Beyond transcription--new mechanisms for the regulation of molecular chaperones

Molecular chaperones are an essential part of the universal heat shock response that allows organisms to survive stress conditions that cause intracellular protein unfolding. During the past few years, two new mechanisms have been found to control the activity of several chaperones under stress conditions-the regulation of chaperone activity by the redox state and by the temperature of the environment. Hsp33, for example, is redox-regulated. Hsp33 is specifically activated by disulfide bond formation during oxidative stress, where it becomes a highly efficient chaperone holdase that binds tightly to unfolding proteins. Certain small heat shock proteins, such as Hsp26 and Hsp16.9, on the other hand, are temperature regulated. Exposure to heat shock temperatures causes these oligomeric proteins to disassemble, thereby changing them into highly efficient chaperones. The ATP-dependent chaperone folding system DnaK/DnaJ/GrpE also appears to be temperature regulated, switching from a folding to a holding mode during heat stress. Both of these novel post-translational regulatory strategies appear to have one ultimate goal: to significantly increase the substrate binding affinity of the affected chaperones under exactly those stress conditions that require their highest chaperone activity. This ensures that protein folding intermediates remain bound to the chaperones under stress conditions and are released only after the cells return to non-stress conditions.     

Beyond Transcription—New Mechanisms for the Regulation of Molecular Chaperones

Molecular chaperones are an essential part of the universal heat shock response that allows organisms to survive stress conditions that cause intracellular protein unfolding. During the past few years, two new mechanisms have been found to control the activity of several chaperones under stress conditions—the regulation of chaperone activity by the redox state and by the temperature of the environment. Hsp33, for example, is redox-regulated. Hsp33 is specifically activated by disulfide bond formation during oxidative stress, where it becomes a highly efficient chaperone holdase that binds tightly to unfolding proteins. Certain small heat shock proteins, such as Hsp26 and Hsp16.9, on the other hand, are temperature regulated. Exposure to heat shock temperatures causes these oligomeric proteins to disassemble, thereby changing them into highly efficient chaperones. The ATP-dependent chaperone folding system DnaK/DnaJ/GrpE also appears to be temperature regulated, switching from a folding to a holding mode during heat stress. Both of these novel post-translational regulatory strategies appear to have one ultimate goal: to significantly increase the substrate binding affinity of the affected chaperones under exactly those stress conditions that require their highest chaperone activity. This ensures that protein folding intermediates remain bound to the chaperones under stress conditions and are released only after the cells return to non-stress conditions.     

The Anti-Aggregation Holdase Hsp33 Promotes the Formation of Folded Protein Structures

Holdase chaperones are known to be central to suppressing aggregation, but how they affect substrate conformations remains poorly understood. Here, we use optical tweezers to study how the holdase Hsp33 alters folding transitions within single maltose binding proteins and aggregation transitions between maltose binding protein substrates. Surprisingly, we find that Hsp33 not only suppresses aggregation but also guides the folding process. Two modes of action underlie these effects. First, Hsp33 binds unfolded chains, which suppresses aggregation between substrates and folding transitions within substrates. Second, Hsp33 binding promotes substrate states in which most of the chain is folded and modifies their structure, possibly by intercalating its intrinsically disordered regions. A statistical ensemble model shows how Hsp33 function results from the competition between these two contrasting effects. Our findings reveal an unexpectedly comprehensive functional repertoire for Hsp33 that may be more prevalent among holdases and dispels the notion of a strict chaperone hierarchy.     

The redox-switch domain of Hsp33 functions as dual stress sensor

The redox-regulated chaperone Hsp33 is specifically activated upon exposure of cells to peroxide stress at elevated temperatures. Here we show that Hsp33 harbors two interdependent stress-sensing regions located in the C-terminal redox-switch domain of Hsp33: a zinc center sensing peroxide stress conditions and an adjacent linker region responding to unfolding conditions. Neither of these sensors works sufficiently in the absence of the other, making the simultaneous presence of both stress conditions a necessary requirement for Hsp33's full activation. Upon activation, Hsp33's redox-switch domain adopts a natively unfolded conformation, thereby exposing hydrophobic surfaces in its N-terminal substrate-binding domain. The specific activation of Hsp33 by the oxidative unfolding of its redox-switch domain makes this chaperone optimally suited to quickly respond to oxidative stress conditions that lead to protein unfolding.     

