Numerous human diseases are associated with the chronic expression of misfolded and aggregation-prone proteins. The expansion of polyglutamine residues in unrelated proteins is associated with the early onset of neurodegenerative disease. To understand how the presence of misfolded proteins leads to cellular dysfunction, we employed Caenorhabditis elegans polyglutamine aggregation models. Here, we find that polyglutamine expansions disrupted the global balance of protein folding quality control, resulting in the loss of function of diverse metastable proteins with destabilizing temperature-sensitive mutations. In turn, these proteins, although innocuous under normal physiological conditions, enhanced the aggregation of polyglutamine proteins. Thus, weak folding mutations throughout the genome can function as modifiers of polyglutamine phenotypes and toxicity.

Protein damage contributes prominently to cellular aging. To address whether this occurs at a specific period during aging or accumulates gradually, we monitored the biochemical, cellular, and physiological properties of folding sensors expressed in different tissues of C. elegans. We observed the age-dependent misfolding and loss of function of diverse proteins harboring temperature-sensitive missense mutations in all somatic tissues at the permissive condition. This widespread failure in proteostasis occurs rapidly at an early stage of adulthood, and coincides with a severely reduced activation of the cytoprotective heat shock response and the unfolded protein response. Enhancing stress responsive factors HSF-1 or DAF-16 suppresses misfolding of these metastable folding sensors and restores the ability of the cell to maintain a functional proteome. This suggests that a compromise in the regulation of proteostatic stress responses occurs early in adulthood and tips the balance between the load of damaged proteins and the proteostasis machinery. We propose that the collapse of proteostasis represents an early molecular event of aging that amplifies protein damage in age-associated diseases of protein conformation.daf-16 ͉ folding sensors ͉ hsf-1 ͉ protein misfolding ͉ stress response T he stability of the proteome is challenged by conditions that cause proteotoxic stress including errors during protein synthesis, oxidant-induced covalent modifications, inherited polymorphisms, and misfolding (1-3). Consequently, all cells have highly conserved stress-inducible pathways that detect, prevent, and resolve such damage (4, 5). Accumulation of misfolded proteins titrate the negative regulation of chaperones from HSF-1 (4, 6, 7). Likewise, an E3 ubiquitin ligase was shown to regulate the levels of DAF-16, also indicating a link between stress response activation to the balance between proteostasis capacity and the load of damaged proteins (8). The activation of cellular stress responses limits the accumulation of damaged proteins by suppression of misfolding and enhancing chaperonemediated folding, promoting the enzymatic removal of covalent modifications, and stimulating clearance by ubiquitin-mediated, proteosomal, and autophagic processes (1, 4, 5, 9, 10). The dynamics of these processes during aging, however, are poorly understood.Aging can be characterized by the loss of cellular function and increased vulnerability to environmental and physiological stress that results in enhanced susceptibility to disease. Many agedependent changes have been detected at mid-to-late lifespan of C. elegans, including the accumulation of damaged macromolecules, cell and organellar degeneration, and physiological decline (11-17). Likewise, aggregation and toxicity of diseaseassociated aggregation-prone proteins, such as huntingtin and A␤ peptide, is enhanced during aging in C. elegans and other model systems (18-21).The regulation of aging and the maintenance of proteostasis has been recently established in C. elegans. Downregulatio...

A major activity of molecular chaperones is to prevent aggregation and refold misfolded proteins. However, when allowed to form, protein aggregates are refolded poorly by most chaperones. We show here that the sequential action of two Escherichia coli chaperone systems, ClpB and DnaK-DnaJ-GrpE, can efficiently solubilize excess amounts of protein aggregates and refold them into active proteins. Measurements of aggregate turbidity, Congo red, and 4,4-dianilino-1,1-binaphthyl-5,5-disulfonic acid binding, and of the disaggregation͞refolding kinetics by using a specific ClpB inhibitor, suggest a mechanism where (i) ClpB directly binds protein aggregates, ATP induces structural changes in ClpB, which (ii) increase hydrophobic exposure of the aggregates and (iii) allow DnaK-DnaJ-GrpE to bind and mediate dissociation and refolding of solubilized polypeptides into native proteins. This efficient mechanism, whereby chaperones can catalytically solubilize and refold a wide variety of large and stable protein aggregates, is a major addition to the molecular arsenal of the cell to cope with protein damage induced by stress or pathological states.Hsp104 ͉ ClpB ͉ DnaK ͉ Congo red ͉ protein disaggregation

Classic in vitro studies show that the Hsp70 chaperone system from Escherichia coli (DnaK-DnaJ-GrpE, the DnaK system) can bind to proteins, prevent aggregation, and promote the correct refolding of chaperone-bound polypeptides into native proteins. However, little is known about how the DnaK system handles proteins that have already aggregated. In this study, glucose-6-phosphate dehydrogenase was used as a model system to generate stable populations of protein aggregates comprising controlled ranges of particle sizes. The DnaK system recognized the glucose-6-phosphate dehydrogenase aggregates as authentic substrates and specifically solubilized and refolded the protein into a native enzyme. The efficiency of disaggregation by the DnaK system was high with small aggregates, but the efficiency decreased as the size of the aggregates increased. High folding efficiency was restored by either excess DnaK or substoichiometric amounts of the chaperone ClpB. We suggest a mechanism whereby the DnaK system can readily solubilize small aggregates and refold them into active proteins. With large aggregates, however, the binding sites for the DnaK system had to be dynamically exposed with excess DnaK or the catalytic action of ClpB and ATP. Disaggregation by the DnaK machinery in the cell can solubilize early aggregates that formed accidentally during chaperone-assisted protein folding or that escaped the protection of "holding" chaperones during stress.

