# THE HSP70 MOLECULAR CHAPERONE MACHINES

EDITED BY: Matthias P. Mayer and Pierre Goloubinoff PUBLISHED IN: Frontiers in Molecular Biosciences

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ISSN 1664-8714 ISBN 978-2-88945-125-8 DOI 10.3389/978-2-88945-125-8

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# **THE HSP70 MOLECULAR CHAPERONE MACHINES**

Topic Editors:

**Matthias P. Mayer,** Ruprecht-Karls-Universität Heidelberg, Germany **Pierre Goloubinoff,** University of Lausanne, Switzerland

HSP70 is a central hub of the chaperone network (center). It uses the energy of ATP-hydrolysis to modify the conformation and the function of various polypeptides. It is targeted onto its misfolded or alternatively folded substrates by J-domain co-chaperones (HSP40s, magenta). It is dissociated from its modified polypeptide substrates by ADP to ATP exchange, which is accelerated by ADP-release factors ( GrpE, yellow, in prokaryotes and mitochondria and plastides of eukaryotic cells; HspBP1 family proteins, Bag-domain proteins, and Hsp110/Hsp170 proteins in cytosol and endoplasmic reticulum of eukaryotic cells). Small HSPs (cyan) and HSP90s (blue) can maintain various misfolded polypeptides in a nonaggregated conformation poised to be further processed by HSP70. Upon activation by HSP70, the AAA+ disaggregases (HSP104, ClpB, red) can use ATP to forcefully solubilize stable protein aggregates and, together with HSP70, revert them into the native functional state. Some proteins are handed over to GroEL/GroES (green) or Hsp90 (blue) for further maturation or to be kept inactive until activation by cellular signals. Cover figure by Pierre Goloubinoff

Members of the HSP70 family form a central hub of the molecular chaperone network, controlling protein homeostasis in prokaryotes and in the ATP-containing compartments of the eukaryotic cells. The heat-inducible form HSPA1A (HSP70), its constitutive cytosolic cognate HSPA8 (Hsc70), its endoplasmic reticulum form HSPA5 (BiP), and its mitochondrial form HSPA9 (Mortalin), as well as the more distantly related HSPHs (HSP110s), make up 1-2 % of the total mass of proteins in human cells. They use the energy of ATP-hydrolysis to prevent and forcefully revert the process of protein misfolding and aggregation during and following various stresses, presumably by working as unfoldases to lift aberrant conformers out of kinetic traps. As such, HSP70s, in cooperation with their J-domain co-chaperones and nucleotide exchange factors (NEFs) and co-disaggregases, form an efficient network of cellular defenses against the accumulation of cytotoxic misfolded protein conformers, which may cause degenerative diseases such as Parkinson's and Alzheimer's disease, diabetes, and aging in general. In addition to their function in repair of stress-induced damage, HSP70s fulfill many housekeeping functions, including assisting the de novo folding and maturation of proteins, driving the translocation of protein precursors across narrow membrane pores into organelles, and by controlling the oligomeric state of key regulator protein complexes involved in signal transduction and vesicular trafficking. For reasons not well understood, HSP70s are also found on the surface of some animal cells, in particular cancer cells where they may serve as specific targets for cancer immunotherapy. Here, we gathered seven mini reviews, each presenting a complementary aspect of HSP70's structure and function in bacteria and eukaryotes, under physiological and stressful conditions. These articles highlight how, the various members of this conserved family of molecular chaperones, assisted by their various J-domain and NEF cochaperones and co-disaggregases, harness ATP hydrolysis to perform a great diversity of life-sustaining cellular functions using a similar molecular mechanism.

**Citation:** Mayer, M. P., Goloubinoff, P., eds. (2017). The HSP70 Molecular Chaperone Machines. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-125-8

# Table of Contents

*63 Plasmodial HSP70s are functionally adapted to the malaria parasite life cycle* Jude M. Przyborski, Mathias Diehl and Gregory L. Blatch

# Editorial: The HSP70 Molecular Chaperone Machines

### Pierre Goloubinoff\*

*Department of Plant Molecular Biology, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland*

Keywords: unfoldase, disaggregase, HSC70, J- domain cochaperones, nucleotide exchanges factors, Hsp110s, protein misfolding, protein aggregation

**Editorial on the Research Topic**

### **The HSP70 Molecular Chaperone Machines**

The HSP70s belong to a small family of highly conserved ∼70 kDa enzymes that can use the energy of ATP-hydrolysis to modify the structure, and consequently the function, of specific native proteins, and to unfold, solubilize, and thereby reduce the cellular concentration of harmful misfolded proteins (Finka et al., 2016). Particular HSP70s are expressed constitutively in the cytosol of bacteria and in all the ATP-containing compartments of eukaryotic cells. In unstressed bacteria, plant, and animal cells, HSP70s are 0.5–2% of the total protein mass (Finka and Goloubinoff, 2013). They form the central hub of the chaperone network that controls all aspects of cellular protein homeostasis: protein de novo synthesis, protein translocation across membranes, native folding, and oligomer assembly. HSP70s also participate in the active removal of toxic protein aggregates by actively converting them into harmless degraded peptides, or into natively refolded functional proteins (Calamini and Morimoto, 2012; Finka et al., 2016).

