The protein import motor of mitochondria: a targeted molecular ratchet driving unfolding and translocation

Unfolding and import of preproteins into mitochondria are facilitated by a molecular motor in which heat shock protein 70 (Hsp70) in the matrix plays an essential role. Here we present two different experimental approaches to analyze mechanisms underlying this function of Hsp70. First, preproteins containing stretches of glutamic acid (polyE) or glycine (polyG) repeats in front of folded domains were imported into mitochondria. This occurred although Hsp70 cannot pull on these stretches to unfold the folded domains, since it does not bind to polyE and polyG. Secondly, preproteins containing titin immunoglobulin (Ig)-like domains were imported into mitochondria, despite the fact that forces of >200 pN are required to mechanically unfold these domains. Since molecular motors generate forces of approximately 5 pN, Hsp70 could not promote unfolding of the Ig-like domains by mechanical pulling. Our observations suggest that Hsp70 acts as an element of a Brownian ratchet, which mediates unfolding and translocation of preproteins across the mitochondrial membranes.     

Active solubilization and refolding of stable protein aggregates by cooperative unfolding action of individual hsp70 chaperones

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.     

Interaction of plant mitochondrial and chloroplast signal peptides with the Hsp70 molecular chaperone.

Most mitochondrial and chloroplast proteins are synthesized on cytosolic polyribosomes as precursor proteins, with an N-terminal signal sequence that targets the precursor to the correct organelle. In mitochondria, the chaperone Hsp70 functions as a molecular motor, pulling the precursor across the mitochondrial membranes; 97.0% of plant mitochondrial presequences contain an Hsp70 binding site. In chloroplasts, the outer envelope, intermembrane space and a stromal Hsp70 are thought to participate in protein import; 82.5% of chloroplast transit peptides have an Hsp70 binding site. The interaction of signal peptides with Hsp70 during the import process is supported by biochemical and bioinformatic studies.     

Lipid interaction differentiates the constitutive and stress-induced heat shock proteins Hsc70 and Hsp70

Heat shock proteins play a major role in the process of protein folding, and they have been termed molecular chaperones. Two members of the Hsp70 family, Hsc70 and Hsp70, have a high degree of sequence homology. But they differ in their expression pattern. Hsc70 is constitutively expressed, whereas Hsp70 is stress inducible. These 2 proteins are localized in the cytosol and the nucleus. In addition, they have also been observed in close proximity to cellular membranes. We have recently reported that Hsc70 is capable of interacting with a lipid bilayer forming ion-conductance channels. In the present study, we found that both Hsc70 and Hsp70 interact with lipids and can be differentiated by their characteristic induction of liposome aggregation. These proteins promote the aggregation of phosphatidylserine liposomes in a time- and protein concentration-dependent manner. Although both proteins are active in this process, the level and kinetics of aggregation are different between them. Calcium ions enhance Hsc70 and Hsp70 liposome aggregation, but the effect is more dramatic for Hsc70 than for Hsp70. Addition of adenosine triphosphate blocks liposome aggregation induced by both proteins. Adenosine diphosphate (ADP) also blocks Hsp70-mediated liposome aggregation. Micromolar concentrations of ADP enhance Hsc70-induced liposome aggregation, whereas at millimolar concentrations the nucleotide has an inhibitory effect. These results confirm those of previous studies indicating that the Hsp70 family can interact with lipids directly. It is possible that the interaction of Hsp70s with lipids may play a role in the folding of membrane proteins and the translocation of polypeptides across membranes.     