Molecular-level interplay between intrinsically disordered clients and Hsp90

Proteostasis is maintained by a network of molecular chaperones, a prominent member of which is the 90-kilodalton heat shock protein Hsp90. The chaperone function of Hsp90 has been extensively reviewed previously, emphasizing its ATPase activity and remodeling of folded client proteins. Experimental evidence implicating Hsp90 in neurodegenerative diseases has bolstered interest in the noncanonical chaperoning of intrinsically disordered protein (IDPs), however the interplay between Hsp90 and its disordered clients remains poorly understood. In this review we describe recent advances that have contributed to our understanding of the intricate mechanisms characterizing Hsp90-mediated chaperoning of the IDPs tau and α-synuclein and survey emerging insights into the modulation of the chaperone-client interplay in the context of neurodegeneration.     

Binding specificity of an alpha-helical protein sequence to a full-length Hsp70 chaperone and its minimal substrate-binding domain

Hsp70 chaperones are involved in the prevention of misfolding, and possibly the folding, of newly synthesized proteins. The members of this chaperone family are capable of interacting with polypeptide chains both co- and posttranslationally, but it is currently not clear how different structural domains of the chaperone affect binding specificity. We explored the interactions between the bacterial Hsp70, DnaK, and the sequence of a model all-alpha-helical globin (apoMb) by cellulose-bound peptide scanning. The binding specificity of the full-length chaperone was compared with that of its minimal substrate-binding domain, DnaK-beta. Six specific chaperone binding sites evenly distributed along the apoMb sequence were identified. Binding site locations are identical for the full-length chaperone and its substrate-binding domain, but relative affinities differ. The binding specificity of DnaK-beta is only slightly decreased relative to that of full-length DnaK. DnaK's binding motif is known to comprise hydrophobic regions flanked by positively charged residues. We found that the simple fractional mean buried area correlates well with Hsp70's binding site locations along the apoMb sequence. In order to further characterize the properties of the minimal binding host, the stability of DnaK-beta upon chemical denaturation by urea and protons was investigated. Urea unfolding titrations yielded an apparent folding DeltaG degrees of 3.1 +/- 0.9 kcal mol-1 and an m value of 1.7 +/- 0.4 kcal mol-1 M-1.     

Solubility-promoting function of Hsp90 contributes to client maturation and robust cell growth

The Hsp90 chaperone is required for the maturation of signal transduction clients, including many kinases and nuclear steroid hormone receptors. The binding and hydrolysis of ATP by Hsp90 drive conformational rearrangements in three structure domains. Two intrinsically disordered regions of Hsp90 located between these domains and at the C terminus have traditionally been considered to impart flexibility. We discovered that the charged nature of these acid-rich disordered regions imparts a solubility-promoting function to Hsp90 that is important for its cellular activity in yeast. Both the solubility-promoting function and ATPase activity must occur in the same Hsp90 molecule in order to support robust growth, suggesting that the solubility-promoting function is required during the ATP-driven client maturation process. Expression of model clients together with Hsp90 variants indicated interdependent solubilities mediated by the aggregation propensities of both the client and Hsp90. We propose a model whereby the charge-rich disordered regions of Hsp90 serve a solubility-promoting function important for complexes with aggregation-prone clients. These findings demonstrate a novel biological function of the intrinsically disordered regions in Hsp90 and provide a compelling rationale for why their charged properties are conserved throughout eukaryotic evolution.     

Identification of a redox-regulated chaperone network

We have identified and reconstituted a multicomponent redox-chaperone network that appears to be designed to protect proteins against stress-induced unfolding and to refold proteins when conditions return to normal. The central player is Hsp33, a redox-regulated molecular chaperone. Hsp33, which is activated by disulfide bond formation and subsequent dimerization, works as an efficient chaperone holdase that binds to unfolding protein intermediates and maintains them in a folding competent conformation. Reduction of Hsp33 is catalyzed by the glutaredoxin and thioredoxin systems in vivo, and leads to the formation of highly active, reduced Hsp33 dimers. Reduction of Hsp33 is necessary but not sufficient for substrate protein release. Substrate dissociation from Hsp33 is linked to the presence of the DnaK/DnaJ/GrpE foldase system, which alone, or in concert with the GroEL/GroES system, then supports the refolding of the substrate proteins. Upon substrate release, reduced Hsp33 dimers dissociate into inactive monomers. This regulated substrate transfer ultimately links substrate release and Hsp33 inactivation to the presence of available DnaK/DnaJ/GrpE, and, therefore, to the return of cells to non-stress conditions.     