#### Edited by:

*Anat Ben-Zvi, Ben-Gurion University of the Negev, Israel*

### Reviewed by:

*Martin Lothar Duennwald, University of Western Ontario, Canada*

> \*Correspondence: *Pierre Goloubinoff<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Protein Folding, Misfolding and Degradation, a section of the journal Frontiers in Molecular Biosciences*

Received: *14 December 2016* Accepted: *05 January 2017* Published: *24 January 2017*

#### Citation:

*Goloubinoff P (2017) Editorial: The HSP70 Molecular Chaperone Machines. Front. Mol. Biosci. 4:1. doi: 10.3389/fmolb.2017.00001*

In immortalized human cancer cells and in naïve rat livers cells for example, HSPA8 is the major HSP70 species that is constitutively expressed in the cytosol, accounting for about half of the total mass of the HSP70s (Finka and Goloubinoff, 2013). In heat-shocked cells, particular HSP70s accumulate. Hence, following a 4 h mild fever-like heat-shock at 41◦C, the total mass of the HSP70s in Jurkat cells increases 1.6-folds, from ∼0.7% (at 37◦C) to ∼1.1% (Finka et al., 2015). Thus, although sharing 90% sequence identity with HSPA8, the cytosolic HSPA1A, which generally remains undetected in unstressed tissues, strongly accumulates during various abiotic stresses (Finka et al., 2015). Noticeably, non-heat-shocked cancer cells generally express constitutively abnormally elevated levels of HSPA1A that may even exceed the naturally high amounts of HSPA8. High expression levels of HSPA1A in otherwise unstressed tissues is thus a hallmark of malignancy and of poor survival outcome (Feder et al., 1992; Finka and Goloubinoff, 2013; Yang et al., 2015).

The endoplasmic reticulum HSP70, HSPA5 (called BIP), and the mitochondrial HSP70, HSPA9 (called mortalin) are the next most abundant HSP70 species. As in the case of cytosolic HSPA8, their concentration may also increase in response to heat-shock (Finka et al., 2015). In addition to their function in protein quality control, they act as pulling motors that import cytosolic polypeptides into their respective organelles.

Assisted by over 30 different J-domain cochaperones in eukaryotes (Dekker et al.) and various nucleotide exchange factors (NEFs) (Bracher and Verghese), the HSP70s can control a great number of housekeeping cellular processes. They can use the energy of ATP-hydrolysis to convert active (alter)native protein complexes into differently active native polypeptides. Thus,

**Abbreviations:** SBD, Substrate binding domain; NBD, Nucleotide binding domain; NEF, Nucleotide Exchange Factor; Natively folded proteins, proteins folded in a functional native conformation; (Alter)natively folded proteins, proteins folded in a different, albeit still native conformation.

at the exit of ribosomes, specific HSP70s can pull and regulate the de-novo folding of nascent polypeptides. Others can pull and unfold cytosolic polypeptides necessary for their translocation across membranes into organelles. Yet other HSPAs can regulate the post-translocation folding and assembly of (alter)natively folded protein complexes and coordinate their timely disassembly into differently active sub-complexes, as in the case of clathrin cages and triskalions (Sousa and Lafer; De Los Rios and Goloubinoff, 2016). HSP70s can also determine the lifespan of native cellular proteins by timely targeting them to chaperone-gated proteases, as in the case of the bacterial transcription factor σ32, which is targeted by DnaK to the FtsH protease (Rodriguez et al., 2008; VanPelt and Page, 2017). HSP70s also target misfolded proteins to the lysosome in a process called chaperone-mediated-autophagy (Cuervo and Wong, 2014).

Some HSP70s can act as ATP-fuelled molecular motors that, together with specific cochaperones, such as their eukaryotic HSP110 cognates (Mattoo et al., 2013; Nillegoda and Bukau) or the ClpB/HSP104s, which are non-cognate AAA+ ATPases (Mogk et al., 1999), can solubilize stable aggregates and convert them into harmless natively refolded or degraded polypeptides. Thus, together, the HSP70s form a powerful cellular defense network against harmful misfolded proteins. HSP70s failure to be expressed, or to function in aging metazoan cells, directly correlates with the late age onset of severe human degenerative diseases, such as Parkinson, Alzheimer, and type 2 diabetes that are associated to the accumulation of misfolded proteins in the damaged tissues (Hartl, 2016).

In this research topic, several reviews tackled from different complementing angles the central question: how may the members of a small class of relatively simple two-domain 70 kDa molecules use ATP to drive dramatic conformational changes, in so diverse aggregated, misfolded, and alternatively folded polypeptides, and thus control such a great number of different housekeeping and stress related biological functions in the cell?

In their review "Insight into the molecular mechanism of allostery in HSP70s," Mayer and Kityk described the intricate molecular paths of various allosteric signals during the HSP70 ATPase cycle, leading to dramatic movements of the two subdomains that bind the polypeptide substrates. They focused on specific residues that are involved in various key allosteric signals in the chaperone molecule, such as the signal that is issued by the substrate upon binding to the base of the substrate binding domain (SBD) to induce ATP hydrolysis in the nucleotide binding domain (NBD). Another signal is issued by the NBD to induce the closure of the SBD's lid onto its base, and yet another signal that is issued by the NEF-triggered NBD to induce the sequential release of the ADP from the NBD and of the structurally modified polypeptide from the SBD.