Hsp70s are highly conserved ATPase molecular chaperones mediating the correct folding of de novo synthesized proteins, the translocation of proteins across membranes, the disassembly of some native protein oligomers, and the active unfolding and disassembly of stress-induced protein aggregates. Here, we bring thermodynamic arguments and biochemical evidences for a unifying mechanism named entropic pulling, based on entropy loss due to excluded-volume effects, by which Hsp70 molecules can convert the energy of ATP hydrolysis into a force capable of accelerating the local unfolding of various protein substrates and, thus, perform disparate cellular functions. By means of entropic pulling, individual Hsp70 molecules can accelerate unfolding and pulling of translocating polypeptides into mitochondria in the absence of a molecular fulcrum, thus settling former contradictions between the power-stroke and the Brownian ratchet models for Hsp70-mediated protein translocation across membranes. Moreover, in a very different context devoid of membrane and components of the import pore, the same physical principles apply to the forceful unfolding, solubilization, and assisted native refolding of stable protein aggregates by individual Hsp70 molecules, thus providing a mechanism for Hsp70-mediated protein disaggregation.H sp70 is a central component of the chaperone network in the cell with disparate cellular functions. Associated with the ribosome, Hsp70s foster proper de novo protein folding. In the cytoplasm, Hsp70s mediate the deoligomerization and recycling of native protein complexes (1, 2) and control key functions in evolution, cell morphogenesis (2), and apoptosis (3), often in association with Hsp90 (4). Hsp70 also serves as the central translocation motor in the posttranslational import of cytoplasmic proteins into mitochondria (5), chloroplasts (6, 7), and the endoplasmic reticulum (8). Moreover, Hsp70s can actively unfold, solubilize, and reactivate already formed, stable protein aggregates (9, 10) and may participate in targeting proteins to the degradation pathway (11, 12). Existing Models for Hsp70-Mediated Protein Translocation into MitochondriaThe translocation of proteins across the mitochondrial membrane, through the translocase of the outer membrane (TOM) and translocase of the inner membrane (TIM) translocation pores, is mediated by the presequence translocase-associated motor (PAM) complex consisting of matrix-localized Hsp70 (mtHsp70), membrane-associated J domain-containing proteins (three identified so far, PAM16͞Tim16, PAM18͞Tim14, and Mdj2) (13-19) and the nucleotide exchange factor Mge1. In the ATP-bound state, mtHsp70 is in the open (unlocked) state, which is as yet unbound to the translocating protein substrate, whereas mtHsp70 is found anchored to the mitochondrial import channel by way of its transient association with the mitochondrial peripheral inner-membrane protein Tim44. In the ADP-bound state, mtHsp70 is tightly bound (locked) onto the incoming polypeptide and is not associated to the m...

SummaryAll cells rely on highly conserved protein folding and clearance pathways to detect and resolve protein damage and to maintain protein homeostasis (proteostasis). Because age is associated with an imbalance in proteostasis, there is a need to understand how protein folding is regulated in a multicellular organism that undergoes aging. We have observed that the ability of Caenorhabditis elegans to maintain proteostasis declines sharply following the onset of oocyte biomass production, suggesting that a restricted protein folding capacity may be linked to the onset of reproduction. To test this hypothesis, we monitored the effects of different sterile mutations on the maintenance of proteostasis in the soma of C. elegans. We found that germline stem cell (GSC) arrest rescued protein quality control, resulting in maintenance of robust proteostasis in different somatic tissues of adult animals. We further demonstrated that GSC-dependent modulation of proteostasis requires several different signaling pathways, including hsf-1 and daf-16/kri-1/tcer-1, daf-12, daf-9, daf-36, nhr-80, and pha-4 that differentially modulate somatic quality control functions, such that each signaling pathway affects different aspects of proteostasis and cannot functionally complement the other pathways. We propose that the effect of GSCs on the collapse of proteostasis at the transition to adulthood is due to a switch mechanism that links GSC status with maintenance of somatic proteostasis via regulation of the expression and function of different quality control machineries and cellular stress responses that progressively lead to a decline in the maintenance of proteostasis in adulthood, thereby linking reproduction to the maintenance of the soma.

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Hsp70 is a central molecular chaperone that passively prevents protein aggregation and uses the energy of ATP hydrolysis to solubilize, translocate, and mediate the proper refolding of proteins in the cell. Yet, the molecular mechanism by which the active Hsp70 chaperone functions are achieved remains unclear. Here, we show that the bacterial Hsp70 (DnaK) can actively unfold misfolded structures in aggregated polypeptides, leading to gradual disaggregation. We found that the specific unfolding and disaggregation activities of individual DnaK molecules were optimal for large aggregates but dramatically decreased for small aggregates. The active unfolding of the smallest aggregates, leading to proper global refolding, required the cooperative action of several DnaK molecules per misfolded polypeptide. This finding suggests that the unique ATP-fueled locking/unlocking mechanism of the Hsp70 chaperones can recruit random chaperone motions to locally unfold misfolded structures and gradually disentangle stable aggregates into refoldable proteins.