Chaperone‐client interactions between Hsp21 and client proteins monitored in solution by small angle X‐ray scattering and captured by crosslinking mass spectrometry

The small heat shock protein (sHsp) chaperones are important for stress survival, yet the molecular details of how they interact with client proteins are not understood. All sHsps share a folded middle domain to which is appended flexible N‐ and C‐terminal regions varying in length and sequence between different sHsps which, in different ways for different sHsps, mediate recognition of client proteins. In plants there is a chloroplast‐localized sHsp, Hsp21, and a structural model suggests that Hsp21 has a dodecameric arrangement with six N‐terminal arms located on the outside of the dodecamer and six inwardly‐facing. Here, we investigated the interactions between Hsp21 and thermosensitive model substrate client proteins in solution, by small‐angle X‐ray scattering (SAXS) and crosslinking mass spectrometry. The chaperone‐client complexes were monitored and the R_g‐values were found to increase continuously during 20 min at 45°, which could reflect binding of partially unfolded clients to the flexible N‐terminal arms of the Hsp21 dodecamer. No such increase in R_g‐values was observed with a mutational variant of Hsp21, which is mainly dimeric and has reduced chaperone activity. Crosslinking data suggest that the chaperone‐client interactions involve the N‐terminal region in Hsp21 and only certain parts in the client proteins. These parts are peripheral structural elements presumably the first to unfold under destabilizing conditions. We propose that the flexible and hydrophobic N‐terminal arms of Hsp21 can trap and refold early‐unfolding intermediates with or without dodecamer dissociation.     

The ER Chaperones BiP and Grp94 Regulate the Formation of Insulin-Like Growth Factor 2 (IGF2) Oligomers

While cytosolic Hsp90 chaperones have been extensively studied, less is known about how the ER Hsp90 paralog Grp94 recognizes clients and influences client folding. Here, we examine how Grp94 and the ER Hsp70 paralog, BiP, influence the folding of insulin-like growth factor 2 (IGF2), an established client protein of Grp94. ProIGF2 is composed of a disulfide-bonded insulin-like hormone and a C-terminal E-peptide that has sequence characteristics of an intrinsically disordered region. BiP and Grp94 have a minimal influence on folding whereby both chaperones slow proIGF2 folding and do not substantially alter the disulfide-bonded folding intermediates, suggesting that BiP and Grp94 may have an additional influence unrelated to proIGF2 folding. Indeed, we made the unexpected discovery that the E-peptide region allows proIGF2 to form dynamic oligomers. ProIGF2 oligomers can transition from a dynamic state that is capable of exchanging monomers to an irreversibly aggregated state, providing a plausible role for BiP and Grp94 in regulating proIGF2 oligomerization. In contrast to the modest influence on folding, BiP and Grp94 have a stronger influence on proIGF2 oligomerization and these chaperones exert counteracting effects. BiP suppresses proIGF2 oligomerization while Grp94 can enhance proIGF2 oligomerization in a nucleotide-dependent manner. We propose that BiP and Grp94 regulate the assembly and dynamic behavior of proIGF2 oligomers, although the biological role of proIGF2 oligomerization is not yet known.     

Binding of non-native protein to Hsp25 during heat shock creates a reservoir of folding intermediates for reactivation.

Small heat shock proteins (sHsps) are a conserved and ubiquitous protein family. Their ability to convey thermoresistance suggests their participation in protecting the native conformation of proteins. However, the underlying functional principles of their protective properties and their role in concert with other chaperone families remain enigmatic. Here, we analysed the influence of Hsp25 on the inactivation and subsequent aggregation of a model protein, citrate synthase (CS), under heat shock conditions in vitro. We show that stable binding of several non-native CS molecules to one Hsp25 oligomer leads to an accumulation of CS unfolding intermediates, which are protected from irreversible aggregation. Furthermore, a number of different proteins which bind to Hsp25 can be isolated from heat-shocked extracts of cells. Under permissive folding conditions, CS can be released from Hsp25 and, in cooperation with Hsp70, an ATP-dependent chaperone, the native state can be restored. Taken together, our findings allow us to integrate sHsps functionally in the cellular chaperone system operating under heat shock conditions. The task of sHsps in this context is to efficiently trap a large number of unfolding proteins in a folding-competent state and thus create a reservoir of non-native proteins for an extended period of time, allowing refolding after restoration of physiological conditions in cooperation with other chaperones.     