In their review "Multi-layered molecular mechanisms of polypeptide holding, unfolding and disaggregation by HSP70/HSP110chaperones" Finka et al. addressed the apparent consequences of the coordinated subdomain movements in the HSP70 molecule during the ATPase cycle, on the bound misfolded or (alter)natively folded polypeptide substrates, leading to their conversion into native or degraded polypeptides. Two complementing mechanisms are being discussed: (1) unfolding by direct clamping, in which the closure of the SBD lid onto a bulky misfolded protein segment and its consequent squeezing against the SBD base would result in the local unfolding of the bound misfolded segment. (2) Unfolding by entropic pulling, in which the distal binding of several HSP70 molecules onto the same misfolded polypeptide, and their ensuing independent dangling motions would generate a force capable of unfolding by entropic pulling the misfolded segments that are in between two HSP70-bound sites (Goloubinoff and De Los Rios, 2007).

The various allosteric paths leading to sub-domain movements and their possible effects on a bound misfolded substrate can be summarized as follow: In an ATP-bound HSP70, there is a tight crosstalk between a J-domain from a DNAJ co-chaperone anchored in the NBD, and a hydrophobic misfolded polypeptide substrate newly bound to the wide-open SBD. Both then concomitantly trigger ATP hydrolysis in the NBD and the closing of the SBD's lid that applies a local pressure onto a bulky misfolded segment in the bound substrate leading, upon tight engulfment by the SBD, to the extensive unfolding of the substrate (Sharma et al., 2010; Mashaghi et al., 2016). The subsequent transient binding of a NEF to the distal lobes of the NBD then triggers ADP release from the NBD and the opening of the protein-binding lid, allowing the newly unfolded polypeptide to dissociate and spontaneously refold to the native state.

However effective an unfolding nanomachine may the HSP70 molecule be, it remains quiet ineffective at choosing its substrates among the many different polypeptides in need to be structurally modified. Substrate discrimination by the HSP70 system is predominantly achieved by the J-domain co-chaperones that initially interact with the different substrates and at the same time bind the HSP70s through their J-domain.

In their review "DNAJs: more than substrate delivery to HSPA," Dekker et al. highlighted the crucial role of J-domain cochaperones to the protein quality control of metazoan cells. DNAJs can target HSP70s onto misfolded, aggregated, or specific alternatively folded protein substrates, to be unfolded, refolded, or ultimately degraded by chaperone-gated proteases. In addition, specific DNAJs are involved in the unidirectional translocation of cytosolic pre-proteins across membranes to the ER, the mitochondria, the lysosome, and in plants also the chloroplast and the peroxisome. Other DNAJs carry unique antiaggregation functions of their own, as in the case of mammalian DNAJB6, which is a very potent anti-aggregation chaperone that prevents the formation of toxic polyglutamine species that cause severe degenerative diseases, such as Huntington's chorea.

In their review, "Metazoan HSP70-based protein disaggregases: emergence and mechanisms," Nillegoda and Bukau further dwelt on the key role of J-domain cochaperones of HSP70, in particular the human DNAJA2 and DNAJB1, which seem to specifically recruit HSC70 and its sequencerelated HSP110 co-chaperone, to form powerful disaggregating hetero-oligomers capable of solubilizing denatured reporter proteins, among them, stable alfa-synuclein fibrils (Gao et al., 2015; Finka et al., 2016). Two putative "clamp and walk" and "metazoan nucleation" models are discussed for the HSP70- and HSP110-mediated disaggregation mechanism.

In contrast to misfolded protein aggregates serving as substrates to the HSC70-HSP110 disaggregation machinery, in their review "The role of molecular chaperones in clathrin mediated vesicular trafficking," Sousa and Lafer discuss the role of a specific J-domain cochaperone, auxilin, in the recruitment of HSC70 and HSP110 to a specific position onto a natively assembled protein complex: the clathrin cage. There, HSC70 uses ATP to pull and disassemble clathrin coats by a "steric wedge" mechanism, which is very similar to entropic pulling. Recently, the same authors brought evidence that loose HSC70 multimers might form and thus join their "steric wedge" entropic pulling forces to disassemble clathrin cages more efficiency. HSC70 also sequesters the depolymerized clathrin, implying that triskelion release must be regulated by HSP110 (Sousa et al., 2016). Interestingly, in neurons, HSP110 activity as a specific NEF for HSC70 is regulated upon phosphorylation by a Calciumregulated kinase (Ishihara et al., 2003). Thus, following the vesicle fusion with the plasma membrane and the discharge of their neuro-transmitters content in the synapse, the transient entry of Ca2<sup>+</sup> ions in the cytosol would be able to precisely signal the onset of clathrin cage dismantling for triskelion recycling.

In their review "The nucleotide exchange factors of HSP70 molecular chaperones" (Bracher and Verghese), Bracher and Verghese discussed the large diversity of the eukaryotic NEFs. In addition to the HSP110s, which are sequence wise-, structure wise-, and function wise-related to the HSP70s (Mattoo et al., 2013), there are unrelated HSPBP1/Sil1, BAG domain proteins and the M-domain of the HSP104 disaggregase cochaperone (Carroni et al., 2014; Doyle et al., 2015), and the GrpE type of NEFs to be found in prokaryotes and eukaryotic organelles of eubacteria origin. Despite their great disparity of structure, the various NEFs, interact with the same surfaces on the HSP70s: the tips of the two NBD subdomains embracing the ADP-binding pocket.

It should be noted that BAG domain proteins and GrpE do not have nucleotide binding sites. Moreover, both GrpE and HSP110 were shown to induce polypeptide release, respectively, from DnaK and HSP70, in the total absence of ATP (Sharma et al., 2010; Mattoo et al., 2013). Thus, these versatile regulatory cochaperones should be called ADP- and substrate-release factors rather than, nucleotide exchange factors.