Hsp110 Is a Bona Fide Chaperone Using ATP to Unfold Stable Misfolded Polypeptides and Reciprocally Collaborate with Hsp70 to Solubilize Protein Aggregates

Structurally and sequence-wise, the Hsp110s belong to a subfamily of the Hsp70 chaperones. Like the classical Hsp70s, members of the Hsp110 subfamily can bind misfolding polypeptides and hydrolyze ATP. However, they apparently act as a mere subordinate nucleotide exchange factors, regulating the ability of Hsp70 to hydrolyze ATP and convert stable protein aggregates into native proteins. Using stably misfolded and aggregated polypeptides as substrates in optimized in vitro chaperone assays, we show that the human cytosolic Hsp110s (HSPH1 and HSPH2) are bona fide chaperones on their own that collaborate with Hsp40 (DNAJA1 and DNAJB1) to hydrolyze ATP and unfold and thus convert stable misfolded polypeptides into natively refolded proteins. Moreover, equimolar Hsp70 (HSPA1A) and Hsp110 (HSPH1) formed a powerful molecular machinery that optimally reactivated stable luciferase aggregates in an ATP- and DNAJA1-dependent manner, in a disaggregation mechanism whereby the two paralogous chaperones alternatively activate the release of bound unfolded polypeptide substrates from one another, leading to native protein refolding.     

Chaperones in control of protein disaggregation

The chaperone protein network controls both initial protein folding and subsequent maintenance of proteins in the cell. Although the native structure of a protein is principally encoded in its amino-acid sequence, the process of folding in vivo very often requires the assistance of molecular chaperones. Chaperones also play a role in a post-translational quality control system and thus are required to maintain the proper conformation of proteins under changing environmental conditions. Many factors leading to unfolding and misfolding of proteins eventually result in protein aggregation. Stress imposed by high temperature was one of the first aggregation-inducing factors studied and remains one of the main models in this field. With massive protein aggregation occurring in response to heat exposure, the cell needs chaperones to control and counteract the aggregation process. Elimination of aggregates can be achieved by solubilization of aggregates and either refolding of the liberated polypeptides or their proteolysis. Here, we focus on the molecular mechanisms by which heat-shock protein 70 (Hsp70), Hsp100 and small Hsp chaperones liberate and refold polypeptides trapped in protein aggregates.     

Step-size analyses of the mitochondrial Hsp70 import motor reveal the Brownian ratchet in operation

Newly synthesized mitochondrial precursor proteins have to become unfolded by the mitochondrial Hsp70 (mtHsp70) import motor to cross the mitochondrial membranes. To assess the mechanism of unfolding of precursor proteins by mtHsp70, we designed a system to measure step sizes of the mtHsp70 import motor, which are distances at which the motor system moves along polypeptide chains during a single turnover of ATP. We made a series of fusion proteins consisting of a mitochondrial presequence containing the first mtHsp70 binding site, a spacer sequence containing an Hsp70 avoidance segment followed by the second mtHsp70 binding site, and different folded mature domains. Analyses of the dependence of the import rates of those fusion proteins on the lengths of Hsp70 avoidance segments allowed us to estimate the step sizes, which differ for different mature domains and different lengths of the spacers. These results suggest that the mtHsp70 import motor functions at least as a molecular Brownian ratchet to unfold mitochondrial precursor proteins.     

Potentiated Hsp104 variants suppress toxicity of diverse neurodegenerative disease-linked proteins

Protein misfolding is implicated in numerous lethal neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Parkinson disease (PD). There are no therapies that reverse these protein-misfolding events. We aim to apply Hsp104, a hexameric AAA+ protein from yeast, to target misfolded conformers for reactivation. Hsp104 solubilizes disordered aggregates and amyloid, but has limited activity against human neurodegenerative disease proteins. Thus, we have previously engineered potentiated Hsp104 variants that suppress aggregation, proteotoxicity and restore proper protein localization of ALS and PD proteins in Saccharomyces cerevisiae, and mitigate neurodegeneration in an animal PD model. Here, we establish that potentiated Hsp104 variants possess broad substrate specificity and, in yeast, suppress toxicity and aggregation induced by wild-type TDP-43, FUS and α-synuclein, as well as missense mutant versions of these proteins that cause neurodegenerative disease. Potentiated Hsp104 variants also rescue toxicity and aggregation of TAF15 but not EWSR1, two RNA-binding proteins with a prion-like domain that are connected with the development of ALS and frontotemporal dementia. Thus, potentiated Hsp104 variants are not entirely non-specific. Indeed, they do not unfold just any natively folded protein. Rather, potentiated Hsp104 variants are finely tuned to unfold proteins bearing short unstructured tracts that are not recognized by wild-type Hsp104. Our studies establish the broad utility of potentiated Hsp104 variants.KEY WORDS: FUS, Hsp104, TDP-43, α-synuclein, Disaggregase, Neurodegeneration     