Dancing with the Diva: Hsp90–Client Interactions

The molecular chaperone Hsp90 is involved in the folding, maturation, and degradation of a large number structurally and sequentially unrelated clients, often connected to serious diseases. Elucidating the principles of how Hsp90 recognizes this large variety of substrates is essential for comprehending the mechanism of this chaperone machinery, as well as it is a prerequisite for the design of client specific drugs targeting Hsp90. Here, we discuss the recent progress in understanding the substrate recognition principles of Hsp90 and its implications for the role of Hsp90 in the lifecycle of proteins. Hsp90 acts downstream of the chaperone Hsp70, which exposes its substrate to a short and highly hydrophobic cleft. The subsequently acting Hsp90 has an extended client-binding interface that enables a large number of low-affinity contacts. Structural studies show interaction modes of Hsp90 with the intrinsically disordered Alzheimer's disease-causing protein Tau, the kinase Cdk4 in a partially unfolded state and the folded ligand-binding domain of a steroid receptor. Comparing the features shared by these different proteins provides a picture of the substrate-binding principles of Hsp90.     

Grp94 Works Upstream of BiP in Protein Remodeling Under Heat Stress

Hsp90 and Hsp70 are highly conserved molecular chaperones that promote the proper folding and activation of substrate proteins that are often referred to as clients. The two chaperones functionally collaborate to fold specific clients in an ATP-dependent manner. In eukaryotic cytosol, initial client folding is done by Hsp70 and its co-chaperones, followed by a direct transfer of client refolding intermediates to Hsp90 for final client processing. However, the mechanistic details of collaboration of organelle specific Hsp70 and Hsp90 are lacking. This work investigates the collaboration of the endoplasmic reticulum (ER) Hsp70 and Hsp90, BiP and Grp94 respectively, in protein remodeling using in vitro refolding assays. We show that under milder denaturation conditions, BiP collaborates with its co-chaperones to refold misfolded proteins in an ATP-dependent manner. Grp94 does not play a major role in this refolding reaction. However, under stronger denaturation conditions that favor aggregation, Grp94 works in an ATP-independent manner to bind and hold misfolded clients in a folding competent state for subsequent remodeling by the BiP system. We also show that the collaboration of Grp94 and BiP is not simply a reversal of the eukaryotic refolding mechanism since a direct interaction of Grp94 and BiP is not required for client transfer. Instead, ATP binding but not hydrolysis by Grp94 facilitates the release of the bound client, which is then picked up by the BiP system for subsequent refolding in a Grp94-independent manner.     

Conserved,Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

The 70-kDa heat shock proteins (Hsp70s) function as molecular chaperones through the allosteric coupling of their nucleotide- and substrate-binding domains, the structures of which are highly conserved. In contrast, the roles of the poorly structured, variable length C-terminal regions present on Hsp70s remain unclear. In many eukaryotic Hsp70s, the extreme C-terminal EEVD tetrapeptide sequence associates with co-chaperones via binding to tetratricopeptide repeat domains. It is not known whether this is the only function for this region in eukaryotic Hsp70s and what roles this region performs in Hsp70s that do not form complexes with tetratricopeptide repeat domains. We compared C-terminal sequences of 730 Hsp70 family members and identified a novel conservation pattern in a diverse subset of 165 bacterial and organellar Hsp70s. Mutation of conserved C-terminal sequence in DnaK, the predominant Hsp70 in Escherichia coli, results in significant impairment of its protein refolding activity in vitro without affecting interdomain allostery, interaction with co-chaperones DnaJ and GrpE, or the binding of a peptide substrate, defying classical explanations for the chaperoning mechanism of Hsp70. Moreover, mutation of specific conserved sites within the DnaK C terminus reduces the capacity of the cell to withstand stresses on protein folding caused by elevated temperature or the absence of other chaperones. These features of the C-terminal region support a model in which it acts as a disordered tether linked to a conserved, weak substrate-binding motif and that this enhances chaperone function by transiently interacting with folding clients.     

Crowbars and ratchets: hsp100 chaperones as tools in reversing protein aggregation.

Molecular chaperones have the capacity to prevent inappropriate interactions between aggregation-prone folding or unfolding intermediates created in the cell during protein synthesis or in response to physical and chemical stress. What happens when surveillance by molecular chaperones is evaded or overwhelmed and aggregates accumulate? Recent progress in the elucidation of Hsp100/Clp function suggests that intracellular aggregates or stable complexes can be progressively dissolved by the action of chaperones that act as molecular crowbars or ratchets. These insights set the stage for new progress in the understanding and treatment of diseases of protein folding.     