### REFERENCES

Within cells, HSP70s can use ATP to drive structural changes in other cellular proteins. Yet, extracellular HSP70 is also to be found at the surface of cells facing the ATP-less outward milieu. In mammals extracellular HSP70 can stimulate innate immune responses and facilitate the cross-presentation of immunogenic peptides, in association with major histocompatibility complex (MHC) antigens (Shevtsov and Multhoff, 2016).

Providing biological context to this research topic, Przyborski et al. (Bracher and Verghese) discussed in their review "Plasmodial HSP70s are functionally adapted to the malaria parasite life cycle" the role HSP70 in a severe human disease: malaria. The parasite Plasmodium falciparum encodes for half a dozen different HSP70s, which are key to the survival and the pathology of the parasite in its various stages of its development, in particular for its resistance to high temperatures episodes during human fever, as well as of low ambient temperature when dwelling in the salivary glands of the mosquitos.

In conclusion, whereas the lack of an appropriate level of HSP70 expression in metazoan neural and muscle tissues, without stress and following biotic and abiotic stresses, has been associated to the onset of excessive damaging apoptosis, leading to degenerative tissue loss and accelerated aging (Kimura et al., 2007; Ben-Zvi et al., 2009; Morimoto and Cuervo, 2014), excessive accumulation of HSP70s has been linked to therapeutic resistance of carcinomas, increased metastasis, and poor survival outcomes to anti-cancer therapies (Sherman and Gabai, 2015; Figure 1). Yet, compared to terminally differentiated cells, rapidly growing embryonic cells that are not cancerous, also express high HSP70 and HSP90 levels (Jensen et al., 2013). Thus, the effectiveness of the various HSP70s at maintaining cellular protein homeostasis and controlling life span likely depends on yet to be characterized, fine-tuned qualitative differences in the expression and activation levels of the various HSP70s and their many J-domain, NEF, and disaggregase cochaperones.

### AUTHOR CONTRIBUTIONS

The manuscript was written solely by PG.

### FUNDING

Research is supported by Grant 31003A\_156948 from the Swiss National Fund.

Annu. Rev. Biochem. 85, 715–742. doi: 10.1146/annurev-biochem-060815- 014124

**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Goloubinoff. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Insights into the molecular mechanism of allostery in Hsp70s

### Matthias P. Mayer\* and Roman Kityk

Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ/ZMBH Alliance, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany

Hsp70s chaperone an amazing number and variety of cellular protein folding processes. Key to their versatility is the recognition of a short degenerate sequence motif, present in practically all polypeptides, and a bidirectional allosteric intramolecular regulation mechanism linking their N-terminal nucleotide binding domain (NBD) and their C-terminal polypeptide substrate binding domain (SBD). Through this interdomain communication ATP binding to the NBD and ATP hydrolysis control the affinity of the SBD for polypeptide substrates and substrate binding to the SBD triggers ATP hydrolysis. Genetic screens for defective variants of Hsp70s and systematic analysis of available structures of the isolated domains revealed some residues involved in allosteric control. Recent elucidation of the crystal structure of the Hsp70 homolog DnaK in the ATP bound open conformation as well as numerous NMR and mutagenesis studies bring us closer to an understanding of the communication between NBD and SBD. In this review we will discuss our current view of the allosteric control mechanism of Hsp70 chaperones.

#### Edited by:

Paolo De Los Rios, Ecole Polytechnique Fédérale de Lausanne, Switzerland

### Reviewed by:

Dejana Mokranjac, University of Munich, Germany Eileen M. Lafer, University of Texas Health Science Center at San Antonio, USA

\*Correspondence:

Matthias P. Mayer<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Protein Folding, Misfolding and Degradation, a section of the journal Frontiers in Molecular Biosciences

> Received: 20 August 2015 Accepted: 05 October 2015 Published: 20 October 2015

#### Citation:

Mayer MP and Kityk R (2015) Insights into the molecular mechanism of allostery in Hsp70s. Front. Mol. Biosci. 2:58. doi: 10.3389/fmolb.2015.00058 Keywords: Hsp70 heat-shock proteins, allostery, interdomain communication, conformational dynamics, structure-function relationships

### INTRODUCTION

Hsp70s are involved in a large variety of cellular processes. Thereby they interact with substrate proteins that are in many different conformations: with completely extended polypeptides such as nascent chains at the ribosome (Deuerling and Bukau, 2004; Hartl et al., 2011) or during translocation into organelles (Neupert and Herrmann, 2007; Chacinska et al., 2009); with partially folded and misfolded conformations in late folding intermediates, or upon disaggregation and refolding of stress denatured proteins (Tyedmers et al., 2010); and with native regulatory proteins to control their activity and stability (e.g., heat shock transcription factor σ <sup>32</sup> in E. coli or transcription factors, receptors, and kinases in eukaryotes) (Wegele et al., 2004), and while assisting oligomerization or disassembly of oligomeric structures (e.g., clathrin, Sousa and Lafer, 2015). Hsp70s are ATP dependent chaperones that consist of an N-terminal 45 kDa nucleotide binding domain (NBD) and a 25 kDa substrate polypeptide binding domain (SBD). They do not work alone but interact with cochaperones of the J-domain protein (DnaJ, Hsp40) family, which target Hsp70s to substrate proteins, and several families of nucleotide exchange factors. Hsp70s also cooperate with chaperones of other families like small HSPs and Hsp100s for protein disaggregation, with Hsp90 for regulation of native proteins, with ribosome bound chaperones like trigger factor in prokaryotes and specialized Hsp70s (RAC) in eukaryotes and with Hsp60s for de novo folding of proteins. Thus, Hsp70 is probably the most versatile of all chaperones, constituting a central hub of the cellular protein folding network.

