins to mediate the biogenesis of ribosomes themselves. Jjj1 and Zuo1 have overlapping but distinct functions in this complex process involving the coordinated assembly and remodeling of dozens of proteins on the ribosomal RNA (rRNA). Both Jjj1 and Zuo1 associate with nuclear 60S ribosomal biogenesis intermediates and play an important role in nuclear rRNA processing, leading to mature 25S rRNA. In addition, Zuo1, acting together with its Hsp70 partner, SSB (stress 70 B), also participates in maturation of the 35S rRNA. Our results demonstrate that, in addition to their known cytoplasmic roles in de novo protein folding, some ribosome-anchored CLIPS chaperones play a critical role in nuclear steps of ribosome biogenesis.

  View details for DOI 10.1083/jcb.201001054

  View details for Web of Science ID 000276336400009

  View details for PubMedID 20368619

  View details for PubMedCentralID PMC2854368

• Mechanism of folding chamber closure in a group II chaperonin NATURE Zhang, J., Baker, M. L., Schroeder, G. F., Douglas, N. R., Reissmann, S., Jakana, J., Dougherty, M., Fu, C. J., Levitt, M., Ludtke, S. J., Frydman, J., Chiu, W. 2010; 463 (7279): 379-U130

  Abstract

  Group II chaperonins are essential mediators of cellular protein folding in eukaryotes and archaea. These oligomeric protein machines, approximately 1 megadalton, consist of two back-to-back rings encompassing a central cavity that accommodates polypeptide substrates. Chaperonin-mediated protein folding is critically dependent on the closure of a built-in lid, which is triggered by ATP hydrolysis. The structural rearrangements and molecular events leading to lid closure are still unknown. Here we report four single particle cryo-electron microscopy (cryo-EM) structures of Mm-cpn, an archaeal group II chaperonin, in the nucleotide-free (open) and nucleotide-induced (closed) states. The 4.3 A resolution of the closed conformation allowed building of the first ever atomic model directly from the single particle cryo-EM density map, in which we were able to visualize the nucleotide and more than 70% of the side chains. The model of the open conformation was obtained by using the deformable elastic network modelling with the 8 A resolution open-state cryo-EM density restraints. Together, the open and closed structures show how local conformational changes triggered by ATP hydrolysis lead to an alteration of intersubunit contacts within and across the rings, ultimately causing a rocking motion that closes the ring. Our analyses show that there is an intricate and unforeseen set of interactions controlling allosteric communication and inter-ring signalling, driving the conformational cycle of group II chaperonins. Beyond this, we anticipate that our methodology of combining single particle cryo-EM and computational modelling will become a powerful tool in the determination of atomic details involved in the dynamic processes of macromolecular machines in solution.

  View details for DOI 10.1038/nature08701

  View details for Web of Science ID 000273748100049

  View details for PubMedID 20090755

  View details for PubMedCentralID PMC2834796

• The chaperonin TRiC blocks a huntingtin sequence element that promotes the conformational switch to aggregation NATURE STRUCTURAL & MOLECULAR BIOLOGY Tam, S., Spiess, C., Auyeung, W., Joachimiak, L., Chen, B., Poirier, M. A., Frydman, J. 2009; 16 (12): 1279-U98

  Abstract

  Aggregation of proteins containing polyglutamine (polyQ) expansions characterizes many neurodegenerative disorders, including Huntington's disease. Molecular chaperones modulate the aggregation and toxicity of the huntingtin (Htt) protein by an ill-defined mechanism. Here we determine how the chaperonin TRiC suppresses Htt aggregation. Unexpectedly, TRiC does not physically block the polyQ tract itself, but rather sequesters a short Htt sequence element, N-terminal to the polyQ tract, that promotes the amyloidogenic conformation. The residues of this element essential for rapid Htt aggregation are directly bound by TRiC. Our findings illustrate how molecular chaperones, which recognize hydrophobic determinants, can prevent aggregation of polar polyQ tracts associated with neurodegenerative diseases. The observation that short endogenous sequence elements can accelerate the switch of polyQ tracts to an amyloidogenic conformation provides a novel target for therapeutic strategies.

  View details for DOI 10.1038/nsmb.1700

  View details for Web of Science ID 000272609200016

  View details for PubMedID 19915590

  View details for PubMedCentralID PMC2788664

• The Hsp90 mosaic: a picture emerges NATURE STRUCTURAL & MOLECULAR BIOLOGY Mayer, M. P., Prodromou, C., Frydman, J. 2009; 16 (1): 2-6

  View details for DOI 10.1038/nsmb0109-2

  View details for Web of Science ID 000262267600002

  View details for PubMedID 19125165

• Defining the TRiC/CCT interactome links chaperonin function to stabilization of newly made proteins with complex topologies NATURE STRUCTURAL & MOLECULAR BIOLOGY Yam, A. Y., Xia, Y., Lin, H. J., Burlingame, A., Gerstein, M., Frydman, J. 2008; 15 (12): 1255-1262

  Abstract

  Folding within the crowded cellular milieu often requires assistance from molecular chaperones that prevent inappropriate interactions leading to aggregation and toxicity. The contribution of individual chaperones to folding the proteome remains elusive. Here we demonstrate that the eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) has broad binding specificity in vitro, similar to the prokaryotic chaperonin GroEL. However, in vivo, TRiC substrate selection is not based solely on intrinsic determinants; instead, specificity is dictated by factors present during protein biogenesis. The identification of cellular substrates revealed that TRiC interacts with folding intermediates of a subset of structurally and functionally diverse polypeptides. Bioinformatics analysis revealed an enrichment in multidomain proteins and regions of beta-strand propensity that are predicted to be slow folding and aggregation prone. Thus, TRiC may have evolved to protect complex protein topologies within its central cavity during biosynthesis and folding.

  View details for DOI 10.1038/nsmb.1515

  View details for Web of Science ID 000261383900011

  View details for PubMedID 19011634

  View details for PubMedCentralID PMC2658641

• Misfolded proteins partition between two distinct quality control compartments NATURE Kaganovich, D., Kopito, R., Frydman, J. 2008; 454 (7208): 1088-U36

  Abstract

  The accumulation of misfolded proteins in intracellular amyloid inclusions, typical of many neurodegenerative disorders including Huntington's and prion disease, is thought to occur after failure of the cellular protein quality control mechanisms. Here we examine the formation of misfolded protein inclusions in the eukaryotic cytosol of yeast and mammalian cell culture models. We identify two intracellular compartments for the sequestration of misfolded cytosolic proteins. Partition of quality control substrates to either compartment seems to depend on their ubiquitination status and aggregation state. Soluble ubiquitinated misfolded proteins accumulate in a juxtanuclear compartment where proteasomes are concentrated. In contrast, terminally aggregated proteins are sequestered in a perivacuolar inclusion. Notably, disease-associated Huntingtin and prion proteins are preferentially directed to the perivacuolar compartment. Enhancing ubiquitination of a prion protein suffices to promote its delivery to the juxtanuclear inclusion. Our findings provide a framework for understanding the preferential accumulation of amyloidogenic proteins in inclusions linked to human disease.

  View details for DOI 10.1038/nature07195

  View details for Web of Science ID 000258719600031

  View details for PubMedID 18756251

  View details for PubMedCentralID PMC2746971

• Mechanism of lid closure in the eukaryotic chaperonin TRiC/CCT NATURE STRUCTURAL & MOLECULAR BIOLOGY Booth, C. R., Meyer, A. S., Cong, Y., Topf, M., Sali, A., Ludtke, S. J., Chiu, W., Frydman, J. 2008; 15 (7): 746-753

  Abstract

  All chaperonins mediate ATP-dependent polypeptide folding by confining substrates within a central chamber. Intriguingly, the eukaryotic chaperonin TRiC (also called CCT) uses a built-in lid to close the chamber, whereas prokaryotic chaperonins use a detachable lid. Here we determine the mechanism of lid closure in TRiC using single-particle cryo-EM and comparative protein modeling. Comparison of TRiC in its open, nucleotide-free, and closed, nucleotide-induced states reveals that the interdomain motions leading to lid closure in TRiC are radically different from those of prokaryotic chaperonins, despite their overall structural similarity. We propose that domain movements in TRiC are coordinated through unique interdomain contacts within each subunit and, further, these contacts are absent in prokaryotic chaperonins. Our findings show how different mechanical switches can evolve from a common structural framework through modification of allosteric networks.

  View details for DOI 10.1038/nsmb.1436

  View details for Web of Science ID 000257412500018

  View details for PubMedID 18536725

  View details for PubMedCentralID PMC2546500

• Diverse cellular functions of the Hsp90 molecular chaperone uncovered using systems approaches CELL McClellan, A. J., Xia, Y., Deutschbauer, A. M., Davis, R. W., Gerstein, M., Frydman, J. 2007; 131 (1): 121-135

  Abstract

  A comprehensive understanding of the cellular functions of the Hsp90 molecular chaperone has remained elusive. Although Hsp90 is essential, highly abundant under normal conditions, and further induced by environmental stress, only a limited number of Hsp90 "clients" have been identified. To define Hsp90 function, a panel of genome-wide chemical-genetic screens in Saccharomyces cerevisiae were combined with bioinformatic analyses. This approach identified several unanticipated functions of Hsp90 under normal conditions and in response to stress. Under normal growth conditions, Hsp90 plays a major role in various aspects of the secretory pathway and cellular transport; during environmental stress, Hsp90 is required for the cell cycle, meiosis, and cytokinesis. Importantly, biochemical and cell biological analyses validated several of these Hsp90-dependent functions, highlighting the potential of our integrated global approach to uncover chaperone functions in the cell.

  View details for DOI 10.1016/j.cell.2007.07.036

  View details for Web of Science ID 000249934700016

  View details for PubMedID 17923092

• Essential function of the built-in lid in the allosteric regulation of eukaryotic and archaeal chaperonins NATURE STRUCTURAL & MOLECULAR BIOLOGY Reissmann, S., Parnot, C., Booth, C. R., Chiu, W., Frydman, J. 2007; 14 (5): 432-440

  Abstract

  Chaperonins are allosteric double-ring ATPases that mediate cellular protein folding. ATP binding and hydrolysis control opening and closing of the central chaperonin chamber, which transiently provides a protected environment for protein folding. During evolution, two strategies to close the chaperonin chamber have emerged. Archaeal and eukaryotic group II chaperonins contain a built-in lid, whereas bacterial chaperonins use a ring-shaped cofactor as a detachable lid. Here we show that the built-in lid is an allosteric regulator of group II chaperonins, which helps synchronize the subunits within one ring and, to our surprise, also influences inter-ring communication. The lid is dispensable for substrate binding and ATP hydrolysis, but is required for productive substrate folding. These regulatory functions of the lid may serve to allow the symmetrical chaperonins to function as 'two-stroke' motors and may also provide a timer for substrate encapsulation within the closed chamber.

  View details for DOI 10.1038/nsmb1236

  View details for Web of Science ID 000246187400017

  View details for PubMedID 17460696

  View details for PubMedCentralID PMC3339572

• Evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance GENES & DEVELOPMENT Geller, R., Vignuzzi, M., Andino, R., Frydman, J. 2007; 21 (2): 195-205

  Abstract

  The genome diversity of RNA viruses allows for rapid adaptation to a wide variety of adverse conditions. Accordingly, viruses can escape inhibition by most antiviral compounds targeting either viral or host factors. Here we exploited the capacity of RNA viruses for rapid adaptation to explore the evolutionary constraints of chaperone-mediated protein folding. We hypothesized that inhibiting a host molecular chaperone required for folding of a viral protein would force the virus to evolve an alternate folding strategy. We identified the chaperone Hsp90 as an essential factor for folding and maturation of picornavirus capsid proteins. Pharmacological inhibition of Hsp90 impaired the replication of poliovirus, rhinovirus, and coxsackievirus in cell culture. Strikingly, anti-Hsp90 treatment did not yield drug-resistant viruses, suggesting that the complexity of capsid folding precludes the emergence of alternate folding pathways. These results reveal tight evolutionary constraints on chaperone-mediated protein folding, which may be exploited for viral inhibition in vivo. Indeed, Hsp90 inhibitors drastically reduced poliovirus replication in infected animals without the emergence of drug-resistant escape mutants. We propose that targeting folding of viral proteins may provide a general antiviral strategy that is refractory to development of drug resistance.

  View details for DOI 10.1101/gad.1505307

  View details for Web of Science ID 000243565400007

  View details for PubMedID 17234885

  View details for PubMedCentralID PMC1770902

• Identification of the TRiC/CCT substrate binding sites uncovers the function of subunit diversity in eukaryotic MOLECULAR CELL Spiess, C., Miller, E. J., McClellan, A. J., Frydman, J. 2006; 24 (1): 25-37

  Abstract

  The ring-shaped hetero-oligomeric chaperonin TRiC/CCT uses ATP to fold a diverse subset of eukaryotic proteins. To define the basis of TRiC/CCT substrate recognition, we mapped the chaperonin interactions with the VHL tumor suppressor. VHL has two well-defined TRiC binding determinants. Each determinant contacts a specific subset of chaperonin subunits, indicating that TRiC paralogs exhibit distinct but overlapping specificities. The substrate binding site in these subunits localizes to a helical region in the apical domains that is structurally equivalent to that of bacterial chaperonins. Transferring the distal portion of helix 11 between TRiC subunits suffices to transfer specificity for a given substrate motif. We conclude that the architecture of the substrate binding domain is evolutionarily conserved among eukaryotic and bacterial chaperonins. The unique combination of specificity and plasticity in TRiC substrate binding may diversify the range of motifs recognized by this chaperonin and contribute to its unique ability to fold eukaryotic proteins.

  View details for DOI 10.1016/j.molcel.2006.09.003

  View details for Web of Science ID 000241407100003

  View details for PubMedID 17018290

  View details for PubMedCentralID PMC3339573

• The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions NATURE CELL BIOLOGY Tam, S., Geller, R., Spiess, C., Frydman, J. 2006; 8 (10): 1155-U211

  Abstract

  Misfolding and aggregation of proteins containing expanded polyglutamine repeats underlie Huntington's disease and other neurodegenerative disorders. Here, we show that the hetero-oligomeric chaperonin TRiC (also known as CCT) physically interacts with polyglutamine-expanded variants of huntingtin (Htt) and effectively inhibits their aggregation. Depletion of TRiC enhances polyglutamine aggregation in yeast and mammalian cells. Conversely, overexpression of a single TRiC subunit, CCT1, is sufficient to remodel Htt-aggregate morphology in vivo and in vitro, and reduces Htt-induced toxicity in neuronal cells. Because TRiC acts during de novo protein biogenesis, this chaperonin may have an early role preventing Htt access to pathogenic conformations. Based on the specificity of the Htt-CCT1 interaction, the CCT1 substrate-binding domain may provide a versatile scaffold for therapeutic inhibitors of neurodegenerative disease.

  View details for DOI 10.1038/ncb1477

  View details for Web of Science ID 000241395300021

  View details for PubMedID 16980959

  View details for PubMedCentralID PMC2829982

• Systems analyses reveal two chaperone networks with distinct functions in eukaryotic cells CELL Albanese, V., Yam, A. Y., Baughman, J., Parnot, C., Frydman, J. 2006; 124 (1): 75-88

  Abstract

  Molecular chaperones assist the folding of newly translated and stress-denatured proteins. In prokaryotes, overlapping sets of chaperones mediate both processes. In contrast, we find that eukaryotes evolved distinct chaperone networks to carry out these functions. Genomic and functional analyses indicate that in addition to stress-inducible chaperones that protect the cellular proteome from stress, eukaryotes contain a stress-repressed chaperone network that is dedicated to protein biogenesis. These stress-repressed chaperones are transcriptionally, functionally, and physically linked to the translational apparatus and associate with nascent polypeptides emerging from the ribosome. Consistent with a function in de novo protein folding, impairment of the translation-linked chaperone network renders cells sensitive to misfolding in the context of protein synthesis but not in the context of environmental stress. The emergence of a translation-linked chaperone network likely underlies the elaborate cotranslational folding process necessary for the evolution of larger multidomain proteins characteristic of eukaryotic cells.

  View details for DOI 10.1016/j.cell.2005.11.039

  View details for Web of Science ID 000234969600011

  View details for PubMedID 16413483

• Protein quality control: chaperones culling corrupt conformations NATURE CELL BIOLOGY McClellan, A. J., Tam, S., Kaganovich, D., Frydman, J. 2005; 7 (8): 736-741

  Abstract

  Achieving the correct balance between folding and degradation of misfolded proteins is critical for cell viability. The importance of defining the mechanisms and factors that mediate cytoplasmic quality control is underscored by the growing list of diseases associated with protein misfolding and aggregation. Molecular chaperones assist protein folding and also facilitate degradation of misfolded polypeptides by the ubiquitin-proteasome system. Here we discuss emerging links between folding and degradation machineries and highlight challenges for future research.