Hsp104 and Potentiated Variants Can Operate as Distinct Nonprocessive Translocases

Heat shock protein (Hsp) 104 is a hexameric ATPases associated with diverse cellular activities motor protein that enables cells to survive extreme stress. Hsp104 couples the energy of ATP binding and hydrolysis to solubilize proteins trapped in aggregated structures. The mechanism by which Hsp104 disaggregates proteins is not completely understood but may require Hsp104 to partially or completely translocate polypeptides across its central channel. Here, we apply transient state, single turnover kinetics to investigate the ATP-dependent translocation of soluble polypeptides by Hsp104 and Hsp104^A503S, a potentiated variant developed to resolve misfolded conformers implicated in neurodegenerative disease. We establish that Hsp104 and Hsp104^A503S can operate as nonprocessive translocases for soluble substrates, indicating a “partial threading” model of translocation. Remarkably, Hsp104^A503S exhibits altered coupling of ATP binding to translocation and decelerated dissociation from polypeptide substrate compared to Hsp104. This altered coupling and prolonged substrate interaction likely increases entropic pulling forces, thereby enabling more effective aggregate dissolution by Hsp104^A503S.     

Roles of cytosolic Hsp70 and Hsp40 molecular chaperones in post-translational translocation of presecretory proteins into the endoplasmic reticulum

Hsp70 molecular chaperones and their co-chaperones work together in various cellular compartments to guide the folding of proteins and to aid the translocation of proteins across membranes. Hsp70s stimulate protein folding by binding exposed hydrophobic sequences thereby preventing irreversible aggregation. Hsp40s stimulate the ATPase activity of Hsp70s and target unfolded proteins to Hsp70s. Genetic and biochemical evidence supports a role for cytosolic Hsp70s and Hsp40s in the post-translational translocation of precursor proteins into endoplasmic reticulum and mitochondria. To gain mechanistic insight, we measured the effects of Saccharomyces cerevisiae Ssa1p (Hsp70) and Ydj1p (Hsp40) on the translocation of histidine-tagged prepro-alpha-factor (ppalphaF6H) into microsomes. Radiolabeled ppalphaF6H was affinity purified from wheat germ translation reactions (or Escherichia coli) to remove endogenous chaperones. We demonstrated that either Ssa1p or Ydj1p stimulates post-translational translocation by preventing ppalphaF6H aggregation. The binding and/or hydrolysis of ATP by Ssa1p were required to maintain the translocation competence of ppalphaF6H. To clarify the contributions of membrane-bound and cytosolic Ydj1p, we compared the efficiency of chaperone-dependent translocation into wild-type and Ydj1p-deficient microsomes. Neither soluble nor membrane-bound Ydj1p was essential for post-translational protein translocation. The ability of Ssa1p, Ydj1p, or both chaperones to restore the translocation competence of aggregated ppalphaF6H was negligible.     

Molecular analysis of Hsp70 mechanisms in plants and their function in response to stress

Studying the strategies of improving abiotic stress tolerance is quite imperative and research under this field will increase our understanding of response mechanisms to abiotic stress such as heat. The Hsp70 is an essential regulator of protein having the tendency to maintain internal cell stability like proper folding protein and breakdown of unfolded proteins. Hsp70 holds together protein substrates to help in movement, regulation, and prevent aggregation under physical and or chemical pressure. However, this review reports the molecular mechanism of heat shock protein 70 kDa (Hsp70) action and its structural and functional analysis, research progress on the interaction of Hsp70 with other proteins and their interaction mechanisms as well as the involvement of Hsp70 in abiotic stress responses as an adaptive defense mechanism.     

