Current Research and Scholarly Interests
The long term goal of our research is to understand how proteins fold in living cells. My lab uses a multidisciplinary approach to address fundamental questions about molecular chaperones, protein folding and degradation. In addition to basic mechanistic principles, we aim to define how impairment of cellular folding and quality control are linked to disease, including cancer and neurodegenerative diseases and examine whether reengineering chaperone networks can provide therapeutic strategies.
Independent Studies (11)
- Advanced Research Laboratory in Experimental Biology
BIO 199 (Aut, Win, Spr, Sum)
- Directed Reading in Biology
BIO 198 (Aut, Win, Spr, Sum)
- Directed Reading in Biophysics
BIOPHYS 399 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Win, Spr, Sum)
- Graduate Research
BIO 300 (Aut, Win, Spr, Sum)
- Graduate Research
BIOPHYS 300 (Aut, Win, Spr, Sum)
- Graduate Research
CBIO 399 (Aut, Win, Spr, Sum)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Sum)
- Out-of-Department Directed Reading
BIO 198X (Aut, Win, Spr, Sum)
- Out-of-Department Graduate Research
BIO 300X (Aut, Win, Spr, Sum)
- Teaching of Biology
BIO 290 (Aut, Win, Spr)
- Advanced Research Laboratory in Experimental Biology
- Prior Year Courses
- The Structural Basis of Substrate Recognition by the Eukaryotic Chaperonin TRiC/CCT CELL 2014; 159 (5): 1042-1055
- 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 2014; 111 (26): 9455-9460
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
2014; 111 (26): 9455-9460
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
Interplay between chaperones and protein disorder promotes the evolution of protein networks.
PLoS computational biology
2014; 10 (6)
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
Modulation of STAT3 Folding and Function by TRiC/CCT Chaperonin
2014; 12 (4)
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 Web of Science ID 000335458800010
View details for PubMedID 24756126
Sorting out the trash: the spatial nature of eukaryotic protein quality control
CURRENT OPINION IN CELL BIOLOGY
2014; 26: 139-146
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
2013; 15 (10): 1231-1243
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 2013; 15 (10): 1231-U253
- TRiC's tricks inhibit huntingtin aggregation ELIFE 2013; 2
Principles of Cotranslational Ubiquitination and Quality Control at the Ribosome
2013; 50 (3): 379-393
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 Web of Science ID 000319183500009
The role of mutational robustness in RNA virus evolution
NATURE REVIEWS MICROBIOLOGY
2013; 11 (5): 327-336
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 Web of Science ID 000318142600012
View details for PubMedID 23524517
Hsp90 Inhibitors Exhibit Resistance-Free Antiviral Activity against Respiratory Syncytial Virus
2013; 8 (2)
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
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
2013; 110 (8): 3077-3082
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
The Ribosome as a Hub for Protein Quality Control
2013; 49 (3): 411-421
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
Evolutionary conservation of codon optimality reveals hidden signatures of cotranslational folding.
Nature structural & molecular biology
2013; 20 (2): 237-243
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
- Evolutionary conservation of codon optimality reveals hidden signatures of cotranslational folding NATURE STRUCTURAL & MOLECULAR BIOLOGY 2013; 20 (2): 237-243
The Cotranslational Function of Ribosome-Associated Hsp70 in Eukaryotic Protein Homeostasis
2013; 152 (1-2): 196-209
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
TRiC's tricks inhibit huntingtin aggregation.