  View details for DOI 10.1038/ncb0805-736

  View details for Web of Science ID 000230881500003

  View details for PubMedID 16056264

• Folding and quality control of the VHL tumor suppressor proceed through distinct chaperone pathways CELL McClellan, A. J., Scott, M. D., Frydman, J. 2005; 121 (5): 739-748

  Abstract

  The mechanisms by which molecular chaperones assist quality control of cytosolic proteins are poorly understood. Analysis of the chaperone requirements for degradation of misfolded variants of a cytosolic protein, the VHL tumor suppressor, reveals that distinct chaperone pathways mediate its folding and quality control. While both folding and degradation of VHL require Hsp70, the chaperonin TRiC is essential for folding but is dispensable for degradation. Conversely, the chaperone Hsp90 neither participates in VHL folding nor is required to maintain misfolded VHL solubility but is essential for its degradation. The cochaperone HOP/Sti1p also participates in VHL quality control and may direct the triage decision by bridging the Hsp70-Hsp90 interaction. Our finding that a distinct chaperone complex is uniquely required for quality control provides evidence for active and specific chaperone participation in triage decisions and suggests that a hierarchy of chaperone interactions can control the alternate fates of a cytosolic protein.

  View details for DOI 10.1016/j.cell.2005.03.024

  View details for Web of Science ID 000229658000012

  View details for PubMedID 15935760

• Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets TRENDS IN CELL BIOLOGY Spiess, C., Meyer, A. S., Reissmann, S., Frydman, J. 2004; 14 (11): 598-604

  Abstract

  Chaperonins are key components of the cellular chaperone machinery. These large, cylindrical complexes contain a central cavity that binds to unfolded polypeptides and sequesters them from the cellular environment. Substrate folding then occurs in this central cavity in an ATP-dependent manner. The eukaryotic chaperonin TCP-1 ring complex (TRiC, also called CCT) is indispensable for cell survival because the folding of an essential subset of cytosolic proteins requires TRiC, and this function cannot be substituted by other chaperones. This specificity indicates that TRiC has evolved structural and mechanistic features that distinguish it from other chaperones. Although knowledge of this unique complex is in its infancy, we review recent advances that open the way to understanding the secrets of its folding chamber.

  View details for DOI 10.1016/j.tcb.2004.09.015

  View details for Web of Science ID 000225270700003

  View details for PubMedID 15519848

  View details for PubMedCentralID PMC2812437

• Tumorigenic mutations in VHL disrupt folding in vivo by interfering with chaperonin binding MOLECULAR CELL Feldman, D. E., Spiess, C., Howard, D. E., Frydman, J. 2003; 12 (5): 1213-1224

  Abstract

  The eukaryotic chaperonin TRiC/CCT mediates folding of an essential subset of newly synthesized proteins, including the tumor suppressor VHL. Here we show that chaperonin binding is specified by two short hydrophobic beta strands in VHL that, upon folding, become buried within the native structure. These TRiC binding determinants are disrupted by tumor-causing point mutations that interfere with chaperonin association and lead to misfolding. Strikingly, while unable to fold correctly in vivo, some of these VHL mutants can reach the native state when refolded in a chaperonin-independent manner. The specificity of TRiC/CCT for extended hydrophobic beta strands may help explain its role in folding aggregation-prone polypeptides. Our findings reveal a class of disease-causing mutations that inactivate protein function by disrupting chaperone-mediated folding in vivo.

  View details for Web of Science ID 000186764700017

  View details for PubMedID 14636579

• Closing the folding chamber of the eukaryotic chaperonin requires the transition state of ATP hydrolysis CELL Meyer, A. S., Gillespie, J. R., Walther, D., Millet, I. S., Doniach, S., Frydman, J. 2003; 113 (3): 369-381

  Abstract

  Chaperonins use ATPase cycling to promote conformational changes leading to protein folding. The prokaryotic chaperonin GroEL requires a cofactor, GroES, which serves as a "lid" enclosing substrates in the central cavity and confers an asymmetry on GroEL required for cooperative transitions driving the reaction. The eukaryotic chaperonin TRiC/CCT does not have such a cofactor but appears to have a "built-in" lid. Whether this seemingly symmetric chaperonin also operates through an asymmetric cycle is unclear. We show that unlike GroEL, TRiC does not close its lid upon nucleotide binding, but instead responds to the trigonal-bipyramidal transition state of ATP hydrolysis. Further, nucleotide analogs inducing this transition state confer an asymmetric conformation on TRiC. Similar to GroEL, lid closure in TRiC confines the substrates in the cavity and is essential for folding. Understanding the distinct mechanisms governing eukaryotic and bacterial chaperonin function may reveal how TRiC has evolved to fold specific eukaryotic proteins.

  View details for Web of Science ID 000182640800011

  View details for PubMedID 12732144

• Where chaperones and nascent polypeptides meet NATURE STRUCTURAL BIOLOGY Albanese, V., Frydman, J. 2002; 9 (10): 716-718

  View details for DOI 10.1038/nsb1002-716

  View details for Web of Science ID 000178242300003

  View details for PubMedID 12352951

• Molecular chaperones and the art of recognizing a lost cause NATURE CELL BIOLOGY McClellan, A. J., Frydman, J. 2001; 3 (2): E51-E53

  View details for Web of Science ID 000166793000007

  View details for PubMedID 11175763

• Folding of newly translated proteins in vivo: The role of molecular chaperones ANNUAL REVIEW OF BIOCHEMISTRY Frydman, J. 2001; 70: 603-647

  Abstract

  Recent years have witnessed dramatic advances in our understanding of how newly translated proteins fold in the cell and the contribution of molecular chaperones to this process. Folding in the cell must be achieved in a highly crowded macromolecular environment, in which release of nonnative polypeptides into the cytosolic solution might lead to formation of potentially toxic aggregates. Here I review the cellular mechanisms that ensure efficient folding of newly translated proteins in vivo. De novo protein folding appears to occur in a protected environment created by a highly processive chaperone machinery that is directly coupled to translation. Genetic and biochemical analysis shows that several distinct chaperone systems, including Hsp70 and the cylindrical chaperonins, assist the folding of proteins upon translation in the cytosol of both prokaryotic and eukaryotic cells. The cellular chaperone machinery is specifically recruited to bind to ribosomes and protects nascent chains and folding intermediates from nonproductive interactions. In addition, initiation of folding during translation appears to be important for efficient folding of multidomain proteins.

  View details for Web of Science ID 000170012100018

  View details for PubMedID 11395418

• The interaction of the chaperonin tailless complex polypeptide 1 (TCP1) ring complex (TRiC) with ribosome-bound nascent chains examined using photo-cross-linking JOURNAL OF CELL BIOLOGY McCallum, C. D., Do, H., Johnson, A. E., Frydman, J. 2000; 149 (3): 591-601

  Abstract

  The eukaryotic chaperonin tailless complex polypeptide 1 (TCP1) ring complex (TRiC) (also called chaperonin containing TCP1 [CCT]) is a hetero-oligomeric complex that facilitates the proper folding of many cellular proteins. To better understand the manner in which TRiC interacts with newly translated polypeptides, we examined its association with nascent chains using a photo-cross-linking approach. To this end, a series of ribosome-bound nascent chains of defined lengths was prepared using truncated mRNAs. Photoactivatable probes were incorporated into these (35)S- labeled nascent chains during translation. Upon photolysis, TRiC was cross-linked to ribosome-bound polypeptides exposing at least 50-90 amino acids outside the ribosomal exit channel, indicating that the chaperonin associates with much shorter nascent chains than indicated by previous studies. Cross-links were observed for nascent chains of the cytosolic proteins actin, luciferase, and enolase, but not to ribosome-bound preprolactin. The pattern of cross-links became more complex as the nascent chain increased in length. These results suggest a chain length-dependent increase in the number of TRiC subunits involved in the interaction that is consistent with the idea that the substrate participates in subunit-specific contacts with the chaperonin. Both ribosome isolation by centrifugation through sucrose cushions and immunoprecipitation with anti-puromycin antibodies demonstrated that the photoadducts form on ribosome-bound polypeptides. Our results indicate that TRiC/CCT associates with the translating polypeptide shortly after it emerges from the ribosome and suggest a close association between the chaperonin and the translational apparatus.

  View details for Web of Science ID 000086831700008

  View details for PubMedID 10791973

  View details for PubMedCentralID PMC2174856

• Formation of the VHL-elongin BC tumor suppressor complex is mediated by the chaperonin TRiC MOLECULAR CELL Feldman, D. E., Thulasiraman, V., Ferreyra, R. G., Frydman, J. 1999; 4 (6): 1051-1061

  Abstract

  von Hippel-Lindau (VHL) disease is caused by loss of function of the VHL tumor suppressor protein. Here, we demonstrate that the folding and assembly of VHL into a complex with its partner proteins, elongin B and elongin C (herein, elongin BC), is directly mediated by the chaperonin TRiC/CCT. Association of VHL with TRiC is required for formation of the VHL-elongin BC complex. A 55-amino acid domain of VHL is both necessary and sufficient for binding to TRiC. Importantly, mutation or deletion of this domain is associated with VHL disease. We identified two mutations that disrupt the normal interaction with TRiC and impair VHL folding. Our results define a novel role for TRiC in mediating oligomerization and suggest that inactivating mutations can impair polypeptide function by interfering with chaperone-mediated folding.

  View details for Web of Science ID 000084485900016

  View details for PubMedID 10635329

• Co-translational domain folding as the structural basis for the rapid de novo folding of firefly luciferase NATURE STRUCTURAL BIOLOGY Frydman, J., Erdjument-Bromage, H., Tempst, P., Hartl, F. U. 1999; 6 (7): 697-705

  Abstract

  The 62 kDa protein firefly luciferase folds very rapidly upon translation on eukaryotic ribosomes. In contrast, the chaperone-mediated refolding of chemically denatured luciferase occurs with significantly slower kinetics. Here we investigate the structural basis for this difference in folding kinetics. We find that an N-terminal domain of luciferase (residues 1-190) folds co-translationally, followed by rapid formation of native protein upon release of the full-length polypeptide from the ribosome. In contrast sequential domain formation is not observed during in vitro refolding. Discrete unfolding steps, corresponding to domain unfolding, are however observed when the native protein is exposed to increasing concentrations of denaturant. Thus, the co-translational folding reaction bears more similarities to the unfolding reaction than to refolding from denaturant. We propose that co-translational domain formation avoids intramolecular misfolding and may be critical in the folding of multidomain proteins.

  View details for Web of Science ID 000081217500026

  View details for PubMedID 10404229

• In vivo newly translated polypeptides are sequestered in a protected folding environment EMBO JOURNAL Thulasiraman, V., Yang, C. F., Frydman, J. 1999; 18 (1): 85-95

  Abstract

  Molecular chaperones play a fundamental role in cellular protein folding. Using intact mammalian cells we examined the contribution of cytosolic chaperones to de novo folding. A large fraction of newly translated polypeptides associate transiently with Hsc70 and the chaperonin TRiC/CCT during their biogenesis. The substrate repertoire observed for Hsc70 and TRiC is not identical: Hsc70 interacts with a wide spectrum of polypeptides larger than 20 kDa, while TRiC associates with a diverse set of proteins between 30 and 60 kDa. Overexpression of a bacterial chaperonin 'trap' that irreversibly captures unfolded polypeptides did not interrupt the productive folding pathway. The trap was unable to bind newly translated polypeptides, indicating that folding in mammalian cells occurs without the release of non-native folding intermediates into the bulk cytosol. We conclude that de novo protein folding occurs in a protected environment created by a highly processive chaperone machinery and is directly coupled to translation.

  View details for Web of Science ID 000077875400010

  View details for PubMedID 9878053

• Principles of chaperone-assisted protein folding: Differences between in vitro and in vivo mechanisms SCIENCE Frydman, J., Hartl, F. U. 1996; 272 (5267): 1497-1502

  Abstract

  Molecular chaperones in the eukaryotic cytosol were shown to interact differently with chemically denatured proteins and their newly translated counterparts. During refolding from denaturant, actin partitioned freely between 70-kilodalton heat shock protein, the bulk cytosol, and the chaperonin TCP1-ring complex. In contrast, during cell-free translation, the chaperones were recruited to the elongating polypeptide and protected it from exposure to the bulk cytosol during folding. Posttranslational cycling between chaperone-bound and free states was observed with subunits of oligomeric proteins and with aberrant polypeptides; this cycling allowed the subunits to assemble and the aberrant polypeptides to be degraded. Thus, folding, oligomerization, and degradation are linked hierarchically to ensure the correct fate of newly synthesized polypeptides.

  View details for Web of Science ID A1996UP89900053

  View details for PubMedID 8633246

• FOLDING OF NASCENT POLYPEPTIDE-CHAINS IN A HIGH-MOLECULAR-MASS ASSEMBLY WITH MOLECULAR CHAPERONES NATURE Frydman, J., Nimmesgern, E., Ohtsuka, K., Hartl, F. U. 1994; 370 (6485): 111-117

  Abstract

  The folding of polypeptides emerging from ribosomes was analysed in a mammalian translation system using firefly luciferase as a model protein. The growing polypeptide interacts with a specific set of molecular chaperones, including Hsp70, the DnaJ homologue Hsp40 and the chaperonin TRiC. The ordered assembly of these components on the nascent chain forms a high molecular mass complex that allows the cotranslational formation of protein domains and the completion of folding once the chain is released from the ribosome.

  View details for Web of Science ID A1994NW80400047

  View details for PubMedID 8022479

• Human tNeurons reveal aging-linked proteostasis deficits driving Alzheimer's phenotypes. Research square Chou, C. C., Vest, R., Prado, M. A., Wilson-Grady, J., Paulo, J. A., Shibuya, Y., Moran-Losada, P., Lee, T. T., Luo, J., Gygi, S. P., Kelly, J. W., Finley, D., Wernig, M., Wyss-Coray, T., Frydman, J. 2024

  Abstract

  Aging is a prominent risk factor for Alzheimer's disease (AD), but the cellular mechanisms underlying neuronal phenotypes remain elusive. Both accumulation of amyloid plaques and neurofibrillary tangles in the brain1 and age-linked organelle deficits2-7 are proposed as causes of AD phenotypes but the relationship between these events is unclear. Here, we address this question using a transdifferentiated neuron (tNeuron) model directly from human dermal fibroblasts. Patient-derived tNeurons retain aging hallmarks and exhibit AD-linked deficits. Quantitative tNeuron proteomic analyses identify aging and AD-linked deficits in proteostasis and organelle homeostasis, particularly affecting endosome-lysosomal components. The proteostasis and lysosomal homeostasis deficits in aged tNeurons are exacerbated in sporadic and familial AD tNeurons, promoting constitutive lysosomal damage and defects in ESCRT-mediated repair. We find deficits in neuronal lysosomal homeostasis lead to inflammatory cytokine secretion, cell death and spontaneous development of Aß and phospho-Tau deposits. These proteotoxic inclusions co-localize with lysosomes and damage markers and resemble inclusions in brain tissue from AD patients and APP-transgenic mice. Supporting the centrality of lysosomal deficits driving AD phenotypes, lysosome-function enhancing compounds reduce AD-associated cytokine secretion and Aβ deposits. We conclude that proteostasis and organelle deficits are upstream initiating factors leading to neuronal aging and AD phenotypes.

  View details for DOI 10.21203/rs.3.rs-4407236/v1

  View details for PubMedID 38853828

  View details for PubMedCentralID PMC11160905

• BDNF and TRiC-inspired reagent rescue cortical synaptic deficits in a mouse model of Huntington's disease. Neurobiology of disease Gu, Y., Pope, A., Smith, C., Carmona, C., Johnstone, A., Shi, L., Chen, X., Santos, S., Bacon-Brenes, C. C., Shoff, T., Kleczko, K. M., Frydman, J., Thompson, L. M., Mobley, W. C., Wu, C. 2024: 106502

  Abstract

  Synaptic changes are early manifestations of neuronal dysfunction in Huntington's disease (HD). However, the mechanisms by which mutant HTT protein impacts synaptogenesis and function are not well understood. Herein we explored HD pathogenesis in the BACHD mouse model by examining synaptogenesis and function in long term primary cortical cultures. At DIV14 (days in vitro), BACHD cortical neurons showed no difference from WT neurons in synaptogenesis as revealed by colocalization of a pre-synaptic (Synapsin I) and a post-synaptic (PSD95) marker. From DIV21 to DIV35, BACHD neurons showed progressively reduced colocalization of Synapsin I and PSD95 relative to WT neurons. The deficits were effectively rescued by treatment of BACHD neurons with BDNF. The recombinant apical domain of CCT1 (ApiCCT1) yielded a partial rescuing effect. BACHD neurons also showed culture age-related significant functional deficits as revealed by multielectrode arrays (MEAs). These deficits were prevented by BDNF, whereas ApiCCT1 showed a less potent effect. These findings are evidence that deficits in BACHD synapse and function can be replicated in vitro and that BDNF or a TRiC-inspired reagent can potentially be protective against these changes in BACHD neurons. Our findings support the use of cellular models to further explicate HD pathogenesis and potential treatments.

