Frydman Lab | Stanford University

Lab interests

The long term goal of our research is to understand how proteins fold in living cells. The Frydman 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.

Function and mechanism of the eukaryotic chaperonin TRiC
Chaperone networks, ribosome biogenesis and co-translational folding
Protein homeostasis, quality-control and selective degradation
Misfolding and Aggregation Diseases

Funding

Function and mechanism of the eukaryotic chaperonin TRiC

Douglas NR, Reissmann S, Zhang J, Chen B, Jakana J, Kumar R, Chiu W, Frydman J. Dual Action of ATP Hydrolysis Couples Lid Closure to Substrate Release into the Group II Chaperonin Chamber. Cell 144:240-52 (2011).

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.

Zhang J, Baker ML, Schröder GF, Douglas NR, Reissmann S, Jakana J, Dougherty M, Fu CJ, Levitt M, Ludtke SJ, Frydman J, Chiu W. Mechanism of folding chamber closure in a group II chaperonin. Nature 463:379-83 (2010)

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.

Yam AY, Xia Y, Lin HT, Burlingame A, Gerstein M, Frydman J. Defining the TRiC/CCT interactome links chaperonin function to stabilization of newly made proteins with complex topologies. Nat Struct Mol Biol 15:1255-62 (2008)

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.

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Chaperone networks, ribosome biogenesis and co-translational folding

Albanese V, Reissmann S, Frydman J. A ribosome-anchored chaperone network that facilitates eukaryotic ribosome biogenesis. J Cell Biol 189:69-81 (2010).

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.

McClellan AJ, Xia Y, Deutschbauer AM, Davis RW, Gerstein M, Frydman J. Diverse cellular functions of the Hsp90 molecular chaperone uncovered using systems approaches. Cell 131:121-35 (2007)

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.

Albanèse V, Yam AY, Baughman J, Parnot C, Frydman . Systems analyses reveal two chaperone networks with distinct functions in eukaryotic cells. Cell 124:75-88 (2006)

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.

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Protein homeostasis, quality-control and selective degradation

Kaganovich D, Kopito R, Frydman J. Misfolded proteins partition between two distinct quality control compartments. Nature 454:1088-95 (2008)

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.

McClellan AJ, Scott MD, Frydman J. Folding and quality control of the VHL tumor suppressor proceed through distinct chaperone pathways. Cell 121:739-48 (2005)

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.

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Misfolding and Aggregation Diseases

Tam S, Spiess C, Auyeung W, Joachimiak L, Chen B, Poirier MA, Frydman J. The chaperonin TRiC blocks a huntingtin sequence element that promotes the conformational switch to aggregation. Nat Struct Mol Biol 16:1279-85 (2009)

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 pr ovides a novel target for therapeutic strategies.

Tam S, Geller R, Spiess C, Frydman J. The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions. Nat Cell Biol 8:1155-62 (2006)

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.

Feldman DE, Spiess C, Howard DE, Frydman J. Tumorigenic mutations in VHL disrupt folding in vivo by interfering with chaperonin binding. Mol Cell 12:1213-24 (2003)

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.

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