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
Cellular Inclusion Bodies of Mutant Huntingtin Exon 1 Obscure Small Fibrillar Aggregate Species
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
A Gradient of ATP Affinities Generates an Asymmetric Power Stroke Driving the Chaperonin TRIC/CCT Folding Cycle
2012; 2 (4): 866-877
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
Systematic Functional Prioritization of Protein Posttranslational Modifications
2012; 150 (2): 413-425
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
State of the Science: An Update on Renal Cell Carcinoma
MOLECULAR CANCER RESEARCH
2012; 10 (7): 859-880
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
The Molecular Architecture of the Eukaryotic Chaperonin TRiC/CCT
2012; 20 (5): 814-825
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
Broad action of Hsp90 as a host chaperone required for viral replication
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH
2012; 1823 (3): 698-706
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
Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycle
2012; 31 (3): 720-730
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
Heterozygous Yeast Deletion Collection Screens Reveal Essential Targets of Hsp90
2011; 6 (11)
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
Sensing cooperativity in ATP hydrolysis for single multisubunit enzymes in solution
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (41): 16962-16967
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 Web of Science ID 000295973800024
View details for PubMedID 21896715
- Sub-diffraction imaging of huntingtin protein aggregates by fluorescence blink-microscopy and atomic force microscopy. Chemphyschem 2011; 12 (13): 2387-2390
- Sub-Diffraction Imaging of Huntingtin Protein Aggregates by Fluorescence Blink-Microscopy and Atomic Force Microscopy CHEMPHYSCHEM 2011; 12 (13): 2386-2389
Cellular Strategies of Protein Quality Control
COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY
2011; 3 (8)
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
2011; 9 (7)
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
Cryo-EM Structure of a Group II Chaperonin in the Prehydrolysis ATP-Bound State Leading to Lid Closure
2011; 19 (5): 633-639
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
Dual Action of ATP Hydrolysis Couples Lid Closure to Substrate Release into the Group II Chaperonin Chamber
2011; 144 (2): 240-252
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
Trivalent Arsenic Inhibits the Functions of Chaperonin Complex
2010; 186 (2): 725-U434
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
2010; 285 (36): 27958-27966
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
2010; 401 (4): 553-563
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
A ribosome-anchored chaperone network that facilitates eukaryotic ribosome biogenesis
JOURNAL OF CELL BIOLOGY
2010; 189 (1): 69-U105
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
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
2010; 107 (11): 4967-4972
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
Mechanism of folding chamber closure in a group II chaperonin
2010; 463 (7279): 379-U130
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
The chaperonin TRiC blocks a huntingtin sequence element that promotes the conformational switch to aggregation
NATURE STRUCTURAL & MOLECULAR BIOLOGY
2009; 16 (12): 1279-U98
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
The Predicted Structure of the Headpiece of the Huntingtin Protein and Its Implications on Huntingtin Aggregation
JOURNAL OF MOLECULAR BIOLOGY
2009; 388 (5): 919-927
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
- The Hsp90 mosaic: a picture emerges NATURE STRUCTURAL & MOLECULAR BIOLOGY 2009; 16 (1): 2-6
Defining the TRiC/CCT interactome links chaperonin function to stabilization of newly made proteins with complex topologies
NATURE STRUCTURAL & MOLECULAR BIOLOGY
2008; 15 (12): 1255-1262
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
Misfolded proteins partition between two distinct quality control compartments
2008; 454 (7208): 1088-U36
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
Mechanism of lid closure in the eukaryotic chaperonin TRiC/CCT
NATURE STRUCTURAL & MOLECULAR BIOLOGY
2008; 15 (7): 746-753
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
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
2008; 7038: 1-12
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
Diverse cellular functions of the Hsp90 molecular chaperone uncovered using systems approaches
2007; 131 (1): 121-135
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
2007; 14 (5): 432-440
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
Evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance
GENES & DEVELOPMENT
2007; 21 (2): 195-205
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
Identification of the TRiC/CCT substrate binding sites uncovers the function of subunit diversity in eukaryotic
2006; 24 (1): 25-37
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
The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions
NATURE CELL BIOLOGY
2006; 8 (10): 1155-U211
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
Modeling of possible subunit arrangements in the eukaryotic chaperonin TRiC
2006; 15 (6): 1522-1526
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
Chaperonin GroEL and its mutant D87K protect from ischemia in vivo and in vitro
NEUROBIOLOGY OF AGING
2006; 27 (4): 562-569
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
Systems analyses reveal two chaperone networks with distinct functions in eukaryotic cells
2006; 124 (1): 75-88
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
Probing the sequence of conformationally induced polarity changes in the molecular chaperonin GroEL with fluorescence spectroscopy
JOURNAL OF PHYSICAL CHEMISTRY B
2005; 109 (51): 24517-24525
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
Hsp110 cooperates with different cytosolic Hsp70 systems in a pathway for de novo folding
JOURNAL OF BIOLOGICAL CHEMISTRY
2005; 280 (50): 41252-41261
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
Protein quality control: chaperones culling corrupt conformations
NATURE CELL BIOLOGY
2005; 7 (8): 736-741
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
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
2005; 280 (30): 28118-28126
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
Folding and quality control of the VHL tumor suppressor proceed through distinct chaperone pathways
2005; 121 (5): 739-748
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
Actin mutations in hypertrophic and dilated cardiomyopathy cause inefficient protein folding and perturbed filament formation
2005; 272 (8): 2037-2049
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
Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets
TRENDS IN CELL BIOLOGY
2004; 14 (11): 598-604
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
Tumorigenic mutations in VHL disrupt folding in vivo by interfering with chaperonin binding
2003; 12 (5): 1213-1224
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
2003; 113 (3): 369-381
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
The Hsp70 and TRiC/CCT chaperone systems cooperate in vivo to assemble the von Hippel-Lindau tumor suppressor complex
MOLECULAR AND CELLULAR BIOLOGY
2003; 23 (9): 3141-3151
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
Aberrant protein folding as the molecular basis of cancer.