  View details for DOI 10.1016/j.nbd.2024.106502

  View details for PubMedID 38608784

• SARS-CoV-2 Nsp1 cooperates with initiation factors EIF1 and 1A to selectively enhance translation of viral RNA. PLoS pathogens Aviner, R., Lidsky, P. V., Xiao, Y., Tassetto, M., Kim, D., Zhang, L., McAlpine, P. L., Elias, J., Frydman, J., Andino, R. 2024; 20 (2): e1011535

  Abstract

  A better mechanistic understanding of virus-host dependencies can help reveal vulnerabilities and identify opportunities for therapeutic intervention. Of particular interest are essential interactions that enable production of viral proteins, as those could target an early step in the virus lifecycle. Here, we use subcellular proteomics, ribosome profiling analyses and reporter assays to detect changes in protein synthesis dynamics during SARS-CoV-2 (CoV2) infection. We identify specific translation factors and molecular chaperones that are used by CoV2 to promote the synthesis and maturation of its own proteins. These can be targeted to inhibit infection, without major toxicity to the host. We also find that CoV2 non-structural protein 1 (Nsp1) cooperates with initiation factors EIF1 and 1A to selectively enhance translation of viral RNA. When EIF1/1A are depleted, more ribosomes initiate translation from a conserved upstream CUG start codon found in all genomic and subgenomic viral RNAs. This results in higher translation of an upstream open reading frame (uORF1) and lower translation of the main ORF, altering the stoichiometry of viral proteins and attenuating infection. Replacing the upstream CUG with AUG strongly inhibits translation of the main ORF independently of Nsp1, EIF1, or EIF1A. Taken together, our work describes multiple dependencies of CoV2 on host biosynthetic networks and proposes a model for dosage control of viral proteins through Nsp1-mediated control of translation start site selection.

  View details for DOI 10.1371/journal.ppat.1011535

  View details for PubMedID 38335237

• A structural vista of phosducin-like PhLP2A-chaperonin TRiC cooperation during the ATP-driven folding cycle. Nature communications Park, J., Kim, H., Gestaut, D., Lim, S., Opoku-Nsiah, K. A., Leitner, A., Frydman, J., Roh, S. 2024; 15 (1): 1007

  Abstract

  Proper cellular proteostasis, essential for viability, requires a network of chaperones and cochaperones. ATP-dependent chaperonin TRiC/CCT partners with cochaperones prefoldin (PFD) and phosducin-like proteins (PhLPs) to facilitate folding of essential eukaryotic proteins. Using cryoEM and biochemical analyses, we determine the ATP-driven cycle of TRiC-PFD-PhLP2A interaction. PhLP2A binds to open apo-TRiC through polyvalent domain-specific contacts with its chamber's equatorial and apical regions. PhLP2A N-terminal H3-domain binding to subunits CCT3/4 apical domains displace PFD from TRiC. ATP-induced TRiC closure rearranges the contacts of PhLP2A domains within the closed chamber. In the presence of substrate, actin and PhLP2A segregate into opposing chambers, each binding to positively charged inner surface residues from CCT1/3/6/8. Notably, actin induces a conformational change in PhLP2A, causing its N-terminal helices to extend across the inter-ring interface to directly contact a hydrophobic groove in actin. Our findings reveal an ATP-driven PhLP2A structural rearrangement cycle within the TRiC chamber to facilitate folding.

  View details for DOI 10.1038/s41467-024-45242-x

  View details for PubMedID 38307855

• Human transdifferentiated neurons reveal lysosomal repair deficits in Alzheimer's disease Chou, C., Frydman, J. ACADEMIC PRESS INC ELSEVIER SCIENCE. 2024

  View details for DOI 10.1016/j.ymgme.2023.107794

  View details for Web of Science ID 001192169300056

• A lethal mitonuclear incompatibility in complex I of natural hybrids. Nature Moran, B. M., Payne, C. Y., Powell, D. L., Iverson, E. N., Donny, A. E., Banerjee, S. M., Langdon, Q. K., Gunn, T. R., Rodriguez-Soto, R. A., Madero, A., Baczenas, J. J., Kleczko, K. M., Liu, F., Matney, R., Singhal, K., Leib, R. D., Hernandez-Perez, O., Corbett-Detig, R., Frydman, J., Gifford, C., Schartl, M., Havird, J. C., Schumer, M. 2024

  Abstract

  The evolution of reproductive barriers is the first step in the formation of new species and can help us understand the diversification of life on Earth. These reproductive barriers often take the form of hybrid incompatibilities, in which alleles derived from two different species no longer interact properly in hybrids1-3. Theory predicts that hybrid incompatibilities may be more likely to arise at rapidly evolving genes4-6 and that incompatibilities involving multiple genes should be common7,8, but there has been sparse empirical data to evaluate these predictions. Here we describe a mitonuclear incompatibility involving three genes whose protein products are in physical contact within respiratory complex I of naturally hybridizing swordtail fish species. Individuals homozygous for mismatched protein combinations do not complete embryonic development or die as juveniles, whereas those heterozygous for the incompatibility have reduced complex I function and unbalanced representation of parental alleles in the mitochondrial proteome. We find that the effects of different genetic interactions on survival are non-additive, highlighting subtle complexity in the genetic architecture of hybrid incompatibilities. Finally, we document the evolutionary history of the genes involved, showing signals of accelerated evolution and evidence that an incompatibility has been transferred between species via hybridization.

  View details for DOI 10.1038/s41586-023-06895-8

  View details for PubMedID 38200310

  View details for PubMedCentralID 6502311

• Transdifferentiation: A Novel Tool for Disease Modeling and Translational Applications in Alzheimer's Disease Chou, C., Vest, R., Prado, M. A., Wilson-Grady, J., Paulo, J. A., Shibuya, Y., Moran-Losada, P., Lee, T., Luo, J., Gygi, S. P., Kelly, J. W., Finley, D. P., Wernig, M., Wyss-Coray, T., Frydman, J. WILEY. 2023: S205-S206

  View details for Web of Science ID 001084474200356

• SARS-CoV-2 Nsp1 regulates translation start site fidelity to promote infection. bioRxiv : the preprint server for biology Aviner, R., Lidsky, P. V., Xiao, Y., Tasseto, M., Zhang, L., McAlpine, P. L., Elias, J., Frydman, J., Andino, R. 2023

  Abstract

  A better mechanistic understanding of virus-host interactions can help reveal vulnerabilities and identify opportunities for therapeutic interventions. Of particular interest are essential interactions that enable production of viral proteins, as those could target an early step in the virus lifecycle. Here, we use subcellular proteomics, ribosome profiling analyses and reporter assays to detect changes in polysome composition and protein synthesis during SARS-CoV-2 (CoV2) infection. We identify specific translation factors and molecular chaperones whose inhibition impairs infectious particle production without major toxicity to the host. We find that CoV2 non-structural protein Nsp1 selectively enhances virus translation through functional interactions with initiation factor EIF1A. When EIF1A is depleted, more ribosomes initiate translation from an upstream CUG start codon, inhibiting translation of non-structural genes and reducing viral titers. Together, our work describes multiple dependencies of CoV2 on host biosynthetic networks and identifies druggable targets for potential antiviral development.

  View details for DOI 10.1101/2023.07.05.547902

  View details for PubMedID 37461541

  View details for PubMedCentralID PMC10350044

• The unfolded protein response of the endoplasmic reticulum supports mitochondrial biogenesis by buffering non-imported proteins. Molecular biology of the cell Knöringer, K., Groh, C., Krämer, L., Stein, K. C., Hansen, K. G., Zimmermann, J., Morgan, B., Herrmann, J. M., Frydman, J., Boos, F. 2023: mbcE23050205

  Abstract

  Almost all mitochondrial proteins are synthesized in the cytosol and subsequently targeted to mitochondria. The accumulation of non-imported precursor proteins occurring upon mitochondrial dysfunction can challenge cellular protein homeostasis. Here we show that blocking protein translocation into mitochondria results in the accumulation of mitochondrial membrane proteins at the endoplasmic reticulum, thereby triggering the unfolded protein response (UPRER). Moreover, we find that mitochondrial membrane proteins are also routed to the ER under physiological conditions. The level of ER-resident mitochondrial precursors is enhanced by import defects as well as metabolic stimuli that increase the expression of mitochondrial proteins. Under such conditions, the UPRER is crucial to maintain protein homeostasis and cellular fitness. We propose the ER serves as a physiological buffer zone for those mitochondrial precursors that cannot be immediately imported into mitochondria while engaging the UPRER to adjust the ER proteostasis capacity to the extent of precursor accumulation.

  View details for DOI 10.1091/mbc.E23-05-0205

  View details for PubMedID 37379206

• A structural vista of phosducin-like PhLP2A-chaperonin TRiC cooperation during the ATP-driven folding cycle. bioRxiv : the preprint server for biology Park, J., Kim, H., Gestaut, D., Lim, S., Leitner, A., Frydman, J., Roh, S. H. 2023

  Abstract

  Proper cellular proteostasis, essential for viability, requires a network of chaperones and cochaperones. ATP-dependent chaperonin TRiC/CCT partners with cochaperones prefoldin (PFD) and phosducin-like proteins (PhLPs) to facilitate the folding of essential eukaryotic proteins. Using cryoEM and biochemical analyses, we determine the ATP-driven cycle of TRiC-PFD-PhLP2A interaction. In the open TRiC state, PhLP2A binds to the chamber's equator while its N-terminal H3-domain binds to the apical domains of CCT3/4, thereby displacing PFD from TRiC. ATP-induced TRiC closure rearranges the contacts of PhLP2A domains within the closed chamber. In the presence of substrate, actin and PhLP2A segregate into opposing chambers, each binding to the positively charged inner surfaces formed by CCT1/3/6/8. Notably, actin induces a conformational change in PhLP2A, causing its N-terminal helices to extend across the inter-ring interface to directly contact a hydrophobic groove in actin. Our findings reveal an ATP-driven PhLP2A structural rearrangement cycle within the TRiC chamber to facilitate folding.

  View details for DOI 10.1101/2023.03.25.534239

  View details for PubMedID 37016670

  View details for PubMedCentralID PMC10071816

• A Comprehensive Enumeration of the Human Proteostasis Network. 2. Components of the Autophagy-Lysosome Pathway. bioRxiv : the preprint server for biology Elsasser, S., Elia, L. P., Morimoto, R. I., Powers, E. T., Finley, D., Costa, B., Budron, M., Tokuno, Z., Wang, S., Iyer, R. G., Barth, B., Mockler, E., Finkbeiner, S., Gestwicki, J. E., Richardson, R. A., Stoeger, T., Tan, E. P., Xiao, Q., Cole, C. M., Massey, L. A., Garza, D., Kelly, J. W., Rainbolt, T. K., Chou, C. C., Masto, V. B., Frydman, J., Nixon, R. A. 2023

  Abstract

  The condition of having a healthy, functional proteome is known as protein homeostasis, or proteostasis. Establishing and maintaining proteostasis is the province of the proteostasis network, approximately 2,700 components that regulate protein synthesis, folding, localization, and degradation. The proteostasis network is a fundamental entity in biology that is essential for cellular health and has direct relevance to many diseases of protein conformation. However, it is not well defined or annotated, which hinders its functional characterization in health and disease. In this series of manuscripts, we aim to operationally define the human proteostasis network by providing a comprehensive, annotated list of its components. We provided in a previous manuscript a list of chaperones and folding enzymes as well as the components that make up the machineries for protein synthesis, protein trafficking into and out of organelles, and organelle-specific degradation pathways. Here, we provide a curated list of 838 unique high-confidence components of the autophagy-lysosome pathway, one of the two major protein degradation systems in human cells.

  View details for DOI 10.1101/2023.03.22.533675

  View details for PubMedID 36993380

  View details for PubMedCentralID PMC10055369

• CryoET reveals organelle phenotypes in huntington disease patient iPSC-derived and mouse primary neurons. Nature communications Wu, G. H., Smith-Geater, C., Galaz-Montoya, J. G., Gu, Y., Gupte, S. R., Aviner, R., Mitchell, P. G., Hsu, J., Miramontes, R., Wang, K. Q., Geller, N. R., Hou, C., Danita, C., Joubert, L. M., Schmid, M. F., Yeung, S., Frydman, J., Mobley, W., Wu, C., Thompson, L. M., Chiu, W. 2023; 14 (1): 692

  Abstract

  Huntington's disease (HD) is caused by an expanded CAG repeat in the huntingtin gene, yielding a Huntingtin protein with an expanded polyglutamine tract. While experiments with patient-derived induced pluripotent stem cells (iPSCs) can help understand disease, defining pathological biomarkers remains challenging. Here, we used cryogenic electron tomography to visualize neurites in HD patient iPSC-derived neurons with varying CAG repeats, and primary cortical neurons from BACHD, deltaN17-BACHD, and wild-type mice. In HD models, we discovered sheet aggregates in double membrane-bound organelles, and mitochondria with distorted cristae and enlarged granules, likely mitochondrial RNA granules. We used artificial intelligence to quantify mitochondrial granules, and proteomics experiments reveal differential protein content in isolated HD mitochondria. Knockdown of Protein Inhibitor of Activated STAT1 ameliorated aberrant phenotypes in iPSC- and BACHD neurons. We show that integrated ultrastructural and proteomic approaches may uncover early HD phenotypes to accelerate diagnostics and the development of targeted therapeutics for HD.

  View details for DOI 10.1038/s41467-023-36096-w

  View details for PubMedID 36754966

• Fragment-based computational design of antibodies targeting structured epitopes. Science advances Aguilar Rangel, M., Bedwell, A., Costanzi, E., Taylor, R. J., Russo, R., Bernardes, G. J., Ricagno, S., Frydman, J., Vendruscolo, M., Sormanni, P. 2022; 8 (45): eabp9540

  Abstract

  De novo design methods hold the promise of reducing the time and cost of antibody discovery while enabling the facile and precise targeting of predetermined epitopes. Here, we describe a fragment-based method for the combinatorial design of antibody binding loops and their grafting onto antibody scaffolds. We designed and tested six single-domain antibodies targeting different epitopes on three antigens, including the receptor-binding domain of the SARS-CoV-2 spike protein. Biophysical characterization showed that all designs are stable and bind their intended targets with affinities in the nanomolar range without in vitro affinity maturation. We further discuss how a high-resolution input antigen structure is not required, as similar predictions are obtained when the input is a crystal structure or a computer-generated model. This computational procedure, which readily runs on a laptop, provides a starting point for the rapid generation of lead antibodies binding to preselected epitopes.

  View details for DOI 10.1126/sciadv.abp9540

  View details for PubMedID 36367941

• Small molecule C381 targets the lysosome to reduce inflammation and ameliorate disease in models of neurodegeneration. Proceedings of the National Academy of Sciences of the United States of America Vest, R. T., Chou, C. C., Zhang, H., Haney, M. S., Li, L., Laqtom, N. N., Chang, B., Shuken, S., Nguyen, A., Yerra, L., Yang, A. C., Green, C., Tanga, M., Abu-Remaileh, M., Bassik, M. C., Frydman, J., Luo, J., Wyss-Coray, T. 2022; 119 (11): e2121609119

  Abstract

  SignificanceNeurodegenerative diseases are poorly understood and difficult to treat. One common hallmark is lysosomal dysfunction leading to the accumulation of aggregates and other undegradable materials, which cause damage to brain resident cells. Lysosomes are acidic organelles responsible for breaking down biomolecules and recycling their constitutive parts. In this work, we find that the antiinflammatory and neuroprotective compound, discovered via a phenotypic screen, imparts its beneficial effects by targeting the lysosome and restoring its function. This is established using a genome-wide CRISPRi target identification screen and then confirmed using a variety of lysosome-targeted studies. The resulting small molecule from this study represents a potential treatment for neurodegenerative diseases as well as a research tool for the study of lysosomes in disease.

  View details for DOI 10.1073/pnas.2121609119

  View details for PubMedID 35259016

• Dissecting the structural basis of Huntingtin pathogenesis: one molecule at the time Rodriguez-Aliaga, P., Sosa, R. P., Bustamante, C., Frydman, J. CELL PRESS. 2022: 22

  View details for Web of Science ID 000759523000103

• Targeted protein degradation: from small molecules to complex organelles-a Keystone Symposia report ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Cable, J., Weber-Ban, E., Clausen, T., Walters, K. J., Sharon, M., Finley, D. J., Gu, Y., Hanna, J., Feng, Y., Martens, S., Simonsen, A., Hansen, M., Zhang, H., Goodwin, J. M., Reggio, A., Chang, C., Ge, L., Schulman, B. A., Deshaies, R. J., Dikic, I., Harper, J., Wertz, I. E., Thoma, N. H., Slabicki, M., Frydman, J., Jakob, U., David, D. C., Bennett, E. J., Bertozzi, C. R., Sardana, R., Eapen, V. V., Carra, S. 2022

  Abstract

  Targeted protein degradation is critical for proper cellular function and development. Protein degradation pathways, such as the ubiquitin proteasomes system, autophagy, and endosome-lysosome pathway, must be tightly regulated to ensure proper elimination of misfolded and aggregated proteins and regulate changing protein levels during cellular differentiation, while ensuring that normal proteins remain unscathed. Protein degradation pathways have also garnered interest as a means to selectively eliminate target proteins that may be difficult to inhibit via other mechanisms. On June 7 and 8, 2021, several experts in protein degradation pathways met virtually for the Keystone eSymposium "Targeting protein degradation: from small molecules to complex organelles." The event brought together researchers working in different protein degradation pathways in an effort to begin to develop a holistic, integrated vision of protein degradation that incorporates all the major pathways to understand how changes in them can lead to disease pathology and, alternatively, how they can be leveraged for novel therapeutics.