One reason for this versatility is most likely the degenerate recognition motif of Hsp70s, which consists of a core of five preferentially hydrophobic amino acids flanked by regions in which positively charged residues are favorable for binding (Rüdiger et al., 1997). Such motifs occur frequently in most proteins. In the folded state these motifs are generally found in the hydrophobic core of the proteins and are exposed only during synthesis when emerging from the ribosomal exit tunnel, during translocation through membranes or during stress denaturation. This explains why Hsp70s interact with most proteins when they are in the denatured but not in the native state. Substrate proteins which interact with Hsp70 in their native conformation apparently expose such sequence motifs even in the completely folded state. Recognition of a short degenerative motif in substrate proteins eliminates any size limitations and Hsp70 can interact with very large proteins and protein complexes, like clathrin cages, or protein aggregates. Another reason for the versatility of the Hsp70 system is certainly the number of J-domain proteins which has increased in the course of evolution from six in E. coli and 22 in S. cerevisiae to 47 in humans (Kampinga and Craig, 2010). J-domain proteins either bind prospective protein substrates themselves or bind to structures like the ribosomal exit tunnel (e.g., zuotin) or translocation pores (e.g., Sec63, Pam18) where substrates for Hsp70 emerge, and recruit Hsp70 for specific protein folding tasks. Similarly, the different nucleotide exchange factors of three distinct families in the eukaryotic cytosol the modular multidomain Bag family, the HspBP1 family and the Hsp110 family, which are Hsp70 homologs themselves (Bracher and Verghese, 2015)—may harness Hsp70s for diverse functions.

Finally and most importantly, the intricate mechanism of the Hsp70 machine itself makes it such a versatile tool. In contrast to ATP-independent chaperones, the affinity of Hsp70 for substrates is regulated by nucleotide, the substrate itself, the J-domain cochaperones and nucleotide exchange factors (**Figure 1**). In a

bound open conformation (low-affinity state, PDB ID 4B9Q, Kityk et al., 2012) in cartoon representation with NBD subdomains IA, IB, IIA, IIB in different shades of green, SBDβ in dark red and SBDα in orange. Right, solution structure of DnaK in the ADP bound/nucleotide-free state as derived from residual dipolar coupling NMR experiments and crystal structures of the isolated domains (high-affinity state, PDB IDs 2KHO, Bertelsen et al., 2009) colored as in the ATP bound state and NBDs in identical orientation. (B) Overlay of the crystal structures of DnaK·ATP (PDB ID 4B9Q; gray) and Sse1 (PDB ID 2QXL, Liu and Hendrickson, 2007); NBD, deep teal; SBDβ, cyan; SBDα, blue. (C) Overlay of the crystal structures of DnaK·ATP (PDB ID 4B9Q; gray) and bovine Hsc70(1-554) (PDB ID 1YUW, Jiang et al., 2005) NBD, deep teal; SBDβ, cyan; SBDα, blue.

nutshell, in the ATP bound state Hsp70 has a low affinity for substrates but high substrate association and dissociation rates. Upon ATP hydrolysis, substrate association and dissociation rates decrease some 100 and 1000-fold, respectively, leading to an increase in affinity of 10 to 50-fold (Schmid et al., 1994; Mayer et al., 2000). However, ATP hydrolysis rates are very low but stimulated synergistically by the substrate itself and the J-domain cochaperone (Karzai and McMacken, 1996; Barouch et al., 1997; Misselwitz et al., 1998; Laufen et al., 1999; Silberg et al., 2004). Thus, Hsp70 acts like a mouse-trap where the substrate itself triggers its capture. The synergism of substrate with J-domain proteins in triggering ATP hydrolysis allows J-domain proteins to target Hsp70 to the proper substrate. At physiological ATP concentrations, nucleotide exchange is rate-limiting for substrate release and thus allows nucleotide exchange factors to regulate the residence time of substrates bound to Hsp70. This mechanism of association of substrates with Hsp70·ATP at high rates and subsequent ATP hydrolysis and transition to the high affinity state creates a non-equilibrium situation resulting in ultra-high affinity that so far has not been found in any other chaperone (De Los Rios and Barducci, 2014). In the following we will discuss the current knowledge of the structural basis for this allosteric mechanism.