Pharmacological tuning of heat shock protein 70 modulates polyglutamine toxicity and aggregation

Nine neurodegenerative disorders are caused by the abnormal expansion of polyglutamine (polyQ) regions within distinct proteins. Genetic and biochemical evidence has documented that the molecular chaperone, heat shock protein 70 (Hsp70), modulates polyQ toxicity and aggregation, yet it remains unclear how Hsp70 might be used as a potential therapeutic target in polyQ-related diseases. We have utilized a pair of membrane-permeable compounds that tune the activity of Hsp70 by either stimulating or by inhibiting its ATPase functions. Using these two pharmacological agents in both yeast and PC12 cell models of polyQ aggregation and toxicity, we were surprised to find that stimulating Hsp70 solubilized polyQ conformers and simultaneously exacerbated polyQ-mediated toxicity. By contrast, inhibiting Hsp70 ATPase activity protected against polyQ toxicity and promoted aggregation. These findings clarify the role of Hsp70 as a possible drug target in polyQ disorders and suggest that Hsp70 uses ATP hydrolysis to help partition polyQ proteins into structures with varying levels of proteotoxicity. Our results thus support an emerging concept in which certain kinds of polyQ aggregates may be protective, while more soluble polyQ species are toxic.     

Molecular chaperones antagonize proteotoxicity by differentially modulating protein aggregation pathways

The self-association of misfolded or damaged proteins into ordered amyloid-like aggregates characterizes numerous neurodegenerative disorders. Insoluble amyloid plaques are diagnostic of many disease states. Yet soluble, oligomeric intermediates in the aggregation pathway appear to represent the toxic culprit. Molecular chaperones regulate the fate of misfolded proteins and thereby influence their aggregation state. Chaperones conventionally antagonize aggregation of misfolded, disease proteins and assist in refolding or degradation pathways. Recent work suggests that chaperones may also suppress neurotoxicity by converting toxic, soluble oligomers into benign aggregates. Chaperones can therefore suppress or promote aggregation of disease proteins to ameliorate the proteotoxic accumulation of soluble, assembly intermediates.Key words: chaperone, heat shock protein, protein aggregation, amyloid, Hsp70, Hsp40, prion     

Ordered assembly of heat shock proteins, Hsp26, Hsp70, Hsp90, and Hsp104, on expanded polyglutamine fragments revealed by chemical probes

In Saccharomyces cerevisae, expanded polyglutamine (polyQ) fragments are assembled into discrete cytosolic aggregates in a process regulated by the molecular chaperones Hsp26, Hsp70, Hsp90, and Hsp104. To better understand how the different chaperones might cooperate during polyQ aggregation, we used sequential immunoprecipitations and mass spectrometry to identify proteins associated with either soluble (Q25) or aggregation-prone (Q103) fragments at both early and later times after induction of their expression. We found that Hsp26, Hsp70, Hsp90, and other chaperones interact with Q103, but not Q25, within the first 2 h. Further, Hsp70 and Hsp90 appear to be partially released from Q103 prior to the maturation of the aggregates and before the recruitment of Hsp104. To test the importance of this seemingly ordered process, we used a chemical probe to artificially enhance Hsp70 binding to Q103. This treatment retained both Hsp70 and Hsp90 on the polyQ fragment and, interestingly, limited subsequent exchange for Hsp26 and Hsp104, resulting in incomplete aggregation. Together, these results suggest that partial release of Hsp70 may be an essential step in the continued processing of expanded polyQ fragments in yeast.     