  View details for DOI 10.1111/nyas.14745

  View details for Web of Science ID 000740430000001

  View details for PubMedID 35000205

• Cotranslational Mechanisms of Protein Biogenesis and Complex Assembly in Eukaryotes Annual Reviews of Biomedical Data Science Morales-Polanco, F., Lee, J. H., Barbosa, N. M., Frydman, J. 2022; 5

  View details for DOI 10.1146/annurev-biodatasci-121721-095858

• Small molecule C381 targets the lysosome to reduce inflammation and ameliorate disease in models of neurodegeneration Proc Natl Acad Sci U S A . Vest*, R. T., Chou*, C., Zhang, H., Haney, M. S., Li, L., Laqtom, N. N., Chang, B., Shuken, S., Nguyen, A., Yerra, L., Yang, A. C., Green, C., Tanga, M., Abu-Remaileh, M., Bassik, M. C., Frydman, J., Luo, J., Wyss-Coray, T. 2022; 119 (11): e2121609119

  View details for DOI 10.1073/pnas.2121609119

• A defective viral genome strategy elicits broad protective immunity against respiratory viruses. Cell Xiao, Y., Lidsky, P. V., Shirogane, Y., Aviner, R., Wu, C., Li, W., Zheng, W., Talbot, D., Catching, A., Doitsh, G., Su, W., Gekko, C. E., Nayak, A., Ernst, J. D., Brodsky, L., Brodsky, E., Rousseau, E., Capponi, S., Bianco, S., Nakamura, R., Jackson, P. K., Frydman, J., Andino, R. 2021

  Abstract

  RNA viruses generate defective viral genomes (DVGs) that can interfere with replication of the parental wild-type virus. To examine their therapeutic potential, we created a DVG by deleting the capsid-coding region of poliovirus. Strikingly, intraperitoneal or intranasal administration of this genome, which we termed eTIP1, elicits an antiviral response, inhibits replication, and protects mice from several RNA viruses, including enteroviruses, influenza, and SARS-CoV-2. While eTIP1 replication following intranasal administration is limited to the nasal cavity, its antiviral action extends non-cell-autonomously to the lungs. eTIP1 broad-spectrum antiviral effects are mediated by both local and distal type I interferon responses. Importantly, while a single eTIP1 dose protects animals from SARS-CoV-2 infection, it also stimulates production of SARS-CoV-2 neutralizing antibodies that afford long-lasting protection from SARS-CoV-2 reinfection. Thus, eTIP1 is a safe and effective broad-spectrum antiviral generating short- and long-term protection against SARS-CoV-2 and other respiratory infections in animal models.

  View details for DOI 10.1016/j.cell.2021.11.023

  View details for PubMedID 34852237

• A very special chaperonin: How does TRiC/CCT achieve tubulin folding? Gestaut, D., Zhao, Y., Park, J., Ma, B., Leitner, A., Collier, M., Aebersold, R., Roh, S., Chiu, W., Frydman, J. WILEY. 2021: 149

  View details for Web of Science ID 000711562500319

• CryoEM reveals the stochastic nature of individual ATP binding events in a group II chaperonin. Zhao, Y., Schmid, M. F., Frydman, J., Chiu, W. WILEY. 2021: 144

  View details for Web of Science ID 000711562500308

• Native mass spectrometry analyses of chaperonin complex TRiC/CCT reveal subunit N-terminal processing and re-association patterns. Scientific reports Collier, M. P., Moreira, K. B., Li, K. H., Chen, Y., Itzhak, D., Samant, R., Leitner, A., Burlingame, A., Frydman, J. 2021; 11 (1): 13084

  Abstract

  The eukaryotic chaperonin TRiC/CCT is a large ATP-dependent complex essential for cellular protein folding. Its subunit arrangement into two stacked eight-membered hetero-oligomeric rings is conserved from yeast to man. A recent breakthrough enables production of functional human TRiC (hTRiC) from insect cells. Here, we apply a suite of mass spectrometry techniques to characterize recombinant hTRiC. We find all subunits CCT1-8 are N-terminally processed by combinations of methionine excision and acetylation observed in native human TRiC. Dissociation by organic solvents yields primarily monomeric subunits with a small population of CCT dimers. Notably, some dimers feature non-canonical inter-subunit contacts absent in the initial hTRiC. This indicates individual CCT monomers can promiscuously re-assemble into dimers, and lack the information to assume the specific interface pairings in the holocomplex. CCT5 is consistently the most stable subunit and engages in the greatest number of non-canonical dimer pairings. These findings confirm physiologically relevant post-translational processing and function of recombinant hTRiC and offer quantitative insight into the relative stabilities of TRiC subunits and interfaces, a key step toward reconstructing its assembly mechanism. Our results also highlight the importance of assigning contacts identified by native mass spectrometry after solution dissociation as canonical or non-canonical when investigating multimeric assemblies.

  View details for DOI 10.1038/s41598-021-91086-6

  View details for PubMedID 34158536

• Structural and functional dissection of reovirus capsid folding and assembly by the prefoldin-TRiC/CCT chaperone network. Proceedings of the National Academy of Sciences of the United States of America Knowlton, J. J., Gestaut, D., Ma, B., Taylor, G., Seven, A. B., Leitner, A., Wilson, G. J., Shanker, S., Yates, N. A., Prasad, B. V., Aebersold, R., Chiu, W., Frydman, J., Dermody, T. S. 2021; 118 (11)

  Abstract

  Intracellular protein homeostasis is maintained by a network of chaperones that function to fold proteins into their native conformation. The eukaryotic TRiC chaperonin (TCP1-ring complex, also called CCT for cytosolic chaperonin containing TCP1) facilitates folding of a subset of proteins with folding constraints such as complex topologies. To better understand the mechanism of TRiC folding, we investigated the biogenesis of an obligate TRiC substrate, the reovirus σ3 capsid protein. We discovered that the σ3 protein interacts with a network of chaperones, including TRiC and prefoldin. Using a combination of cryoelectron microscopy, cross-linking mass spectrometry, and biochemical approaches, we establish functions for TRiC and prefoldin in folding σ3 and promoting its assembly into higher-order oligomers. These studies illuminate the molecular dynamics of σ3 folding and establish a biological function for TRiC in virus assembly. In addition, our findings provide structural and functional insight into the mechanism by which TRiC and prefoldin participate in the assembly of protein complexes.

  View details for DOI 10.1073/pnas.2018127118

  View details for PubMedID 33836586

  View details for PubMedCentralID PMC7980406

• Disease-related Huntingtin seeding activities in cerebrospinal fluids of Huntington's disease patients. Scientific reports Lee, C. Y., Wang, N., Shen, K., Stricos, M., Langfelder, P., Cheon, K. H., Cortes, E. P., Vinters, H. V., Vonsattel, J. P., Wexler, N. S., Damoiseaux, R., Frydman, J., Yang, X. W. 2020; 10 (1): 20295

  Abstract

  In Huntington's disease (HD), the mutant Huntingtin (mHTT) is postulated to mediate template-based aggregation that can propagate across cells. It has been difficult to quantitatively detect such pathological seeding activities in patient biosamples, e.g. cerebrospinal fluids (CSF), and study their correlation with the disease manifestation. Here we developed a cell line expressing a domain-engineered mHTT-exon1 reporter, which showed remarkably high sensitivity and specificity in detecting mHTT seeding species in HD patient biosamples. We showed that the seeding-competent mHTT species in HD CSF are significantly elevated upon disease onset and with the progression of neuropathological grades. Mechanistically, we showed that mHTT seeding activities in patient CSF could be ameliorated by the overexpression of chaperone DNAJB6 and by antibodies against the polyproline domain of mHTT. Together, our study developed a selective and scalable cell-based tool to investigate mHTT seeding activities in HD CSF, and demonstrated that the CSF mHTT seeding species are significantly associated with certain disease states. This seeding activity can be ameliorated by targeting specific domain or proteostatic pathway of mHTT, providing novel insights into such pathological activities.

  View details for DOI 10.1038/s41598-020-77164-1

  View details for PubMedID 33219289

• REP-X: An Evolution-guided Strategy for the Rational Design of Cysteine-less Protein Variants. Scientific reports Dalton, K., Lopez, T., Pande, V., Frydman, J. 2020; 10 (1): 2193

  Abstract

  Site-specific labeling of proteins is often a prerequisite for biophysical and biochemical characterization. Chemical modification of a unique cysteine residue is among the most facile methods for site-specific labeling of proteins. However, many proteins have multiple reactive cysteines, which must be mutated to other residues to enable labeling of unique positions. This trial-and-error process often results in cysteine-free proteins with reduced activity or stability. Herein we describe a general methodology to rationally engineer cysteine-less proteins. Briefly, natural variation across orthologues is exploited to identify suitable cysteine replacements compatible with protein activity and stability. As a proof-of-concept, we recount the successful engineering of a cysteine-less mutant of the group II chaperonin from methanogenic archaeon Methanococcus maripaludis. A webapp, REP-X (Replacement at Endogenous Positions from eXtant sequences), which enables users to design their own cysteine-less protein variants, will make this rational approach widely available.

  View details for DOI 10.1038/s41598-020-58794-x

  View details for PubMedID 32042106

• Dynamics and clustering of IRE1alpha during ER stress. Proceedings of the National Academy of Sciences of the United States of America Rainbolt, T. K., Frydman, J. 2020

  View details for DOI 10.1073/pnas.1921799117

  View details for PubMedID 32019880

• Multi-scale 3D Cryo-Correlative Microscopy for Vitrified Cells. Structure (London, England : 1993) Wu, G. H., Mitchell, P. G., Galaz-Montoya, J. G., Hecksel, C. W., Sontag, E. M., Gangadharan, V. n., Marshman, J. n., Mankus, D. n., Bisher, M. E., Lytton-Jean, A. K., Frydman, J. n., Czymmek, K. n., Chiu, W. n. 2020

  Abstract

  Three-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging modalities to visualize the same specimen at cryogenic temperature at different scales: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast cells, including the ultrastructure of protein inclusions that recruit fluorescently-labeled chaperone Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be applied to other types of cells, it could be used for large-scale phenotypic studies of frozen-hydrated specimens in a variety of healthy and diseased conditions with and without treatments.

  View details for DOI 10.1016/j.str.2020.07.017

  View details for PubMedID 32814034

• ALTERNATIVE FORMS OF ENERGY MODULATE GROUP II CHAPERONIN ACTIVITY Goncalves, K., Lopez, T., Frydman, J. WILEY. 2019: 94

  View details for Web of Science ID 000488134600159

• Methods for measuring misfolded protein clearance in the budding yeast Saccharomyces cerevisiae. Methods in enzymology Samant, R. S., Frydman, J. 2019; 619: 27-45

  Abstract

  Protein misfolding in the cell is linked to an array of diseases, including cancers, cardiovascular disease, type II diabetes, and numerous neurodegenerative disorders. Therefore, investigating cellular pathways by which misfolded proteins are trafficked and cleared ("protein quality control") is of both mechanistic and therapeutic importance. The clearance of most misfolded proteins involves the covalent attachment of one or more ubiquitin molecules; however, the precise fate of the ubiquitinated protein varies greatly, depending on the linkages present in the ubiquitin chain. Here, we discuss approaches for quantifying linkage-specific ubiquitination and clearance of misfolded proteins in the budding yeast Saccharomyces cerevisiae-a model organism used extensively for interrogation of protein quality control pathways, but which presents its own unique challenges for cell and molecular biology experiments. We present a fluorescence microscopy-based assay for monitoring the clearance of misfolded protein puncta, a cycloheximide-chase assay for calculating misfolded protein half-life, and two antibody-based methods for quantifying specific ubiquitin linkages on tagged misfolded proteins, including a 96-well plate-based ELISA. We hope these methods will be of use to the protein quality control, protein degradation, and ubiquitin biology communities.

  View details for DOI 10.1016/bs.mie.2018.12.039

  View details for PubMedID 30910025

• Dosage compensation plans: protein aggregation provides additional insurance against aneuploidy. Genes & development Samant, R. S., Masto, V. B., Frydman, J. n. 2019; 33 (15-16): 1027–30

  Abstract

  Gene dosage alterations caused by aneuploidy are a common feature of most cancers yet pose severe proteotoxic challenges. Therefore, cells have evolved various dosage compensation mechanisms to limit the damage caused by the ensuing protein level imbalances. For instance, for heteromeric protein complexes, excess nonstoichiometric subunits are rapidly recognized and degraded. In this issue of Genes & Development, Brennan et al. (pp. 1031-1047) reveal that sequestration of nonstoichiometric subunits into aggregates is an alternative mechanism for dosage compensation in aneuploid budding yeast and human cell lines. Using a combination of proteomic and genetic techniques, they found that excess proteins undergo either degradation or aggregation but not both. Which route is preferred depends on the half-life of the protein in question. Given the multitude of diseases linked to either aneuploidy or protein aggregation, this study could serve as a springboard for future studies with broad-spanning implications.

  View details for DOI 10.1101/gad.329383.119

  View details for PubMedID 31371460

• Methods for measuring misfolded protein clearance in the budding yeast Saccharomyces cerevisiae Methods in Enzymology Samant, R. S., Frydman, J. 2019; in press

  View details for DOI 10.1016/bs.mie.2018.12.039

• Huntingtin's N-Terminus Rearrangements in the Presence of Membranes: A Joint Spectroscopic and Computational Perspective ACS CHEMICAL NEUROSCIENCE Levy, G. R., Shen, K., Gavrilov, Y., Smith, P. S., Levy, Y., Chan, R., Frydman, J., Frydman, L. 2019; 10 (1): 472–81

  Abstract

  Huntington's disease is a neurodegenerative disorder resulting from an expanded polyglutamine (polyQ) repeat of the Huntingtin (Htt) protein. Affected tissues often contain aggregates of the N-terminal Htt exon 1 (Htt-Ex1) fragment. The N-terminal N17 domain proximal to the polyQ tract is key to enhance aggregation and modulate Htt toxicity. Htt-Ex1 is intrinsically disordered, yet it has been postulated that under physiological conditions membranes induce the N17 to adopt an α-helical structure, which then plays a key role in regulating Htt protein aggregation. The present study leverages the recently available assignment of NMR peaks in an N17Q17 construct, in order to provide a look into the changes occurring in vitro upon exposing this fragment to various brain extract fragments as well as to synthetic bilayers. Residue-specific changes were observed by 3D HNCO NMR, whose nature was further clarified with ancillary CD and aggregation studies, as well as with molecular dynamic calculations. From this combination of measurements and computations, a unified picture emerges, whereby transient structures consisting of α-helices spanning a fraction of the N17 residues form during N17Q17-membrane interactions. These interactions are fairly dynamic, but they qualitatively mimic more rigid variants that have been discussed in the literature. The nature of these interactions and their potential influence on the aggregation process of these kinds of constructs under physiological conditions are briefly assessed.

  View details for PubMedID 30149694

• The stop and go traffic regulating protein biogenesis: how translation kinetics control proteostasis. The Journal of biological chemistry Stein, K. C., Frydman, J. 2018

  Abstract

  Generating a functional proteome requires the ribosome to carefully regulate disparate co-translational processes that determine the fate of nascent polypeptides. With protein synthesis being energetically expensive, the ribosome must balance the costs of efficiently making a protein with those of properly folding it. Emerging as a primary means of regulating this trade-off are the non-uniform rates of translation elongation that define translation kinetics. The varying speeds with which the ribosome progresses along a transcript have been implicated in several aspects of protein biogenesis, including co-translational protein folding and translational fidelity, as well as gene expression by mediating mRNA decay and protein quality control pathways. The optimal translation kinetics required to efficiently execute these processes can be distinct. Thus, the ribosome is tasked with tightly regulating translation kinetics to balance these processes while maintaining adaptability for changing cellular conditions. In this review, we first discuss the regulatory role of translation elongation in protein biogenesis and what factors influence elongation kinetics. We then describe how changes in translation kinetics signal downstream pathways that dictate the fate of nascent polypeptides. By regulating these pathways, the kinetics of translation elongation has emerged as a critical tool for driving gene expression and maintaining proteostasis through varied mechanisms, including nascent chain folding and binding different ribosome-associated machinery. Indeed, a growing number of examples demonstrate the important role of local changes in elongation kinetics in modulating the pathophysiology of human disease.