### STRUCTURAL BASIS FOR ALLOSTERY IN Hsp70s

A thorough understanding of the structural basis for allostery in Hsp70s was hampered for many years by the lack of a structure of the full-length protein. Structures of isolated domains (Flaherty et al., 1990; Zhu et al., 1996) were available for many years but information on the assembly of NBD and SBD in ADP or ATP states is rather recent (Jiang et al., 2005; Chang et al., 2008; Bertelsen et al., 2009; Kityk et al., 2012; Qi et al., 2013). The NBD shares structural homology with actin and sugar kinases (Flaherty et al., 1991), and can be divided into two lobes (I and II) with two subdomains each (IA, IB, IIA, IIB), which form a deep cleft at the bottom of which nucleotides bind, contacting all four subdomains (Flaherty et al., 1990, **Figure 1**). The SBD is composed of a two-layered β-sandwich subdomain (SBDβ), which contains the substrate binding channel with a central pocket capable of binding a single hydrophobic residue of the substrate; an α-helical subdomain (SBDα), consisting of five helices; and a C-terminal intrinsically disordered segment of about 30 residues of unclear function, which seems to be involved in chaperone activity and, in the eukaryotic cytosol, contains the EEVD motif at the very C-terminus, serving as docking site for the cochaperones Hop/Sti1 and Chip (Zhu et al., 1996; Demand et al., 1998; Ballinger et al., 1999; Scheufler et al., 2000; Zhang et al., 2005; Smock et al., 2011). In the isolated SBD, helices A and B of the SBDα are tightly packed onto the SBDβ, enclosing the substrate binding channel like a lid. This also seems to be the most prevalent conformation of the fulllength protein in the nucleotide-free and ADP bound states (Jiang et al., 2005; Chang et al., 2008; Bertelsen et al., 2009; Marcinowski et al., 2011; Schlecht et al., 2011). The crystal structure of Geobacillus kaustophilus DnaK and NMR studies on E. coli DnaK suggest that NBD and SBD are rather separated, independently tumbling units in the nucleotide-free and ADP states only connected by the flexible linker (Swain et al., 2007; Chang et al., 2008; Bertelsen et al., 2009; Zhuravleva et al., 2012). In contrast, the crystal structure of nucleotide-free bovine Hsc70 shows NBD and SBD in a docked conformation (Jiang et al., 2005).

Recently, the crystal structure of DnaK in the ATP-bound open conformation was solved, which significantly broadened our knowledge about allostery in Hsp70s (Kityk et al., 2012; Qi et al., 2013). Comparison of the DnaK·ATP structure with the solution structure of DnaK·ADP indicates that ATP binding leads to dramatic structural rearrangements in the protein (**Figure 1A**). DnaK·ATP has a more compact structure; SBDα and SBDβ are completely detached from each other and docked onto two sides of the NBD; and the interdomain linker is buried in the lower crevice of the NBD. This structure is similar to the structure of the Hsp110 Sse1, which serves as nucleotide exchange factor for Hsp70s (Dragovic et al., 2006; Raviol et al., 2006; Liu and Hendrickson, 2007; Polier et al., 2008; Schuermann et al., 2008) but has clear differences in the structure and orientation of the SBDβ and SBDα (**Figure 1B**). Differences are more striking when compared to the structure of a twodomain construct of bovine Hsc70, which was crystallized in the nucleotide-free state (Jiang et al., 2005) (**Figure 1C**). An overlay of the NBD of DnaK·ATP with all previously solved crystal structures of isolated NBDs in complex with different nucleotides and the solution structure of the full-length protein in the ADP state (e.g., Flaherty et al., 1990; Wilbanks et al., 1994; O'Brien et al., 1996; Jiang et al., 2005; Bertelsen et al., 2009) reveals that ATP binding leads to the rotation of the NBD lobes toward each other (**Figure 2A** and **Supplemental Movie 1**). This leads to a widening of the lower crevice of the NBD, enabling the linker to insert between subdomains IA and IIA. The surface rearrangements of the NBD allow SBDα and SBDβ docking on the NBD. A number of residues (e.g., Arg151, Arg167, Asp326, Asp393, Lys414, Asp481; all numbers refers to residues in E. coli DnaK), which are part of an extensive Hbond network at the NBD-SBDβ interface, were found in genetic and biochemical studies to be important for allosteric signal transmission between the two domains (Montgomery et al., 1999; Vogel et al., 2006a,b; Smock et al., 2010; Kityk et al., 2015) (**Figure 2B**). Thus, interface stabilization by an H-bond network plays a pivotal role in interdomain communication in Hsp70s. In particular, Asp481, which contacts the NBD subdomain IA, and K414, which contacts NBD subdomain IIA, act like a clamp, fixing the NBD in the ATP bound state and strongly reducing basal ATPase activity in the absence of a trigger provided by substrate binding and interaction with a J-domain protein (Kityk et al., 2015).

ATP-induced docking of the SBD to the NBD leads to the stabilization of the open conformation of the SBD. In SBDα, detached from SBDβ, helices A and B form a continuous helix. The substrate binding cleft in SBDβ is wider as compared to the structure of the isolated DnaK-SBD with bound peptide substrate (PDB code 1DKX; **Figures 2C,D**), which is consistent with low affinity for polypeptide substrates and high substrate