A review of multi-domain and flexible molecular chaperones studies by small-angle X-ray scattering

Intrinsic flexibility is closely related to protein function, and a plethora of important regulatory proteins have been found to be flexible, multi-domain or even intrinsically disordered. On the one hand, understanding such systems depends on how these proteins behave in solution. On the other, small-angle X-ray scattering (SAXS) is a technique that fulfills the requirements to study protein structure and dynamics relatively quickly with few experimental limitations. Molecular chaperones from Hsp70 and Hsp90 families are multi-domain proteins containing flexible and/or disordered regions that play central roles in cellular proteostasis. Here, we review the structure and function of these proteins by SAXS. Our general approach includes the use of SAXS data to determine size and shape parameters, as well as protein shape reconstruction and their validation by using accessory biophysical tools. Some remarkable examples are presented that exemplify the potential of the SAXS technique. Protein structure can be determined in solution even at limiting protein concentrations (for example, human mortalin, a mitochondrial Hsp70 chaperone). The protein organization, flexibility and function (for example, the J-protein co-chaperones), oligomeric status, domain organization, and flexibility (for the Hsp90 chaperone and the Hip and Hep1 co-chaperones) may also be determined. Lastly, the shape, structural conservation, and protein dynamics (for the Hsp90 chaperone and both p23 and Aha1 co-chaperones) may be studied by SAXS. We believe this review will enhance the application of the SAXS technique to the study of the molecular chaperones.     

Folding or holding?—Hsp70 and Hsp90 chaperoning of misfolded proteins in neurodegenerative disease

The toxic accumulation of misfolded proteins as inclusions, fibrils, or aggregates is a hallmark of many neurodegenerative diseases. However, how molecular chaperones, such as heat shock protein 70 kDa (Hsp70) and heat shock protein 90 kDa (Hsp90), defend cells against the accumulation of misfolded proteins remains unclear. The ATP-dependent foldase function of both Hsp70 and Hsp90 actively transitions misfolded proteins back to their native conformation. By contrast, the ATP-independent holdase function of Hsp70 and Hsp90 prevents the accumulation of misfolded proteins. Foldase and holdase functions can protect against the toxicity associated with protein misfolding, yet we are only beginning to understand the mechanisms through which they modulate neurodegeneration. This review compares recent structural findings regarding the binding of Hsp90 to misfolded and intrinsically disordered proteins, such as tau, α-synuclein, and Tar DNA-binding protein 43. We propose that Hsp90 and Hsp70 interact with these proteins through an extended and dynamic interface that spans the surface of multiple domains of the chaperone proteins. This contrasts with many other Hsp90–client protein interactions for which only a single bound conformation of Hsp90 is proposed. The dynamic nature of these multidomain interactions allows for polymorphic binding of multiple conformations to vast regions of Hsp90. The holdase functions of Hsp70 and Hsp90 may thus allow neuronal cells to modulate misfolded proteins more efficiently by reducing the long-term ATP running costs of the chaperone budget. However, it remains unclear whether holdase functions protect cells by preventing aggregate formation or can increase neurotoxicity by inadvertently stabilizing deleterious oligomers.     

Hsp31, the Escherichia coli yedU gene product,is a molecular chaperone whose activity is inhibited by ATP at high temperatures

The Escherichia coli chromosome contains several uncharacterized heat-inducible loci that may encode novel molecular chaperones or proteases. Here we show that the 31-kDa product of the yedU gene is an efficient homodimeric molecular chaperone that is conserved in a number of pathogenic eubacteria and fungi. Heat shock protein (Hsp) 31 relies on temperature-driven conformational changes to expose structured hydrophobic domains that are likely responsible for substrate binding. Complementing the function of refolding, remodeling, and holding chaperones, Hsp 31 preferentially interacts with early unfolding intermediates and rapidly releases them in an active form after transfer to low temperatures. Although Hsp 31 does not appear to exhibit intrinsic ATPase activity, binding of ATP at high temperatures restricts the size or availability of the substrate binding site, thereby modulating chaperone activity. The possible role of ATP in coordinating the function of the cellular complement of molecular chaperones is discussed.     

Molecular chaperones,stress proteins and redox homeostasis

Protection against oxidative stress is highly interrelated with the function of the most ancient cellular defense system, the network of molecular chaperones, heat shock, or stress-proteins. These ubiquitous, conserved proteins help other proteins and macromolecules to fold or re-fold and reach their final, native conformation. Redox regulation of protein folding becomes especially important during the preparation of extracellular proteins to the outside oxidative milieu, which should take place in a gradual and step-by-step controlled manner in the endoplasmic reticulum or in the periplasm. Several chaperones, such as members of the Hsp33 family in yeast and the plethora of small heat shock proteins as well as one of the major chaperones, Hsp70 are able to act against cytoplasmic oxidative damage. Abrupt changes of cellular redox status lead to chaperone induction. The function of several chaperones is tightly regulated by the surrounding redox conditions. Moreover, our recent data suggest that chaperones may act as a central switchboard for the transmission of redox changes in the life of the cell.