  View details for PubMedID 30504455

• A Viral Protein Restricts Drosophila RNAi Immunity by Regulating Argonaute Activity and Stability CELL HOST & MICROBE Nayak, A., Kim, D., Trnka, M. J., Kerr, C. H., Lidsky, P. V., Stanley, D. J., Rivera, B., Li, K. H., Burlingame, A. L., Jan, E., Frydman, J., Gross, J. D., Andino, R. 2018; 24 (4): 542-+

  Abstract

  The dicistrovirus, Cricket paralysis virus (CrPV) encodes an RNA interference (RNAi) suppressor, 1A, which modulates viral virulence. Using the Drosophila model, we combined structural, biochemical, and virological approaches to elucidate the strategies by which CrPV-1A restricts RNAi immunity. The atomic resolution structure of CrPV-1A uncovered a flexible loop that interacts with Argonaute 2 (Ago-2), thereby inhibiting Ago-2 endonuclease-dependent immunity. Mutations disrupting Ago-2 binding attenuates viral pathogenesis in wild-type but not Ago-2-deficient flies. CrPV-1A also contains a BC-box motif that enables the virus to hijack a host Cul2-Rbx1-EloBC ubiquitin ligase complex, which promotes Ago-2 degradation and virus replication. Our study uncovers a viral-based dual regulatory program that restricts antiviral immunity by direct interaction with and modulation of host proteins. While the direct inhibition of Ago-2 activity provides an efficient mechanism to establish infection, the recruitment of a ubiquitin ligase complex enables CrPV-1A to amplify Ago-2 inactivation to restrict further antiviral RNAi immunity.

  View details for PubMedID 30308158

• The TRiC chaperonin controls reovirus replication through outer-capsid folding NATURE MICROBIOLOGY Knowlton, J. J., Fernandez de Castro, I., Ashbrook, A. W., Gestaut, D. R., Zamora, P. F., Bauer, J. A., Forrest, J., Frydman, J., Risco, C., Dermody, T. S. 2018; 3 (4): 481–93

  Abstract

  Viruses are molecular machines sustained through a life cycle that requires replication within host cells. Throughout the infectious cycle, viral and cellular components interact to advance the multistep process required to produce progeny virions. Despite progress made in understanding the virus-host protein interactome, much remains to be discovered about the cellular factors that function during infection, especially those operating at terminal steps in replication. In an RNA interference screen, we identified the eukaryotic chaperonin T-complex protein-1 (TCP-1) ring complex (TRiC; also called CCT for chaperonin containing TCP-1) as a cellular factor required for late events in the replication of mammalian reovirus. We discovered that TRiC functions in reovirus replication through a mechanism that involves folding the viral σ3 major outer-capsid protein into a form capable of assembling onto virus particles. TRiC also complexes with homologous capsid proteins of closely related viruses. Our data define a critical function for TRiC in the viral assembly process and raise the possibility that this mechanism is conserved in related non-enveloped viruses. These results also provide insight into TRiC protein substrates and establish a rationale for the development of small-molecule inhibitors of TRiC as potential antiviral therapeutics.

  View details for PubMedID 29531365

• Time-Resolved Measurement of the ATP-Dependent Motion of the Group II Chaperonin by Diffracted Electron Tracking INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Ogawa, N., Yamamoto, Y. Y., Abe, K., Sekiguchi, H., Sasaki, Y. C., Ishikawa, A., Frydman, J., Yohda, M. 2018; 19 (4)

  Abstract

  Previously, we demonstrated the ATP-dependent dynamics of a group II chaperonin at the single-molecule level by diffracted X-ray tracking (DXT). The disadvantage of DXT is that it requires a strong X-ray source and also perfect gold nano-crystals. To resolve this problem, we developed diffracted electron tracking (DET). Electron beams have scattering cross-sections that are approximately 1000 times larger than those of X-rays. Thus, DET enables us to perform super-accurate measurements of the time-resolved 3D motion of proteins labeled with commercially available gold nanorods using a scanning electron microscope. In this study, we compared DXT and DET using the group II chaperonin from Methanococcus maripaludis (MmCpn) as a model protein. In DET, the samples are prepared in an environmental cell (EC). To reduce the electron beam-induced protein damage, we immobilized MmCpn on the bottom of the EC to expose gold nanorods close to the carbon thin film. The sample setup worked well, and the motions of gold nanorods were clearly traced. Compared with the results of DXT, the mobility in DET was significantly higher, which is probably due to the difference in the method for immobilization. In DET, MmCpn was immobilized on a film of triacetyl cellulose. Whereas proteins are directly attached on the surface of solid support in DXT. Therefore, MmCpn could move relatively freely in DET. DET will be a state-of-the-art technology for analyzing protein dynamics.

  View details for PubMedID 29565826

• Proteostasis Function and Disfunction: The Folding Machines that Maintain Proteome Health Frydman, J. CELL PRESS. 2018: 544A–545A

  View details for Web of Science ID 000430563200475

• Protein misfolding in neurodegenerative diseases: implications and strategies. Translational neurodegeneration Sweeney, P., Park, H., Baumann, M., Dunlop, J., Frydman, J., Kopito, R., McCampbell, A., LeBlanc, G., Venkateswaran, A., Nurmi, A., Hodgson, R. 2017; 6: 6-?

  Abstract

  A hallmark of neurodegenerative proteinopathies is the formation of misfolded protein aggregates that cause cellular toxicity and contribute to cellular proteostatic collapse. Therapeutic options are currently being explored that target different steps in the production and processing of proteins implicated in neurodegenerative disease, including synthesis, chaperone-assisted folding and trafficking, and degradation via the proteasome and autophagy pathways. Other therapies, like mTOR inhibitors and activators of the heat shock response, can rebalance the entire proteostatic network. However, there are major challenges that impact the development of novel therapies, including incomplete knowledge of druggable disease targets and their mechanism of action as well as a lack of biomarkers to monitor disease progression and therapeutic response. A notable development is the creation of collaborative ecosystems that include patients, clinicians, basic and translational researchers, foundations and regulatory agencies to promote scientific rigor and clinical data to accelerate the development of therapies that prevent, reverse or delay the progression of neurodegenerative proteinopathies.

  View details for DOI 10.1186/s40035-017-0077-5

  View details for PubMedID 28293421

• Multivalent contacts of the Hsp70 Ssb contribute to its architecture on ribosomes and nascent chain interaction NATURE COMMUNICATIONS Hanebuth, M. A., Kityk, R., Fries, S. J., Jain, A., Kriel, A., Albanese, V., Frickey, T., Peter, C., Mayer, M. P., Frydman, J., Deuerling, E. 2016; 7

  Abstract

  Hsp70 chaperones assist de novo folding of newly synthesized proteins in all cells. In yeast, the specialized Hsp70 Ssb directly binds to ribosomes. The structural basis and functional mode of recruitment of Ssb to ribosomes is not understood. Here, we present the molecular details underlying ribosome binding of Ssb in Saccharomyces cerevisiae. This interaction is multifaceted, involving the co-chaperone RAC and two specific regions within Ssb characterized by positive charges. The C-terminus of Ssb mediates the key contact and a second attachment point is provided by a KRR-motif in the substrate binding domain. Strikingly, ribosome binding of Ssb is not essential. Autonomous ribosome attachment becomes necessary if RAC is absent, suggesting a dual mode of Ssb recruitment to nascent chains. We propose, that the multilayered ribosomal interaction allows positioning of Ssb in an optimal orientation to the tunnel exit guaranteeing an efficient nascent polypeptide interaction.

  View details for DOI 10.1038/ncomms13695

  View details for PubMedID 27917864

• xTract: software for characterizing conformational changes of protein complexes by quantitative cross-linking mass spectrometry. Nature methods Walzthoeni, T., Joachimiak, L. A., Rosenberger, G., Röst, H. L., Malmström, L., Leitner, A., Frydman, J., Aebersold, R. 2015; 12 (12): 1185-1190

  View details for DOI 10.1038/nmeth.3631

  View details for PubMedID 26501516

• The Mechanism and Function of Group II Chaperonins. Journal of molecular biology Lopez, T., Dalton, K., Frydman, J. 2015; 427 (18): 2919-2930

  Abstract

  Protein folding in the cell requires the assistance of enzymes collectively called chaperones. Among these, the chaperonins are 1-MDa ring-shaped oligomeric complexes that bind unfolded polypeptides and promote their folding within an isolated chamber in an ATP-dependent manner. Group II chaperonins, found in archaea and eukaryotes, contain a built-in lid that opens and closes over the central chamber. In eukaryotes, the chaperonin TRiC/CCT is hetero-oligomeric, consisting of two stacked rings of eight paralogous subunits each. TRiC facilitates folding of approximately 10% of the eukaryotic proteome, including many cytoskeletal components and cell cycle regulators. Folding of many cellular substrates of TRiC cannot be assisted by any other chaperone. A complete structural and mechanistic understanding of this highly conserved and essential chaperonin remains elusive. However, recent work is beginning to shed light on key aspects of chaperonin function and how their unique properties underlie their contribution to maintaining cellular proteostasis.

  View details for DOI 10.1016/j.jmb.2015.04.013

  View details for PubMedID 25936650

• The Dynamic Conformational Cycle of the Group I Chaperonin C-Termini Revealed via Molecular Dynamics Simulation PLOS ONE Dalton, K. M., Frydman, J., Pande, V. S. 2015; 10 (3)

  Abstract

  Chaperonins are large ring shaped oligomers that facilitate protein folding by encapsulation within a central cavity. All chaperonins possess flexible C-termini which protrude from the equatorial domain of each subunit into the central cavity. Biochemical evidence suggests that the termini play an important role in the allosteric regulation of the ATPase cycle, in substrate folding and in complex assembly and stability. Despite the tremendous wealth of structural data available for numerous orthologous chaperonins, little structural information is available regarding the residues within the C-terminus. Herein, molecular dynamics simulations are presented which localize the termini throughout the nucleotide cycle of the group I chaperonin, GroE, from Escherichia coli. The simulation results predict that the termini undergo a heretofore unappreciated conformational cycle which is coupled to the nucleotide state of the enzyme. As such, these results have profound implications for the mechanism by which GroE utilizes nucleotide and folds client proteins.

  View details for DOI 10.1371/journal.pone.0117724

  View details for Web of Science ID 000352134700014

  View details for PubMedID 25822285

  View details for PubMedCentralID PMC4379175

• Parkinson's Disease Genes VPS35 and EIF4G1 Interact Genetically and Converge on a-Synuclein. Neuron Dhungel, N., Eleuteri, S., Li, L., Kramer, N. J., Chartron, J. W., Spencer, B., Kosberg, K., Fields, J. A., Stafa, K., Adame, A., Lashuel, H., Frydman, J., Shen, K., Masliah, E., Gitler, A. D. 2015; 85 (1): 76-87

  Abstract

  Parkinson's disease (PD) is a common neurodegenerative disorder. Functional interactions between some PD genes, like PINK1 and parkin, have been identified, but whether other ones interact remains elusive. Here we report an unexpected genetic interaction between two PD genes, VPS35 and EIF4G1. We provide evidence that EIF4G1 upregulation causes defects associated with protein misfolding. Expression of a sortilin protein rescues these defects, downstream of VPS35, suggesting a potential role for sortilins in PD. We also show interactions between VPS35, EIF4G1, and α-synuclein, a protein with a key role in PD. We extend our findings from yeast to an animal model and show that these interactions are conserved in neurons and in transgenic mice. Our studies reveal unexpected genetic and functional interactions between two seemingly unrelated PD genes and functionally connect them to α-synuclein pathobiology in yeast, worms, and mouse. Finally, we provide a resource of candidate PD genes for future interrogation.

  View details for DOI 10.1016/j.neuron.2014.11.027

  View details for PubMedID 25533483

  View details for PubMedCentralID PMC4289081

• The significance of Hsp70 subnetwork for Dengue virus lifecycle. Uirusu Taguwa, S., Frydman, J. 2015; 65 (2): 179-186

  Abstract

  Viruses hijack host machineries for replicating themselves efficiently. Host protein quality control machineries (QC) not only assist protein folding to form bona fide proteins with active functions but also get rid of un/misfolded proteins via degradation to maintain the protein homeostasis. Previous studies have reported that viruses utilize QC at various steps for their lifecycles. Recently we defined Hsp70s and their cochaperones, DnaJs functions on Dengue lifecycle. Here we summarize the significance of QC on Dengue virus.

  View details for PubMedID 27760916

• The dynamic conformational cycle of the group I chaperonin C-termini revealed via molecular dynamics simulation. PloS one Dalton, K. M., Frydman, J., Pande, V. S. 2015; 10 (3)

  Abstract

  Chaperonins are large ring shaped oligomers that facilitate protein folding by encapsulation within a central cavity. All chaperonins possess flexible C-termini which protrude from the equatorial domain of each subunit into the central cavity. Biochemical evidence suggests that the termini play an important role in the allosteric regulation of the ATPase cycle, in substrate folding and in complex assembly and stability. Despite the tremendous wealth of structural data available for numerous orthologous chaperonins, little structural information is available regarding the residues within the C-terminus. Herein, molecular dynamics simulations are presented which localize the termini throughout the nucleotide cycle of the group I chaperonin, GroE, from Escherichia coli. The simulation results predict that the termini undergo a heretofore unappreciated conformational cycle which is coupled to the nucleotide state of the enzyme. As such, these results have profound implications for the mechanism by which GroE utilizes nucleotide and folds client proteins.

  View details for DOI 10.1371/journal.pone.0117724

  View details for PubMedID 25822285

  View details for PubMedCentralID PMC4379175

• Super-resolution fluorescence of huntingtin reveals growth of globular species into short fibers and coexistence of distinct aggregates. ACS chemical biology Duim, W. C., Jiang, Y., Shen, K., Frydman, J., Moerner, W. E. 2014; 9 (12): 2767-2778

  Abstract

  Polyglutamine-expanded huntingtin, the protein encoded by HTT mutations associated with Huntington's disease, forms aggregate species in vitro and in vivo. Elucidation of the mechanism of growth of fibrillar aggregates from soluble monomeric protein is critical to understanding the progression of Huntington's disease and to designing therapeutics for the disease, as well as for aggregates implicated in Alzheimer's and Parkinson's diseases. We used the technique of multicolor single-molecule, super-resolution fluorescence imaging to characterize the growth of huntingtin exon 1 aggregates. The huntingtin exon 1 aggregation followed a pathway from exclusively spherical or globular species of ∼80 nm to fibers ∼1 μm in length that increased in width, but not length, over time with the addition of more huntingtin monomers. The fibers further aggregated with one another into aggregate assemblies of increasing size. Seeds created by sonication, which were comparable in shape and size to the globular species in the pathway, were observed to grow through multidirectional elongation into fibers, suggesting a mechanism for growth of globular species into fibers. The single-molecule sensitivity of our approach made it possible to characterize the aggregation pathway across a large range of size scales, from monomers to fiber assemblies, and revealed the coexistence of different aggregate species (globular species, fibers, fiber assemblies) even at late time points.

  View details for DOI 10.1021/cb500335w

  View details for PubMedID 25330023

• Proteostatic Control of Telomerase Function through TRiC-Mediated Folding of TCAB1 CELL Freund, A., Zhong, F. L., Venteicher, A. S., Meng, Z., Veenstra, T. D., Frydman, J., Artandi, S. E. 2014; 159 (6): 1389-1403

  Abstract

  Telomere maintenance by telomerase is impaired in the stem cell disease dyskeratosis congenita and during human aging. Telomerase depends upon a complex pathway for enzyme assembly, localization in Cajal bodies, and association with telomeres. Here, we identify the chaperonin CCT/TRiC as a critical regulator of telomerase trafficking using a high-content genome-wide siRNA screen in human cells for factors required for Cajal body localization. We find that TRiC is required for folding the telomerase cofactor TCAB1, which controls trafficking of telomerase and small Cajal body RNAs (scaRNAs). Depletion of TRiC causes loss of TCAB1 protein, mislocalization of telomerase and scaRNAs to nucleoli, and failure of telomere elongation. DC patient-derived mutations in TCAB1 impair folding by TRiC, disrupting telomerase function and leading to severe disease. Our findings establish a critical role for TRiC-mediated protein folding in the telomerase pathway and link proteostasis, telomere maintenance, and human disease.

  View details for DOI 10.1016/j.cell.2014.10.059

  View details for Web of Science ID 000346652900017

  View details for PubMedID 25467444

• A Direct Regulatory Interaction between Chaperonin TRiC and Stress-Responsive Transcription Factor HSF1 CELL REPORTS Neef, D. W., Jaeger, A. M., Gomez-Pastor, R., Willmund, F., Frydman, J., Thiele, D. J. 2014; 9 (3): 955-966

  Abstract

  Heat shock transcription factor 1 (HSF1) is an evolutionarily conserved transcription factor that protects cells from protein-misfolding-induced stress and apoptosis. The mechanisms by which cytosolic protein misfolding leads to HSF1 activation have not been elucidated. Here, we demonstrate that HSF1 is directly regulated by TRiC/CCT, a central ATP-dependent chaperonin complex that folds cytosolic proteins. A small-molecule activator of HSF1, HSF1A, protects cells from stress-induced apoptosis, binds TRiC subunits in vivo and in vitro, and inhibits TRiC activity without perturbation of ATP hydrolysis. Genetic inactivation or depletion of the TRiC complex results in human HSF1 activation, and HSF1A inhibits the direct interaction between purified TRiC and HSF1 in vitro. These results demonstrate a direct regulatory interaction between the cytosolic chaperone machine and a critical transcription factor that protects cells from proteotoxicity, providing a mechanistic basis for signaling perturbations in protein folding to a stress-protective transcription factor.