Role of sHsps in organizing cytosolic protein aggregation and disaggregation

Small heat shock proteins (sHsps) exhibit an ATP-independent chaperone activity to prevent the aggregation of misfolded proteins in vitro. The seemingly conflicting presence of sHsps in insoluble protein aggregates in cells obstructs a precise definition of sHsp function in proteostasis networks. Recent findings specify sHsp activities in protein quality control systems. The sHsps of yeast, Hsp42 and Hsp26, interact with early unfolding intermediates of substrates, keeping them in a ready-to-refold conformation close to the native state. This activity facilitates substrate refolding by ATP-dependent Hsp70-Hsp100 disaggregating chaperones. Hsp42 can actively sequester misfolded proteins and promote their deposition at specific cellular sites. This aggregase activity represents a cytoprotective protein quality control strategy. The aggregase function of Hsp42 controls the formation of cytosolic aggregates (CytoQs) under diverse stress regimes and can be reconstituted in vitro, demonstrating that Hsp42 is necessary and sufficient to promote protein aggregation. Substrates sequestered at CytoQs can be dissociated by Hsp70-Hsp100 disaggregases for subsequent triage between refolding and degradation pathways or are targeted for destruction by selective autophagy termed proteophagy.     

In vivo properties of the disaggregase function of J‐proteins and Hsc70 in Caenorhabditis elegans stress and aging

Protein aggregation is enhanced upon exposure to various stress conditions and aging, which suggests that the quality control machinery regulating protein homeostasis could exhibit varied capacities in different stages of organismal lifespan. Recently, an efficient metazoan disaggregase activity was identified in vitro, which requires the Hsp70 chaperone and Hsp110 nucleotide exchange factor, together with single or cooperating J‐protein co‐chaperones of classes A and B. Here, we describe how the orthologous Hsp70s and J‐protein of Caenorhabditis elegans work together to resolve protein aggregates both in vivo and in vitro to benefit organismal health. Using an RNAi knockdown approach, we show that class A and B J‐proteins cooperate to form an interactive flexible network that relocalizes to protein aggregates upon heat shock and preferentially recruits constitutive Hsc70 to disaggregate heat‐induced protein aggregates and polyQ aggregates that form in an age‐dependent manner. Cooperation between class A and B J‐proteins is also required for organismal health and promotes thermotolerance, maintenance of fecundity, and extended viability after heat stress. This disaggregase function of J‐proteins and Hsc70 therefore constitutes a powerful regulatory network that is key to Hsc70‐based protein quality control mechanisms in metazoa with a central role in the clearance of aggregates, stress recovery, and organismal fitness in aging.     

Association of heat-shock proteins in various neurodegenerative disorders: is it a master key to open the therapeutic door?

A number of acute and chronic neurodegenerative disorders are caused due to misfolding and aggregation of many intra- and extracellular proteins. Protein misfolding and aggregation processes in cells are strongly regulated by cellular molecular chaperones known as heat-shock proteins (Hsps) that include Hsp60, Hsp70, Hsp40, and Hsp90. Recent studies have shown the evidences that Hsps are colocalized in protein aggregates in Alzheimer’s disease (AD), Parkinson’s disease (PD), Polyglutamine disease (PGD), Prion disease, and other neurodegenerative disorders. This fact indicates that Hsps might have attempted to prevent aggregate formation in cells and thus to suppress disease conditions. Experimental findings have already established in many cases that selective overexpression of Hsps like Hsp70 and Hsp40 prevented the disease progression in various animal models and cellular models. However, recently, various Hsp modulators like geldanamycin, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin, and celastrol have shown to up-regulate the expression level of Hsp70 and Hsp40, which in turn triggers the solubilization of diseased protein aggregates. Hsps are, therefore, if appropriately selected, an attractive choice for therapeutic targeting in various kinds of neurodegeneration and hence are expected to have strong potential as therapeutic agents in suppressing or curing AD, PD, PGD, and other devastative neurodegenerative disorders. In the present review, we report the experimental findings that describe the implication of Hsps in the development of neurodegeneration and explore the possibility of how Hsps can be used directly or as a target by other agents to prevent various neurodegeneration through preventing aggregation process and thus reducing the toxicity of the oligomers based on the previous reports.     