residues known to be involved in interdomain communication and found in the NBD-SBD interface in space-filling representation with carbon atoms in yellow, oxygen atoms in red and nitrogen atoms in blue. (C) Overlay of the SBD of DnaK in the ATP bound open conformation; SBDβ, dark red; SBDα, orange and cut for space reasons and the structure of the isolated SBD in complex with a substrate peptide (PDB ID 1DKX, Zhu et al., 1996); SBDβ, cyan; SBDα, dark blue; peptide in light blue and stick representation. Arrows indicate ATP-induced changes visible in this orientation. (D) Overlay of SBDβ as in (A), but rotated by 90◦ as indicated. Arrows indicate ATP-induced narrowing of the central substrate binding pocket. (E) Overlay of the SBDβ of the two available structures of DnaK in the ATP bound open conformation (PDB IDs 4B9Q, Kityk et al., 2012; dark red; 4JN4, Qi et al., 2013; green). Indicated are the substrate enclosing loops <sup>L</sup>1,<sup>2</sup> , <sup>L</sup>3,<sup>4</sup> , and <sup>L</sup>5,<sup>6</sup> . (F) In the ADP state Hsp70s are in equilibrium between the closed conformation with NBD (green) and SBD (dark red) only connected via the conserved interdomain linker (black) and substrate (S) tightly enclosed in the substrate binding pocket and a very transient open conformation with NBD and SBDβ (dark red) docked. Since the open conformation is very transient, substrates only dissociate from this state at low rates. Nucleotide exchange factors (NEFs) catalyze ADP dissociation. Subsequent ATP binding to Hsp70 induces rotation of the NBD lobes toward each other, opening of the lower cleft of the NBD, insertion of the conserved interdomain linker, and docking of SBDβ to the NBD, resulting in opening of the α-helical lid (SBDα, magenta) and release of the substrate with high rates. In the ATP state Hsp70s are also in equilibrium between the open and very transient closed conformation. The outer loops of the SBDβ are highly dynamic. Substrates associate with J-domain proteins (JDP) and bind with high rates to the open conformation of Hsp70. Substrate binding induces closing of the SBDα and dissociation of the SBDβ from the NBD, which allows rotation of the NBD lobes to a position optimal for ATP hydrolysis. Substrates stimulate ATP hydrolysis through a distinct pathway (blue) involving a trigger on the NBD (orange). How JDPs act in synergism with substrates is currently not known. Dashed arrows indicate domain movement/dynamics.

association and dissociation rates in the ATP-state (Schmid et al., 1994; Mayer et al., 2000). The substrate binding cleft is also more open than the NMR structure of the substrate-free isolated SBDβ (PDB code 1DG4) (Pellecchia et al., 2000) This observation suggests that the conformational changes in SBDβ of the DnaK·ATP structure are induced by SBDβ-NBD interactions, and not by the absence of substrate or detachment of SBDα, as was demonstrated recently (Zhuravleva and Gierasch, 2015). Notably, the outer loops of the SBDβ are the only parts which differ significantly between the two crystal structures of DnaK·ATP (PDB codes 4B9Q and 4JN4; **Figure 2E**) (Kityk et al., 2012; Qi et al., 2013). While in one structure the outer loops L3,<sup>4</sup> and <sup>L</sup>5,<sup>6</sup> protrude upward from the <sup>β</sup>-sandwich forming a cradle for the substrate (4B9Q), in the other structure they extend the β-strands horizontally (4JN4) (**Figure 2E**). Although the outer loops seem to be very flexible in the first DnaK·ATP structure as well, as indicated by the high b-factor, it is not clear whether the SBDβ opens to the same extent seen in the second structure. The construct used in the second structure had the outer loop L3,<sup>4</sup> replaced by a short MGG-motif, and, in the crystal L5,<sup>6</sup> makes extensive contacts with other molecules that stabilize the extended conformation.

Comparison of the SBDβ of the DnaK·ATP structure with the structure of the isolated SBD with bound substrate (PDB code 1DKX) also suggests a mechanism for how substrates stimulate the ATPase activity. Substrate binding is accompanied by pronounced conformational rearrangements in the SBDβ: (I) the substrate enclosing loop L1,<sup>2</sup> moves toward the substrate and away from the NBD, (II) binding of the central hydrophobic residue requires an expansion of the substrate binding pocket as compared to the ATP-bound state, leading to an overall reorganization of the SBDβ. These changes are transmitted toward the interface through a defined pathway involving residues Val440, Leu484, and Asp148, presumably leading to disruption of some interdomain contacts and release of the linker from the lower crevice of the NBD (Kityk et al., 2015). As a consequence, the NBD subdomains are able to rotate to a degree where the catalytic residues in the ATPase active center reach the optimal position for γ-phosphate cleavage. Such a model is consistent with NMR measurements on a full-length DnaK construct with bound ATP and substrate peptide, which suggest that substrate binding causes dissociation of the SBDβ from the NBD (Zhuravleva et al., 2012).

Although all of these details on the mechanics of allosteric regulation have been elucidated mainly in E. coli DnaK, they are believed to be valid for all Hsp70s due to the high evolutionary conservation of this protein family, albeit, variations of this theme certainly exist, in particular in respect to kinetics of conformational changes.

### INTERPLAY BETWEEN CONFORMATIONAL DYNAMICS AND ALLOSTERY IN Hsp70s

Many different studies have demonstrated that Hsp70s are highly dynamic and undergo transitions between open and closed conformations independent of the nucleotide status (Mapa et al., 2010; Marcinowski et al., 2011; Schlecht et al., 2011; Kityk et al., 2012). Thus, each nucleotide state consists of an ensemble of different conformations as originally proposed (Mayer et al., 2000) and nucleotides modulate the frequency of structural transitions and affect the equilibrium between different conformers of Hsp70s. A recent NMR and molecular dynamics study revealed that the SBDβ is much more dynamic in the ATP state than in the ADP state, and that the SBDβ-NBD contacts influence the dynamics of the substrate binding pocket (Zhuravleva and Gierasch, 2015). Based on their data, the authors propose that the substrate binding loops and SBDβ-NBD interface are dynamically coupled and that this coupling is part of the allosteric mechanism.

The conformational equilibrium is also influenced by substrates. Binding of a substrate to Hsp70·ATP seems to induce the closure of the α-helical lid before ATP hydrolysis occurs. Consistent with this notion is the observation that substrate binding reduces the ATP-induced blueshift of Trp102 fluorescence in DnaK, indicating the undocking of the α-helical lid from the NBD even in the absence of ATP hydrolysis (Slepenkov and Witt, 1998; Vogel et al., 2006a). On the other hand, bound substrates were demonstrated to slow ATP-induced docking of the α-helical lid onto the NBD (Kityk et al., 2012). **Figure 2F** summarizes the current view of allostery and the conformational cycle of Hsp70s.