  View details for DOI 10.1016/j.celrep.2014.09.056

  View details for Web of Science ID 000344470000019

  View details for PubMedID 25437552

• Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Leitner, A., Joachimiak, L. A., Unverdorben, P., Walzthoeni, T., Frydman, J., Foerster, F., Aebersold, R. 2014; 111 (26): 9455-9460

  View details for DOI 10.1073/pnas.1320298111

  View details for Web of Science ID 000338118900039

• Chemical cross-linking/mass spectrometry targeting acidic residues in proteins and protein complexes. Proceedings of the National Academy of Sciences of the United States of America Leitner, A., Joachimiak, L. A., Unverdorben, P., Walzthoeni, T., Frydman, J., Förster, F., Aebersold, R. 2014; 111 (26): 9455-9460

  Abstract

  The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches.

  View details for DOI 10.1073/pnas.1320298111

  View details for PubMedID 24938783

  View details for PubMedCentralID PMC4084482

• Modulation of STAT3 folding and function by TRiC/CCT chaperonin. PLoS biology Kasembeli, M., Lau, W. C., Roh, S., Eckols, T. K., Frydman, J., Chiu, W., Tweardy, D. J. 2014; 12 (4)

  Abstract

  Signal transducer and activator of transcription 3 (Stat3) transduces signals of many peptide hormones from the cell surface to the nucleus and functions as an oncoprotein in many types of cancers, yet little is known about how it achieves its native folded state within the cell. Here we show that Stat3 is a novel substrate of the ring-shaped hetero-oligomeric eukaryotic chaperonin, TRiC/CCT, which contributes to its biosynthesis and activity in vitro and in vivo. TRiC binding to Stat3 was mediated, at least in part, by TRiC subunit CCT3. Stat3 binding to TRiC mapped predominantly to the β-strand rich, DNA-binding domain of Stat3. Notably, enhancing Stat3 binding to TRiC by engineering an additional TRiC-binding domain from the von Hippel-Lindau protein (vTBD), at the N-terminus of Stat3, further increased its affinity for TRiC as well as its function, as determined by Stat3's ability to bind to its phosphotyrosyl-peptide ligand, an interaction critical for Stat3 activation. Thus, Stat3 levels and function are regulated by TRiC and can be modulated by manipulating its interaction with TRiC.

  View details for DOI 10.1371/journal.pbio.1001844

  View details for PubMedID 24756126

  View details for PubMedCentralID PMC3995649

• TRiC's tricks inhibit huntingtin aggregation ELIFE Shahmoradian, S. H., Galaz-Montoya, J. G., Schmid, M. F., Cong, Y., Ma, B., Spiess, C., Frydman, J., Ludtke, S. J., Chiu, W. 2013; 2

  Abstract

  In Huntington's disease, a mutated version of the huntingtin protein leads to cell death. Mutant huntingtin is known to aggregate, a process that can be inhibited by the eukaryotic chaperonin TRiC (TCP1-ring complex) in vitro and in vivo. A structural understanding of the genesis of aggregates and their modulation by cellular chaperones could facilitate the development of therapies but has been hindered by the heterogeneity of amyloid aggregates. Using cryo-electron microscopy (cryoEM) and single particle cryo-electron tomography (SPT) we characterize the growth of fibrillar aggregates of mutant huntingtin exon 1 containing an expanded polyglutamine tract with 51 residues (mhttQ51), and resolve 3-D structures of the chaperonin TRiC interacting with mhttQ51. We find that TRiC caps mhttQ51 fibril tips via the apical domains of its subunits, and also encapsulates smaller mhtt oligomers within its chamber. These two complementary mechanisms provide a structural description for TRiC's inhibition of mhttQ51 aggregation in vitro. DOI:http://dx.doi.org/10.7554/eLife.00710.001.

  View details for DOI 10.7554/eLife.00710

  View details for Web of Science ID 000328620500004

  View details for PubMedID 23853712

  View details for PubMedCentralID PMC3707056

• The role of mutational robustness in RNA virus evolution. Nature reviews. Microbiology Lauring, A. S., Frydman, J., Andino, R. 2013; 11 (5): 327-336

  Abstract

  RNA viruses face dynamic environments and are masters at adaptation. During their short 'lifespans', they must surmount multiple physical, anatomical and immunological challenges. Central to their adaptative capacity is the enormous genetic diversity that characterizes RNA virus populations. Although genetic diversity increases the rate of adaptive evolution, low replication fidelity can present a risk because excess mutations can lead to population extinction. In this Review, we discuss the strategies used by RNA viruses to deal with the increased mutational load and consider how this mutational robustness might influence viral evolution and pathogenesis.

  View details for DOI 10.1038/nrmicro3003

  View details for PubMedID 23524517

• Hsp90 Inhibitors Exhibit Resistance-Free Antiviral Activity against Respiratory Syncytial Virus PLOS ONE Geller, R., Andino, R., Frydman, J. 2013; 8 (2)

  Abstract

  Respiratory syncytial virus (RSV) is a major cause of respiratory illness in young children, leading to significant morbidity and mortality worldwide. Despite its medical importance, no vaccine or effective therapeutic interventions are currently available. Therefore, there is a pressing need to identify novel antiviral drugs to combat RSV infections. Hsp90, a cellular protein-folding factor, has been shown to play an important role in the replication of numerous viruses. We here demonstrate that RSV requires Hsp90 for replication. Mechanistic studies reveal that inhibition of Hsp90 during RSV infection leads to the degradation of a viral protein similar in size to the RSV L protein, the viral RNA-dependent RNA polymerase, implicating it as an Hsp90 client protein. Accordingly, Hsp90 inhibitors exhibit antiviral activity against laboratory and clinical isolates of RSV in both immortalized as well as primary differentiated airway epithelial cells. Interestingly, we find a high barrier to the emergence of drug resistance to Hsp90 inhibitors, as extensive growth of RSV under conditions of Hsp90 inhibition did not yield mutants with reduced sensitivity to these drugs. Our results suggest that Hsp90 inhibitors may present attractive antiviral therapeutics for treatment of RSV infections and highlight the potential of chaperone inhibitors as antivirals exhibiting high barriers to development of drug resistance.

  View details for DOI 10.1371/journal.pone.0056762

  View details for Web of Science ID 000315519000036

  View details for PubMedID 23460813

  View details for PubMedCentralID PMC3584091

• Exogenous delivery of chaperonin subunit fragment ApiCCT1 modulates mutant Huntingtin cellular phenotypes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sontag, E. M., Joachimiak, L. A., Tan, Z., Tomlinson, A., Housman, D. E., Glabe, C. G., Potkin, S. G., Frydman, J., Thompson, L. M. 2013; 110 (8): 3077-3082

  Abstract

  Aggregation of misfolded proteins is characteristic of a number of neurodegenerative diseases, including Huntington disease (HD). The CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring) chaperonin complex can inhibit aggregation and cellular toxicity induced by expanded repeat Huntingtin (mHtt) fragments. The substrate-binding apical domain of CCT/TRiC subunit CCT1, ApiCCT1, is sufficient to inhibit aggregation of expanded repeat mHtt fragments in vitro, providing therapeutic promise for HD. However, a key hurdle in considering ApiCCT1 as a potential treatment is in delivery. Because ApiCCT1 has a region of similarity to the HIV Tat protein cell-transduction domain, we tested whether recombinant ApiCCT1 (ApiCCT1(r)) protein could enter cells following exogenous delivery and modulate an established panel of mHtt-mediated cell-based phenotypes. Cell fractionation studies demonstrate that exogenous ApiCCT1(r) can penetrate cell membranes and can localize to the nucleus, consistent with a strategy that can target both cytosolic and nuclear pathogenic events in HD. ApiCCT1(r) application does indeed modulate HD cellular phenotypes by decreasing formation of visible inclusions, fibrillar oligomers, and insoluble mHtt derived from expression of a truncated mHtt exon 1 fragment. ApiCCT1(r) also delays the onset of inclusion body formation as visualized via live imaging. ApiCCT1(r) reduces mHtt-mediated toxicity in immortalized striatal cells derived from full-length knock-in HD mice, suggesting that therapeutic benefit may extend beyond effects on aggregation. These studies provide the basis for a potentially robust and unique therapeutic strategy to target mHtt-mediated protein pathogenesis.

  View details for DOI 10.1073/pnas.1222663110

  View details for Web of Science ID 000315954400093

  View details for PubMedID 23365139

  View details for PubMedCentralID PMC3581981

• Cellular Inclusion Bodies of Mutant Huntingtin Exon 1 Obscure Small Fibrillar Aggregate Species SCIENTIFIC REPORTS Sahl, S. J., Weiss, L. E., Duim, W. C., Frydman, J., Moerner, W. E. 2012; 2

  Abstract

  The identities of toxic aggregate species in Huntington's disease pathogenesis remain ambiguous. While polyQ-expanded huntingtin (Htt) is known to accumulate in compact inclusion bodies inside neurons, this is widely thought to be a protective coping response that sequesters misfolded conformations or aggregated states of the mutated protein. To define the spatial distributions of fluorescently-labeled Htt-exon1 species in the cell model PC12m, we employed highly sensitive single-molecule super-resolution fluorescence imaging. In addition to inclusion bodies and the diffuse pool of monomers and oligomers, fibrillar aggregates -100 nm in diameter and up to -1-2 µm in length were observed for pathogenic polyQ tracts (46 and 97 repeats) after targeted photo-bleaching of the inclusion bodies. These short structures bear a striking resemblance to fibers described in vitro. Definition of the diverse Htt structures in cells will provide an avenue to link the impact of therapeutic agents to aggregate populations and morphologies.

  View details for DOI 10.1038/srep00895

  View details for Web of Science ID 000311891000001

  View details for PubMedID 23193437

  View details for PubMedCentralID PMC3508451

• State of the Science: An Update on Renal Cell Carcinoma MOLECULAR CANCER RESEARCH Jonasch, E., Futreal, P. A., Davis, I. J., Bailey, S. T., Kim, W. Y., Brugarolas, J., Giaccia, A. J., Kurban, G., Pause, A., Frydman, J., Zurita, A. J., Rini, B. I., Sharma, P., Atkins, M. B., Walker, C. L., Rathmell, W. K. 2012; 10 (7): 859-880

  Abstract

  Renal cell carcinomas (RCC) are emerging as a complex set of diseases that are having a major socioeconomic impact and showing a continued rise in incidence throughout the world. As the field of urologic oncology faces these trends, several major genomic and mechanistic discoveries are altering our core understanding of this multitude of cancers, including several new rare subtypes of renal cancers. In this review, these new findings are examined and placed in the context of the well-established association of clear cell RCC (ccRCC) with mutations in the von Hippel-Lindau (VHL) gene and resultant aberrant hypoxia inducible factor (HIF) signaling. The impact of novel ccRCC-associated genetic lesions on chromatin remodeling and epigenetic regulation is explored. The effects of VHL mutation on primary ciliary function, extracellular matrix homeostasis, and tumor metabolism are discussed. Studies of VHL proteostasis, with the goal of harnessing the proteostatic machinery to refunctionalize mutant VHL, are reviewed. Translational efforts using molecular tools to elucidate discriminating features of ccRCC tumors and develop improved prognostic and predictive algorithms are presented, and new therapeutics arising from the earliest molecular discoveries in ccRCC are summarized. By creating an integrated review of the key genomic and molecular biological disease characteristics of ccRCC and placing these data in the context of the evolving therapeutic landscape, we intend to facilitate interaction among basic, translational, and clinical researchers involved in the treatment of this devastating disease, and accelerate progress toward its ultimate eradication.

  View details for DOI 10.1158/1541-7786.MCR-12-0117

  View details for Web of Science ID 000308027300001

  View details for PubMedID 22638109

• Broad action of Hsp90 as a host chaperone required for viral replication BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH Geller, R., Taguwa, S., Frydman, J. 2012; 1823 (3): 698-706

  Abstract

  Viruses are intracellular pathogens responsible for a vast number of human diseases. Due to their small genome size, viruses rely primarily on the biosynthetic apparatus of the host for their replication. Recent work has shown that the molecular chaperone Hsp90 is nearly universally required for viral protein homeostasis. As observed for many endogenous cellular proteins, numerous different viral proteins have been shown to require Hsp90 for their folding, assembly, and maturation. Importantly, the unique characteristics of viral replication cause viruses to be hypersensitive to Hsp90 inhibition, thus providing a novel therapeutic avenue for the development of broad-spectrum antiviral drugs. The major developments in this emerging field are hereby discussed. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).

  View details for DOI 10.1016/j.bbamcr.2011.11.007

  View details for Web of Science ID 000301628700012

  View details for PubMedID 22154817

  View details for PubMedCentralID PMC3339566

• Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycle EMBO JOURNAL Cong, Y., Schroeder, G. F., Meyer, A. S., Jakana, J., Ma, B., Dougherty, M. T., Schmid, M. F., Reissmann, S., Levitt, M., Ludtke, S. L., Frydman, J., Chiu, W. 2012; 31 (3): 720-730

  Abstract

  The eukaryotic group II chaperonin TRiC/CCT is a 16-subunit complex with eight distinct but similar subunits arranged in two stacked rings. Substrate folding inside the central chamber is triggered by ATP hydrolysis. We present five cryo-EM structures of TRiC in apo and nucleotide-induced states without imposing symmetry during the 3D reconstruction. These structures reveal the intra- and inter-ring subunit interaction pattern changes during the ATPase cycle. In the apo state, the subunit arrangement in each ring is highly asymmetric, whereas all nucleotide-containing states tend to be more symmetrical. We identify and structurally characterize an one-ring closed intermediate induced by ATP hydrolysis wherein the closed TRiC ring exhibits an observable chamber expansion. This likely represents the physiological substrate folding state. Our structural results suggest mechanisms for inter-ring-negative cooperativity, intra-ring-positive cooperativity, and protein-folding chamber closure of TRiC. Intriguingly, these mechanisms are different from other group I and II chaperonins despite their similar architecture.

  View details for DOI 10.1038/emboj.2011.366

  View details for Web of Science ID 000300871700019

  View details for PubMedID 22045336

  View details for PubMedCentralID PMC3273382

• Heterozygous Yeast Deletion Collection Screens Reveal Essential Targets of Hsp90 PLOS ONE Franzosa, E. A., Albanese, V., Frydman, J., Xia, Y., McClellan, A. J. 2011; 6 (11)

  Abstract

  Hsp90 is an essential eukaryotic chaperone with a role in folding specific "client" proteins such as kinases and hormone receptors. Previously performed homozygous diploid yeast deletion collection screens uncovered broad requirements for Hsp90 in cellular transport and cell cycle progression. These screens also revealed that the requisite cellular functions of Hsp90 change with growth temperature. We present here for the first time the results of heterozygous deletion collection screens conducted at the hypothermic stress temperature of 15°C. Extensive bioinformatic analyses were performed on the resulting data in combination with data from homozygous and heterozygous screens previously conducted at normal (30°C) and hyperthermic stress (37°C) growth temperatures. Our resulting meta-analysis uncovered extensive connections between Hsp90 and (1) general transcription, (2) ribosome biogenesis and (3) GTP binding proteins. Predictions from bioinformatic analyses were tested experimentally, supporting a role for Hsp90 in ribosome stability. Importantly, the integrated analysis of the 15°C heterozygous deletion pool screen with previously conducted 30°C and 37°C screens allows for essential genetic targets of Hsp90 to emerge. Altogether, these novel contributions enable a more complete picture of essential Hsp90 functions.

  View details for DOI 10.1371/journal.pone.0028211

  View details for Web of Science ID 000298168100051

  View details for PubMedID 22140548

  View details for PubMedCentralID PMC3227642

• Sensing cooperativity in ATP hydrolysis for single multisubunit enzymes in solution PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jiang, Y., Douglas, N. R., Conley, N. R., Miller, E. J., Frydman, J., Moerner, W. E. 2011; 108 (41): 16962-16967

  Abstract

  In order to operate in a coordinated fashion, multisubunit enzymes use cooperative interactions intrinsic to their enzymatic cycle, but this process remains poorly understood. Accordingly, ATP number distributions in various hydrolyzed states have been obtained for single copies of the mammalian double-ring multisubunit chaperonin TRiC/CCT in free solution using the emission from chaperonin-bound fluorescent nucleotides and closed-loop feedback trapping provided by an Anti-Brownian ELectrokinetic trap. Observations of the 16-subunit complexes as ADP molecules are dissociating shows a peak in the bound ADP number distribution at 8 ADP, whose height falls over time with little shift in the position of the peak, indicating a highly cooperative ADP release process which would be difficult to observe by ensemble-averaged methods. When AlFx is added to produce ATP hydrolysis transition state mimics (ADP·AlFx) locked to the complex, the peak at 8 nucleotides dominates for all but the lowest incubation concentrations. Although ensemble averages of the single-molecule data show agreement with standard cooperativity models, surprisingly, the observed number distributions depart from standard models, illustrating the value of these single-molecule observations in constraining the mechanism of cooperativity. While a complete alternative microscopic model cannot be defined at present, the addition of subunit-occupancy-dependent cooperativity in hydrolysis yields distributions consistent with the data.

