Molecular chaperones and protein folding in the cell

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.

Cryo-EM structure of Mm-Cpn (group II chaperonin)

 

News and recent selected publications

  • Escusa-Toret S, Vonk WI, Frydman J. Spatial sequestration of misfolded proteins by a dynamic chaperone pathway enhances cellular fitness during stress. Nat Cell Biol doi: 10.1038/ncb2838 (2013).
    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.

  • Duttler S, Pechmann S, Frydman J. Principles of Cotranslational Ubiquitination and Quality Control at the Ribosome Mol Cell 50(3): 379-393 (2013).
    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.

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

  • Willmund F, del Alamo M, Pechmann S, Chen T, Albanese V, Dammer EB, Peng J, Frydman J The cotranslational function of ribosome-associated Hsp70 in eukaryotic protein homeostasis Cell 152:196-209 (2013).
    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.
       
  • 'Rhythm' of protein folding encoded in RNA, Stanford biologists find Stanford Report, January, 2013. Read the full story on

    Multiple RNA sequences can code for the same amino acid, but differences in their respective "optimality" slow or accelerate protein translation. Stanford biologists find optimal and non-optimal codons are consistently associated with specific protein structures, suggesting that they influence the mysterious process of protein folding.

       
  • Pechmann S & Frydman J Evolutionary conservation of codon optimality reveals hidden signatures of co-translational folding. Nat Struct Mol Biol (2012) | doi:10.1038/nsmb.2466 [website]
    The choice of codons can influence local translation kinetics during protein synthesis. Whether codon preference is linked to co-translational regulation of polypeptide folding remains unclear. Here, we derive a revised translational efficiency scale that takes into account the competition between tRNA supply and demand, which allows us to uncover the evolutionary conservation of codon optimality across ten yeasts. This analysis reveals distinct and universal patterns of conserved optimal and nonoptimal codons, which associate with the secondary structure of the translated polypeptides independent of the levels of expression. Our analysis thus suggests an evolved function for codon optimality in regulating the rhythm of elongation to facilitate co-translational nascent chain 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 co-translational folding of corresponding polypeptides.
  • Leitner A, Joachimiak LA, Bracher A, Mönkemeyer L, Walzthoeni T, Chen B, Pechmann S, Holmes S, Cong Y, Ma B, Ludtke S, Chiu W, Hartl FU, Aebersold R, Frydman J The Molecular Architecture of the Eukaryotic Chaperonin TRiC/CCT. Structure (2012)
    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.

  • del Alamo M, Hogan DJ, Pechmann S, Albanese V, Brown PO, Frydman J. Defining the Specificity of Cotranslationally Acting Chaperones by Systematic Analysis of mRNAs Associated with Ribosome-Nascent Chain Complexes. PLoS Biol 9(7): e1001100 (2011)
    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.