Metazoan Hsp70 machines use Hsp110 to power protein disaggregation

Accumulation of aggregation‐prone misfolded proteins disrupts normal cellular function and promotes ageing and disease. Bacteria, fungi and plants counteract this by solubilizing and refolding aggregated proteins via a powerful cytosolic ATP‐dependent bichaperone system, comprising the AAA+ disaggregase Hsp100 and the Hsp70‐Hsp40 system. Metazoa, however, lack Hsp100 disaggregases. We show that instead the Hsp110 member of the Hsp70 superfamily remodels the human Hsp70‐Hsp40 system to efficiently disaggregate and refold aggregates of heat and chemically denatured proteins in vitro and in cell extracts. This Hsp110 effect relies on nucleotide exchange, not on ATPase activity, implying ATP‐driven chaperoning is not required. Knock‐down of nematode Caenorhabditis elegans Hsp110, but not an unrelated nucleotide exchange factor, compromises dissolution of heat‐induced protein aggregates and severely shortens lifespan after heat shock. We conclude that in metazoa, Hsp70‐Hsp40 powered by Hsp110 nucleotide exchange represents the crucial disaggregation machinery that reestablishes protein homeostasis to counteract protein unfolding stress.     

The molecular chaperone Hsp70 family members function by a bidirectional heterotrophic allosteric mechanism

The Hsp70 family is one of the most important and conserved molecular chaperone families. It is well documented that Hsp70 family members assist many cellular processes involving protein quality control, as follows: protein folding, transport through membranes, protein degradation, escape from aggregation, intracellular signaling, among several others. The Hsp70 proteins act as a cellular pivot capable of receiving and distributing substrates among the other molecular chaperone families. Despite the high identity of the Hsp70 proteins, there are several homologue Hsp70 members that do not have the same role in the cell, which allow them to develop and participate in such large number of activities. The Hsp70 proteins are composed of two main domains: one that binds ATP and hydrolyses it to ADP and another which directly interacts with substrates. These domains present bidirectional heterotrophic allosteric regulation allowing a fine regulated cycle of substrate binding and release. The general mechanism of the Hsp70s cycle is under the control of ATP hydrolysis that modulates the low (ATP-bound state) and high (ADP-bound state) affinity states of Hsp70 for substrates. An important feature of the Hsp70s cycle is that they have several co-chaperones that modulate their cycle and that can also interact and select substrates. Here, we review some known details of the bidirectional heterotrophic allosteric mechanism and other important features for Hsp70s regulating cycle and function.     

Proteomic data from human cell cultures refine mechanisms of chaperone-mediated protein homeostasis

In the crowded environment of human cells, folding of nascent polypeptides and refolding of stress-unfolded proteins is error prone. Accumulation of cytotoxic misfolded and aggregated species may cause cell death, tissue loss, degenerative conformational diseases, and aging. Nevertheless, young cells effectively express a network of molecular chaperones and folding enzymes, termed here “the chaperome,” which can prevent formation of potentially harmful misfolded protein conformers and use the energy of adenosine triphosphate (ATP) to rehabilitate already formed toxic aggregates into native functional proteins. In an attempt to extend knowledge of chaperome mechanisms in cellular proteostasis, we performed a meta-analysis of human chaperome using high-throughput proteomic data from 11 immortalized human cell lines. Chaperome polypeptides were about 10 % of total protein mass of human cells, half of which were Hsp90s and Hsp70s. Knowledge of cellular concentrations and ratios among chaperome polypeptides provided a novel basis to understand mechanisms by which the Hsp60, Hsp70, Hsp90, and small heat shock proteins (HSPs), in collaboration with cochaperones and folding enzymes, assist de novo protein folding, import polypeptides into organelles, unfold stress-destabilized toxic conformers, and control the conformal activity of native proteins in the crowded environment of the cell. Proteomic data also provided means to distinguish between stable components of chaperone core machineries and dynamic regulatory cochaperones.