  View details for DOI 10.1073/pnas.1112244108

  View details for PubMedID 21896715

• Sub-diffraction imaging of huntingtin protein aggregates by fluorescence blink-microscopy and atomic force microscopy. Chemphyschem Duim, W. C., Chen, B., Frydman, J., Moerner, W. E. 2011; 12 (13): 2387-2390

  View details for DOI 10.1002/cphc.201100392

  View details for PubMedID 21735512

  View details for PubMedCentralID PMC3387990

• Sub-Diffraction Imaging of Huntingtin Protein Aggregates by Fluorescence Blink-Microscopy and Atomic Force Microscopy CHEMPHYSCHEM Duim, W. C., Chen, B., Frydman, J., Moerner, W. E. 2011; 12 (13): 2386-2389

  View details for DOI 10.1002/cphc.201100392

  View details for Web of Science ID 000295235000006

  View details for PubMedCentralID PMC3387990

• Cryo-EM Structure of a Group II Chaperonin in the Prehydrolysis ATP-Bound State Leading to Lid Closure STRUCTURE Zhang, J., Ma, B., DiMaio, F., Douglas, N. R., Joachimiak, L. A., Baker, D., Frydman, J., Levitt, M., Chiu, W. 2011; 19 (5): 633-639

  Abstract

  Chaperonins are large ATP-driven molecular machines that mediate cellular protein folding. Group II chaperonins use their "built-in lid" to close their central folding chamber. Here we report the structure of an archaeal group II chaperonin in its prehydrolysis ATP-bound state at subnanometer resolution using single particle cryo-electron microscopy (cryo-EM). Structural comparison of Mm-cpn in ATP-free, ATP-bound, and ATP-hydrolysis states reveals that ATP binding alone causes the chaperonin to close slightly with a ∼45° counterclockwise rotation of the apical domain. The subsequent ATP hydrolysis drives each subunit to rock toward the folding chamber and to close the lid completely. These motions are attributable to the local interactions of specific active site residues with the nucleotide, the tight couplings between the apical and intermediate domains within the subunit, and the aligned interactions between two subunits across the rings. This mechanism of structural changes in response to ATP is entirely different from those found in group I chaperonins.

  View details for DOI 10.1016/j.str.2011.03.005

  View details for Web of Science ID 000290815500006

  View details for PubMedID 21565698

• Trivalent Arsenic Inhibits the Functions of Chaperonin Complex GENETICS Pan, X., Reissman, S., Douglas, N. R., Huang, Z., Yuan, D. S., Wang, X., McCaffery, J. M., Frydman, J., Boeke, J. D. 2010; 186 (2): 725-U434

  Abstract

  The exact molecular mechanisms by which the environmental pollutant arsenic works in biological systems are not completely understood. Using an unbiased chemogenomics approach in Saccharomyces cerevisiae, we found that mutants of the chaperonin complex TRiC and the functionally related prefoldin complex are all hypersensitive to arsenic compared to a wild-type strain. In contrast, mutants with impaired ribosome functions were highly arsenic resistant. These observations led us to hypothesize that arsenic might inhibit TRiC function, required for folding of actin, tubulin, and other proteins postsynthesis. Consistent with this hypothesis, we found that arsenic treatment distorted morphology of both actin and microtubule filaments. Moreover, arsenic impaired substrate folding by both bovine and archaeal TRiC complexes in vitro. These results together indicate that TRiC is a conserved target of arsenic inhibition in various biological systems.

  View details for DOI 10.1534/genetics.110.117655

  View details for Web of Science ID 000282807400023

  View details for PubMedID 20660648

• Crystal Structures of a Group II Chaperonin Reveal the Open and Closed States Associated with the Protein Folding Cycle JOURNAL OF BIOLOGICAL CHEMISTRY Pereira, J. H., Ralston, C. Y., Douglas, N. R., Meyer, D., Knee, K. M., Goulet, D. R., King, J. A., Frydman, J., Adams, P. D. 2010; 285 (36): 27958-27966

  Abstract

  Chaperonins are large protein complexes consisting of two stacked multisubunit rings, which open and close in an ATP-dependent manner to create a protected environment for protein folding. Here, we describe the first crystal structure of a group II chaperonin in an open conformation. We have obtained structures of the archaeal chaperonin from Methanococcus maripaludis in both a peptide acceptor (open) state and a protein folding (closed) state. In contrast with group I chaperonins, in which the equatorial domains share a similar conformation between the open and closed states and the largest motions occurs at the intermediate and apical domains, the three domains of the archaeal chaperonin subunit reorient as a single rigid body. The large rotation observed from the open state to the closed state results in a 65% decrease of the folding chamber volume and creates a highly hydrophilic surface inside the cage. These results suggest a completely distinct closing mechanism in the group II chaperonins as compared with the group I chaperonins.

  View details for DOI 10.1074/jbc.M110.125344

  View details for Web of Science ID 000281404100050

  View details for PubMedID 20573955

• Action of the Chaperonin GroEL/ES on a Non-native Substrate Observed with Single-Molecule FRET JOURNAL OF MOLECULAR BIOLOGY Kim, S. Y., Miller, E. J., Frydman, J., Moerner, W. E. 2010; 401 (4): 553-563

  Abstract

  The double ring-shaped chaperonin GroEL binds a wide range of non-native polypeptides within its central cavity and, together with its cofactor GroES, assists their folding in an ATP-dependent manner. The conformational cycle of GroEL/ES has been studied extensively but little is known about how the environment in the central cavity affects substrate conformation. Here, we use the von Hippel-Lindau tumor suppressor protein VHL as a model substrate for studying the action of the GroEL/ES system on a bound polypeptide. Fluorescent labeling of pairs of sites on VHL for fluorescence (Förster) resonant energy transfer (FRET) allows VHL to be used to explore how GroEL binding and GroEL/ES/nucleotide binding affect the substrate conformation. On average, upon binding to GroEL, all pairs of labeling sites experience compaction relative to the unfolded protein while single-molecule FRET distributions show significant heterogeneity. Upon addition of GroES and ATP to close the GroEL cavity, on average further FRET increases occur between the two hydrophobic regions of VHL, accompanied by FRET decreases between the N- and C-termini. This suggests that ATP- and GroES-induced confinement within the GroEL cavity remodels bound polypeptides by causing expansion (or racking) of some regions and compaction of others, most notably, the hydrophobic core. However, single-molecule observations of the specific FRET changes for individual proteins at the moment of ATP/GroES addition reveal that a large fraction of the population shows the opposite behavior; that is, FRET decreases between the hydrophobic regions and FRET increases for the N- and C-termini. Our time-resolved single-molecule analysis reveals the underlying heterogeneity of the action of GroES/EL on a bound polypeptide substrate, which might arise from the random nature of the specific binding to the various identical subunits of GroEL, and might help explain why multiple rounds of binding and hydrolysis are required for some chaperonin substrates.

  View details for DOI 10.1016/j.jmb.2010.06.050

  View details for Web of Science ID 000281262400001

  View details for PubMedID 20600107

  View details for PubMedCentralID PMC2927214

• 4.0 angstrom Resolution Cryo-EM Structure of the Mammalian Chaperonin TRiC/CCT Reveals its Unique Subunit Arrangement Cong, Y., Baker, M. L., Jakana, J., Woolford, D., Miller, E. J., Reissmann, S., Kumar, R. N., Redding-Johanson, A. M., Batth, T. S., Mukhopadhyay, A., Ludtke, S. J., Frydman, J., Chiu, W. FEDERATION AMER SOC EXP BIOL. 2010

  View details for Web of Science ID 000208675504142

• 4.0-angstrom resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cong, Y., Baker, M. L., Jakana, J., Woolford, D., Miller, E. J., Reissmann, S., Kumar, R. N., Redding-Johanson, A. M., Batth, T. S., Mukhopadhyay, A., Ludtke, S. J., Frydman, J., Chiu, W. 2010; 107 (11): 4967-4972

  Abstract

  The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of approximately 5-10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC's unique heterooligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7-A resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-A resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a Calpha backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed approximately 95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.

  View details for DOI 10.1073/pnas.0913774107

  View details for Web of Science ID 000275714300032

  View details for PubMedID 20194787

  View details for PubMedCentralID PMC2841888

• Conformational Change of a Group II Chaperonin in Different States Revealed by Single-particle Cryo-EM Zhang, J., Baker, M. L., Schroeder, G., Douglas, N. R., Jakana, J., Fu, C. J., Levitt, M., Ludtke, S. J., Frydman, J., Chiu, W. ADENINE PRESS. 2009: 844–44

  View details for Web of Science ID 000266300700101

• The Predicted Structure of the Headpiece of the Huntingtin Protein and Its Implications on Huntingtin Aggregation JOURNAL OF MOLECULAR BIOLOGY Kelley, N. W., Huang, X., Tam, S., Spiess, C., Frydman, J., Pande, V. S. 2009; 388 (5): 919-927

  Abstract

  We have performed simulated tempering molecular dynamics simulations to study the thermodynamics of the headpiece of the Huntingtin (Htt) protein (N17(Htt)). With converged sampling, we found this peptide is highly helical, as previously proposed. Interestingly, this peptide is also found to adopt two different and seemingly stable states. The region from residue 4 (L) to residue 9 (K) has a strong helicity from our simulations, which is supported by experimental studies. However, contrary to what was initially proposed, we have found that simulations predict the most populated state as a two-helix bundle rather than a single straight helix, although a significant percentage of structures do still adopt a single linear helix. The fact that Htt aggregation is nucleation dependent infers the importance of a critical transition. It has been shown that N17(Htt) is involved in this rate-limiting step. In this study, we propose two possible mechanisms for this nucleating event stemming from the transition between two-helix bundle state and single-helix state for N17(Htt) and the experimentally observed interactions between the N17(Htt) and polyQ domains. More strikingly, an extensive hydrophobic surface area is found to be exposed to solvent in the dominant monomeric state of N17(Htt). We propose the most fundamental role played by N17(Htt) would be initializing the dimerization and pulling the polyQ chains into adequate spatial proximity for the nucleation event to proceed.

  View details for DOI 10.1016/j.jmb.2009.01.032

  View details for Web of Science ID 000266121300001

  View details for PubMedID 19361448

  View details for PubMedCentralID PMC2677131

• Conformational Changes of Eukaryotic Chaperonin TRiC/CCT in the Nucleotide Cycle Revealed by CryoEM Cong, Y., Schroeder, G. F., Jakana, J., Reissmann, S., Levitt, M., Ludtke, S. J., Frydman, J., Chiu, W. FEDERATION AMER SOC EXP BIOL. 2009

  View details for Web of Science ID 000208621505494

• Rocking Motion of the Equatorial Domains of a Group II Chaperonin between Two Biochemical States Revealed by Single-Particle Cryo-EM at Near-atomic and Subnanometer Resolutions Zhang, J., Baker, M., Schroeder, G., Douglas, N., Jakana, J., Fu, C., Levitt, M., Ludtke, S., Frydman, J., Chiu, W. FEDERATION AMER SOC EXP BIOL. 2009

  View details for Web of Science ID 000208621505473

• Hardware-based anti-Brownian electrokinetic trap (ABEL trap) for single molecules: Control loop simulations and application to ATP binding stoichiometry in multi-subunit enzymes. Proceedings - Society of Photo-Optical Instrumentation Engineers Jiang, Y. n., Wang, Q. n., Cohen, A. E., Douglas, N. n., Frydman, J. n., Moerner, W. E. 2008; 7038: 1–12

  Abstract

  The hardware-based Anti-Brownian ELectrokinetic trap (ABEL trap) features a feedback latency as short as 25 µs, suitable for trapping single protein molecules in aqueous solution. The performance of the feedback control loop is analyzed to extract estimates of the position variance for various controller designs. Preliminary data are presented in which the trap is applied to the problem of determining the distribution of numbers of ATP bound for single chaperonin multi-subunit enzymes.

  View details for PubMedID 19823693

• Hardware-based anti-Brownian electrokinetic trap (ABEL trap) for single molecules: Control loop simulations and application to ATP binding stoichiometry in multi-subunit enzymes Conference on Optical Trapped and Optical Micromanipulation V Jiang, Y., Wang, Q., Cohen, A. E., Douglas, N., Frydman, J., Moerner, W. E. SPIE-INT SOC OPTICAL ENGINEERING. 2008

  View details for DOI 10.1117/12.798093

  View details for Web of Science ID 000262711000004

• Modeling of possible subunit arrangements in the eukaryotic chaperonin TRiC PROTEIN SCIENCE Miller, E. J., Meyer, A. S., Frydman, J. 2006; 15 (6): 1522-1526

  Abstract

  The eukaryotic cytosolic chaperonin TRiC (TCP-1 Ring Complex), also known as CCT (Cytosolic Chaperonin containing TCP-1), is a hetero-oligomeric complex consisting of two back-to-back rings of eight different subunits each. The general architecture of the complex has been determined, but the arrangement of the subunits within the complex remains an open question. By assuming that the subunits have a defined arrangement within each ring, we constructed a simple model of TRiC that analyzes the possible arrangements of individual subunits in the complex. By applying the model to existing data, we find that there are only four subunit arrangements consistent with previous observations. Our analysis provides a framework for the interpretation and design of experiments to elucidate the quaternary structure of TRiC/CCT. This in turn will aid in the understanding of substrate binding and allosteric properties of this chaperonin.

  View details for DOI 10.1110/ps.052001606

  View details for Web of Science ID 000237927900030

  View details for PubMedID 16672233

  View details for PubMedCentralID PMC2265097

• Chaperonin GroEL and its mutant D87K protect from ischemia in vivo and in vitro 5th Neurobiology of Aging Conference Xu, L. J., Dayal, M., Ouyang, Y. B., Sun, Y. J., Yang, C. F., Frydman, J., Giffard, R. G. ELSEVIER SCIENCE INC. 2006: 562–69

  Abstract

  Protein aggregation and misfolding are central mechanisms of both acute and chronic neurodegeneration. Overexpression of chaperone Hsp70 protects from stroke in animal and cell culture models. Although it is accepted that chaperones protect cells, the mechanism of protection by chaperones in ischemic injury is poorly understood. In particular, the relative importance of preventing protein aggregation compared to facilitating correct protein folding during ischemia and recovery is not known. To test the importance of protein folding and minimize interaction with co-chaperones we studied the bacterial chaperonin GroEL (HSPD1) and a folding-deficient mutant D87K. Both molecules protected cells from ischemia-like injury, and reduced infarct volume and improved neurological outcome after middle cerebral artery occlusion in rats. Protection was associated with reduced protein aggregation, assessed by ubiquitin immunohistochemistry. Marked neuroprotection by the folding-deficient chaperonin demonstrates that inhibition of aggregation is sufficient to protect the brain from ischemia. This suggests that strategies to maintain protein solubility and inhibit aggregation in the face of acute insults such as stroke may be a useful protective strategy.

  View details for DOI 10.1016/j.neurobiolaging.2005.09.032

  View details for Web of Science ID 000236066200005

  View details for PubMedID 16257478

• Probing the sequence of conformationally induced polarity changes in the molecular chaperonin GroEL with fluorescence spectroscopy JOURNAL OF PHYSICAL CHEMISTRY B Kim, S. Y., Semyonov, A. N., TWIEG, R. J., Horwich, A. L., Frydman, J., Moerner, W. E. 2005; 109 (51): 24517-24525

  Abstract

  Hydrophobic interactions play a major role in binding non-native substrate proteins in the central cavity of the bacterial chaperonin GroEL. The sequence of local conformational changes by which GroEL and its cofactor GroES assist protein folding can be explored using the polarity-sensitive fluorescence probe Nile Red. A specific single-cysteine mutant of GroEL (Cys261), whose cysteine is located inside the central cavity at the apical region of the protein, was covalently labeled with synthetically prepared Nile Red maleimide (NR). Bulk fluorescence spectra of Cys261-NR were measured to examine the effects of binding of the stringent substrate, malate dehydrogenase (MDH), GroES, and nucleotide on the local environment of the probe. After binding denatured substrate, the fluorescence intensity increased by 32 +/- 7%, suggesting enhanced hydrophobicity at the position of the label. On the other hand, in the presence of ATP, the fluorescence intensity decreased by 13 +/- 3%, implying increased local polarity. To explore the sequence of local polarity changes, substrate, GroES, and various nucleotides were added in different orders; the resulting changes in emission intensity provide insight into the sequence of conformational changes occurring during GroEL-mediated protein folding.