Methods in molecular biology (Clifton, N.J.)
2003; 232: 67-76
View details for PubMedID 12840540
- Where chaperones and nascent polypeptides meet NATURE STRUCTURAL BIOLOGY 2002; 9 (10): 716-718
Review: Cellular substrates of the eukaryotic chaperonin TRiC/CCT
JOURNAL OF STRUCTURAL BIOLOGY
2001; 135 (2): 176-184
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
- Molecular chaperones and the art of recognizing a lost cause NATURE CELL BIOLOGY 2001; 3 (2): E51-E53
Folding of newly translated proteins in vivo: The role of molecular chaperones
ANNUAL REVIEW OF BIOCHEMISTRY
2001; 70: 603-647
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
2000; 149 (3): 591-601
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
Protein folding in vivo: the importance of molecular chaperones
CURRENT OPINION IN STRUCTURAL BIOLOGY
2000; 10 (1): 26-33
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.)
2000; 140: 153-160
View details for PubMedID 11484484
Folding assays. Assessing the native conformation of proteins.
Methods in molecular biology (Clifton, N.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.)
2000; 140: 161-167
View details for PubMedID 11484485
Formation of the VHL-elongin BC tumor suppressor complex is mediated by the chaperonin TRiC
1999; 4 (6): 1051-1061
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
1999; 6 (7): 697-705
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
1999; 18 (1): 85-95
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
Directionality of polypeptide transfer in the mitochondrial pathway of chaperone-mediated protein folding
1998; 379 (3): 301-309
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
1997; 22 (3): 87-92
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
Principles of chaperone-assisted protein folding: Differences between in vitro and in vivo mechanisms
1996; 272 (5267): 1497-1502
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
TCP20, A SUBUNIT OF THE EUKARYOTIC TRIC CHAPERONIN FROM HUMANS AND YEAST
JOURNAL OF BIOLOGICAL CHEMISTRY
1994; 269 (28): 18616-18622
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
FOLDING OF NASCENT POLYPEPTIDE-CHAINS IN A HIGH-MOLECULAR-MASS ASSEMBLY WITH MOLECULAR CHAPERONES
1994; 370 (6485): 111-117
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
FUNCTION IN PROTEIN FOLDING OF TRIC, A CYTOSOLIC RING COMPLEX CONTAINING TCP-1 AND STRUCTURALLY RELATED SUBUNITS
1992; 11 (13): 4767-4778
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
1992; 89 (11): 4986-4990
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
4.0 angstrom Resolution Cryo-EM Structure of the Mammalian Chaperonin TRiC/CCT Reveals its Unique Subunit Arrangement
FEDERATION AMER SOC EXP BIOL. 2010
View details for Web of Science ID 000208675504142
Conformational Changes of Eukaryotic Chaperonin TRiC/CCT in the Nucleotide Cycle Revealed by CryoEM
FEDERATION AMER SOC EXP BIOL. 2009
View details for Web of Science ID 000208621505494