  View details for DOI 10.1021/jp0534232

  View details for Web of Science ID 000234259900043

  View details for PubMedID 16375456

  View details for PubMedCentralID PMC1414071

• Hsp110 cooperates with different cytosolic Hsp70 systems in a pathway for de novo folding JOURNAL OF BIOLOGICAL CHEMISTRY Yam, A. Y., Albanese, V., Lin, H. T., Frydman, J. 2005; 280 (50): 41252-41261

  Abstract

  Molecular chaperones such as Hsp70 use ATP binding and hydrolysis to prevent aggregation and ensure the efficient folding of newly translated and stress-denatured polypeptides. Eukaryotic cells contain several cytosolic Hsp70 subfamilies. In yeast, these include the Hsp70s SSB and SSA as well as the Hsp110-like Sse1/2p. The cellular functions and interplay between these different Hsp70 systems remain ill-defined. Here we show that the different cytosolic Hsp70 systems functionally interact with Hsp110 to form a chaperone network that interacts with newly translated polypeptides during their biogenesis. Both SSB and SSA Hsp70s form stable complexes with the Hsp110 Sse1p. Pulse-chase analysis indicates that these Hsp70/Hsp110 teams, SSB/SSE and SSA/SSE, transiently associate with newly synthesized polypeptides with different kinetics. SSB Hsp70s bind cotranslationally to a large fraction of nascent chains, suggesting an early role in the stabilization of nascent chains. SSA Hsp70s bind mostly post-translationally to a more restricted subset of newly translated polypeptides, suggesting a downstream function in the folding pathway. Notably, loss of SSB dramatically enhances the cotranslational association of SSA with nascent chains, suggesting SSA can partially fulfill an SSB-like function. On the other hand, the absence of SSE1 enhances polypeptide binding to both SSB and SSA and impairs cell growth. It, thus, appears that Hsp110 is an important regulator of Hsp70-substrate interactions. Based on our data, we propose that Hsp110 cooperates with the SSB and SSA Hsp70 subfamilies, which act sequentially during de novo folding.

  View details for DOI 10.1074/jbc.M503615200

  View details for Web of Science ID 000233866900019

  View details for PubMedID 16219770

• The cotranslational contacts between ribosome-bound nascent polypeptides and the subunits of the hetero-oligomeric chaperonin TRiC probed by photocross-linking JOURNAL OF BIOLOGICAL CHEMISTRY Etchells, S. A., Meyer, A. S., Yam, A. Y., Roobol, A., Miao, Y. W., Shao, Y. L., Carden, M. J., Skach, W. R., Frydman, J., Johnson, A. E. 2005; 280 (30): 28118-28126

  Abstract

  The hetero-oligomeric eukaryotic chaperonin TRiC (TCP-1-ring complex, also called CCT) interacts cotranslationally with a diverse subset of newly synthesized proteins, including actin, tubulin, and luciferase, and facilitates their correct folding. A photocross-linking approach has been used to map the contacts between individual chaperonin subunits and ribosome-bound nascent chains of increasing length. Whereas a cryo-EM study suggests that chemically denatured actin interacts with only two TRiC subunits (delta and either beta or epsilon), actin and luciferase chains photocross-link to at least six TRiC subunits (alpha, beta, delta, epsilon, xi, and theta) at different stages of translation. Furthermore, the photocross-linking of actin, but not luciferase, nascent chains to TRiC subunits zeta and theta was length-dependent. In addition, a single photoreactive probe incorporated at a unique site in actin nascent chains of different lengths reacted covalently with multiple TRiC subunits, thereby indicating that the nascent chain samples the polypeptide binding sites of different subunits. We conclude that elongating actin and luciferase nascent chains contact multiple TRiC subunits upon emerging from the ribosome, and that the TRiC subunits contacted by nascent actin change as it elongates and starts to fold.

  View details for DOI 10.1074/jbc.M504110200

  View details for Web of Science ID 000230678600075

  View details for PubMedID 15929940

• Actin mutations in hypertrophic and dilated cardiomyopathy cause inefficient protein folding and perturbed filament formation FEBS JOURNAL Vang, S., Corydon, T. J., Borglum, A. D., Scott, M. D., Frydman, J., Mogensen, J., Gregersen, N., Bross, P. 2005; 272 (8): 2037-2049

  Abstract

  Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are the most common hereditary cardiac conditions. Both are frequent causes of sudden death and are often associated with an adverse disease course. Alpha-cardiac actin is one of the disease genes where different missense mutations have been found to cause either HCM or DCM. We have tested the hypothesis that the protein-folding pathway plays a role in disease development for two actin variants associated with DCM and six associated with HCM. Based on a cell-free coupled translation assay the actin variants could be graded by their tendency to associate with the chaperonin TCP-1 ring complex/chaperonin containing TCP-1 (TRiC/CCT) as well as their propensity to acquire their native conformation. Some variant proteins are completely stalled in a complex with TRiC and fail to fold into mature globular actin and some appear to fold as efficiently as the wild-type protein. A fraction of the translated polypeptide became ubiquitinated and detergent insoluble. Variant actin proteins overexpressed in mammalian cell lines fail to incorporate into actin filaments in a manner correlating with the degree of misfolding observed in the cell-free assay; ranging from incorporation comparable to wild-type actin to little or no incorporation. We propose that effects of mutations on folding and fiber assembly may play a role in the molecular disease mechanism.

  View details for DOI 10.1111/j.1742-4658.2005.04630.x

  View details for Web of Science ID 000228266700017

  View details for PubMedID 15819894

• The Hsp70 and TRiC/CCT chaperone systems cooperate in vivo to assemble the von Hippel-Lindau tumor suppressor complex MOLECULAR AND CELLULAR BIOLOGY Melville, M. W., McClellan, A. J., Meyer, A. S., Darveau, A., Frydman, J. 2003; 23 (9): 3141-3151

  Abstract

  The degree of cooperation and redundancy between different chaperones is an important problem in understanding how proteins fold in the cell. Here we use the yeast Saccharomyces cerevisiae as a model system to examine in vivo the chaperone requirements for assembly of the von Hippel-Lindau protein (VHL)-elongin BC (VBC) tumor suppressor complex. VHL and elongin BC expressed in yeast assembled into a correctly folded VBC complex that resembles the complex from mammalian cells. Unassembled VHL did not fold and remained associated with the cytosolic chaperones Hsp70 and TRiC/CCT, in agreement with results from mammalian cells. Analysis of the folding reaction in yeast strains carrying conditional chaperone mutants indicates that incorporation of VHL into VBC requires both functional TRiC and Hsp70. VBC assembly was defective in cells carrying either a temperature-sensitive ssa1 gene as their sole source of cytosolic Hsp70/SSA function or a temperature-sensitive mutation in CCT4, a subunit of the TRiC/CCT complex. Analysis of the VHL-chaperone interactions in these strains revealed that the cct4ts mutation decreased binding to TRiC but did not affect the interaction with Hsp70. In contrast, loss of Hsp70 function disrupted the interaction of VHL with both Hsp70 and TRiC. We conclude that, in vivo, folding of some polypeptides requires the cooperation of Hsp70 and TRiC and that Hsp70 acts to promote substrate binding to TRiC.

  View details for DOI 10.1128/MCB.23.9.3141-3151.2003

  View details for Web of Science ID 000182325500010

  View details for PubMedID 12697815

  View details for PubMedCentralID PMC153194

• Aberrant protein folding as the molecular basis of cancer. Methods in molecular biology (Clifton, N.J.) Scott, M. D., Frydman, J. 2003; 232: 67-76

  View details for PubMedID 12840540

• Review: Cellular substrates of the eukaryotic chaperonin TRiC/CCT JOURNAL OF STRUCTURAL BIOLOGY Dunn, A. Y., Melville, M. W., Frydman, J. 2001; 135 (2): 176-184

  Abstract

  The TCP-1 ring complex (TRiC; also called CCT, for chaperonin containing TCP-1) is a large (approximately 900 kDa) multisubunit complex that mediates protein folding in the eukaryotic cytosol. The physiological substrate spectrum of TRiC is still poorly defined. Genetic and biochemical data show that it is required for the folding of the cytoskeletal proteins actin and tubulin. Recent years have witnessed a steady stream of reports that describe other proteins that require TRiC for proper folding. Furthermore, analysis of the transit of newly synthesized proteins through TRiC in intact cells suggests that the chaperonin contributes to the folding of a distinct subset of cellular proteins. Here we review the current understanding of a role for TRiC in the folding of newly synthesized polypeptides, with a focus on some of the individual proteins that require TRiC.

  View details for Web of Science ID 000171545800011

  View details for PubMedID 11580267

• Protein folding in vivo: the importance of molecular chaperones CURRENT OPINION IN STRUCTURAL BIOLOGY Feldman, D. E., Frydman, J. 2000; 10 (1): 26-33

  Abstract

  The contribution of the two major cytosolic chaperone systems, Hsp70 and the cylindrical chaperonins, to cellular protein folding has been clarified by a number of recent papers. These studies found that, in vivo, a significant fraction of newly synthesized polypeptides transit through these chaperone systems in both prokaryotic and eukaryotic cells. The identification and characterization of the cellular substrates of chaperones will be instrumental in understanding how proteins fold in vivo.

  View details for Web of Science ID 000085529200003

  View details for PubMedID 10679467

• Purification of the cytosolic chaperonin TRiC from bovine testis. Methods in molecular biology (Clifton, N.J.) Ferreyra, R. G., Frydman, J. 2000; 140: 153-160

  View details for PubMedID 11484484

• Folding assays. Assessing the native conformation of proteins. Methods in molecular biology (Clifton, N.J.) Thulasiraman, V., Ferreyra, R. G., Frydman, J. 2000; 140: 169-177

  View details for PubMedID 11484486

• Monitoring actin folding. Purification protocols for labeled proteins and binding to DNase I-sepharose beads. Methods in molecular biology (Clifton, N.J.) Thulasiraman, V., Ferreyra, R. G., Frydman, J. 2000; 140: 161-167

  View details for PubMedID 11484485

• Directionality of polypeptide transfer in the mitochondrial pathway of chaperone-mediated protein folding BIOLOGICAL CHEMISTRY Heyrovska, N., Frydman, J., Hohfeld, J., Hartl, F. U. 1998; 379 (3): 301-309

  Abstract

  Protein folding in mitochondria depends on the functional cooperation of the Hsp70 and Hsp60 chaperone systems, at least for a subset of mitochondrial polypeptides. As suggested previously, Hsp70 and Hsp60 act sequentially. However, recent proposals that the chaperonin Hsp60 functions by releasing substrate protein in an unfolded state would predict a lateral partitioning of folding intermediates between chaperone systems. Firefly luciferase, carrying a mitochondrial targeting signal, was used as a model protein to analyze the degree of coupling and the directionality of substrate transfer between the Hsp70 and Hsp60 chaperones. In vitro, Hsp60 binds unfolded luciferase with high affinity but is unable to promote its folding, whereas the Hsp70 system assists the folding of luciferase efficiently. Upon import into yeast mitochondria, luciferase interacted first with Hsp70. Surprisingly, most of the protein subsequently accumulated in a complex with Hsp60 and never reached the native state. Import into mitochondria that lack a functional Hsp60 did not result in increased folding, but in the aggregation of luciferase. Thus, in intact organelles the two chaperone systems do not function independently in de novo folding of aggregation-sensitive proteins but rather act in an ordered pathway with substrate transfer predominantly in the direction from Hsp70 to Hsp60.

  View details for Web of Science ID 000072865800011

  View details for PubMedID 9563826

• Chaperones get in touch: The hip-hop connection TRENDS IN BIOCHEMICAL SCIENCES Frydman, J., Hohfeld, J. 1997; 22 (3): 87-92

  Abstract

  Recent findings emphasize that different molecular chaperones cooperate during intracellular protein biogenesis. Mechanistic aspects of chaperone cooperation are now emerging from studies on the regulation of certain signal transduction pathways mediated by Hsc70 and Hsp90 in the eukaryotic cytosol. Efficient cooperation appears to be achieved through a defined regulation of Hsc70 activity by the chaperone cofactors Hip and Hop.

  View details for Web of Science ID A1997WM12900005

  View details for PubMedID 9066258

• TCP20, A SUBUNIT OF THE EUKARYOTIC TRIC CHAPERONIN FROM HUMANS AND YEAST JOURNAL OF BIOLOGICAL CHEMISTRY Li, W. Z., Lin, P., Frydman, J., Boal, T. R., Cardillo, T. S., RICHARD, L. M., Toth, D., Lichtman, M. A., Hartl, F. U., Sherman, F., Segel, G. B. 1994; 269 (28): 18616-18622

  Abstract

  Members of the Hsp60 chaperonin family, such as Escherichia coli GroEL/S and the eukaryotic cytosolic chaperonin complex, TRiC (TCP ring complex), are double toroid complexes capable of assisting the folding of proteins in vitro in an ATP-dependent fashion. TRiC differs from the GroEL chaperonin in that it has a hetero rather than homo-oligomeric subunit composition and lacks a GroES-like regulatory cofactor. We have established greater than 57% identity between a protein encoded by the TCP20 gene from a human cDNA library and the newly identified protein encoded by the TCP20 gene located on the right arm of chromosome IV of the yeast Saccharomyces cerevisiae. These Tcp20 proteins showed approximately 30% identity to Tcp1, a known subunit of TRiC. Gel filtration, followed by Western analysis of purified bovine testis TRiC with a Tcp20-specific antibody, indicated that Tcp20 is a subunit of the hetero-oligomeric TRiC. Gene disruption experiments showed that TCP20, like TCP1, is an essential gene in yeast, consistent with the view that TRiC is required for folding of key proteins. The amino acid sequence similarities and the derived evolutionary relationships established that the human and yeast Tcp20 proteins represent members of a new family of subunits of TRiC chaperonins.

  View details for Web of Science ID A1994NW79800061

  View details for PubMedID 8034610

• FUNCTION IN PROTEIN FOLDING OF TRIC, A CYTOSOLIC RING COMPLEX CONTAINING TCP-1 AND STRUCTURALLY RELATED SUBUNITS EMBO JOURNAL Frydman, J., Nimmesgern, E., ERDJUMENTBROMAGE, H., Wall, J. S., Tempst, P., Hartl, F. U. 1992; 11 (13): 4767-4778

  Abstract

  T-complex polypeptide 1 (TCP-1) was analyzed as a potential chaperonin (GroEL/Hsp60) equivalent of the eukaryotic cytosol. We found TCP-1 to be part of a hetero-oligomeric 970 kDa complex containing several structurally related subunits of 52-65 kDa. These members of a new protein family are assembled into a TCP-1 ring complex (TRiC) which resembles the GroEL double ring. The main function of TRiC appears to be in chaperoning monomeric protein folding: TRiC binds unfolded polypeptides, thereby preventing their aggregation, and mediates the ATP-dependent renaturation of unfolded firefly luciferase and tubulin. At least in vitro, TRiC appears to function independently of a small co-chaperonin protein such as GroES. Folding of luciferase is mediated by TRiC but not by GroEL/ES. This suggests that the range of substrate proteins interacting productively with TRiC may differ from that of GroEL. We propose that TRiC mediates the folding of cytosolic proteins by a mechanism distinct from that of the chaperonins in specific aspects.

  View details for Web of Science ID A1992KC83700011

  View details for PubMedID 1361170

• AN ATP-STABILIZED INHIBITOR OF THE PROTEASOME IS A COMPONENT OF THE 1500-KDA UBIQUITIN CONJUGATE-DEGRADING COMPLEX PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Driscoll, J., Frydman, J., Goldberg, A. L. 1992; 89 (11): 4986-4990

  Abstract

  Proteins conjugated to ubiquitin are degraded by a 26S (1500-kDa) proteolytic complex that, in reticulocyte extracts, can be formed by the association of three factors: CF-1, CF-2, and CF-3. One of these factors, CF-3, has been shown to be the proteasome, a 650-kDa multicatalytic protease complex. We have purified a 250-kDa inhibitor of the proteasome and shown that it corresponds to CF-2. In the presence or absence of ATP, this factor inhibited hydrolysis by the proteasome of both fluorogenic tetrapeptides and protein substrates. When the inhibitor, proteasome, and CF-1 were incubated together in the presence of ATP and Mg2+, degradation of ubiquitin-125I-lysozyme occurred. Both the inhibitory activity and the ability to reconstitute ubiquitin-125I-lysozyme degradation were very labile at 42 degrees C, but both activities were stabilized by ATP or a nonhydrolyzable ATP analog. SDS/PAGE indicated that the 250-kDa inhibitor fraction contained a major subunit of 40 kDa (plus some minor bands). The 125I-labeled inhibitor and purified proteasome formed a complex. When CF-1, ATP, and Mg2+ were also present, the 125I-labeled inhibitor along with the proteasome formed a complex of 1500 kDa. The inhibitor (CF-2) thus appears to be an ATP-binding component that regulates proteolysis within the 1500-kDa complex.

  View details for Web of Science ID A1992HX16800043

  View details for PubMedID 1317579