### PERSPECTIVES

Recent years have brought about significant progress in understanding the underlying mechanisms of interdomain communication in Hsp70s. Despite these advances many questions are still not solved. For example, due to the relative scarcity of structural information, the details of the Hsp70- Hsp40 interaction remain elusive. Hence it is not clear how Hsp40s alone, or together with the substrate, influence allosteric signal transmission between the domains. In eukaryotes Hsp70 additionally interacts with other co-chaperones like HOP and CHIP, linking the Hsp70 machinery to the Hsp90 system and the proteasome degradation pathway, respectively. It is not clear whether they alter, either alone or in cooperation with other cochaperones like nucleotide exchange factors, the interdomain communication in Hsp70s to facilitate transfer of the substrate to Hsp90 or to stabilize the Hsp70-substrate complex for timely release at the proteasome. Lastly, it is not known whether interdomain communication is subject to modulation by the post-translational modifications of Hsp70s that occur in eukaryotes (Muller et al., 2012; Truman et al., 2012; Morgner et al., 2015). Interest in the molecular mechanisms of interdomain communication in the Hsp70s is also driven by the prospect of medical applications. Taking into account the important role of Hsp70s in different pathophysiological processes, including cancer, neurodegenerative diseases and autoimmunity, one of the key research areas is development of Hsp70 modulators. The nucleotide binding pocket of Hsp70 was classified as poor inhibitor binding site due to the mostly electrostatic and polar interactions with nucleotide (Halgren, 2009). The polypeptide substrate binding site may be unsui for inhibitors and activators of Hsp70 function and only inhibitors with limited potency have been found so far (Otvos et al., 2000; Otaka et al., 2007; Yamamoto et al., 2010; Knappe et al., 2011). Therefore, allosteric control of the ATPase cycle appears as an attractive target and the first allosteric modulators have already been found (Wisén and Gestwicki, 2008; Kang et al., 2014; Taldone et al., 2014; Hassan et al., 2015).

### ACKNOWLEDGMENTS

The work of the authors was funded by the Deutsche Forschungsgemeinschaft (MA1278/4-1, SFB638 TP13).

### REFERENCES

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmolb. 2015.00058

Supplemental Movie 1 | Lobe rotation in the NBD of Hsp70 upon transition between the ATP and ADP bound state. The structure of the NBD of E. coli DnaK in the ATP bound conformation (PDB ID 4B9Q) was morphed into the structure of the ADP bound conformation (PDB ID 2KHO) using the Yale Morph Server (Krebs and Gerstein, 2000; Flores et al., 2006). Subdomains of the NBD in different shades of green (IA, dark green; IB chartreuse; IIA, dark teal; IIB, lime); NBD-SBD-linker in purple.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Mayer and Kityk. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Multi-layered molecular mechanisms of polypeptide holding, unfolding and disaggregation by HSP70/HSP110 chaperones

#### Andrija Finka1, 2, Sandeep K. Sharma<sup>3</sup> and Pierre Goloubinoff <sup>1</sup> \*

#### Edited by:

James Shorter, University of Pennsylvania, USA

#### Reviewed by:

Kevin Anthony Morano, The University of Texas Health Science Center at Houston, USA Martin Lothar Duennwald, University of Western Ontario, Canada

#### \*Correspondence:

Pierre Goloubinoff, Department of Plant Molecular Biology, Faculty of Biology and Medicine, University of Lausanne, Biophore Building, Lausanne CH-1015, Switzerland<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Protein Folding, Misfolding and Degradation, a section of the journal Frontiers in Molecular Biosciences

> Received: 13 April 2015 Accepted: 19 May 2015 Published: 05 June 2015

#### Citation:

Finka A, Sharma SK and Goloubinoff P (2015) Multi-layered molecular mechanisms of polypeptide holding, unfolding and disaggregation by HSP70/HSP110 chaperones. Front. Mol. Biosci. 2:29. doi: 10.3389/fmolb.2015.00029 <sup>1</sup> Department of Plant Molecular Biology, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland, <sup>2</sup> Laboratoire de Biophysique Statistique, School of Basic Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland, <sup>3</sup> Department of Chemistry, Umeå University, Umeå, Sweden

Members of the HSP70/HSP110 family (HSP70s) form a central hub of the chaperone network controlling all aspects of proteostasis in bacteria and the ATP-containing compartments of eukaryotic cells. The heat-inducible form HSP70 (HSPA1A) and its major cognates, cytosolic HSC70 (HSPA8), endoplasmic reticulum BIP (HSPA5), mitochondrial mHSP70 (HSPA9) and related HSP110s (HSPHs), contribute about 3% of the total protein mass of human cells. The HSP70s carry out a plethora of housekeeping cellular functions, such as assisting proper de novo folding, assembly and disassembly of protein complexes, pulling polypeptides out of the ribosome and across membrane pores, activating and inactivating signaling proteins and controlling their degradation. The HSP70s can induce structural changes in alternatively folded protein conformers, such as clathrin cages, hormone receptors and transcription factors, thereby regulating vesicular trafficking, hormone signaling and cell differentiation in development and cancer. To carry so diverse cellular housekeeping and stress-related functions, the HSP70s act as ATP-fuelled unfolding nanomachines capable of switching polypeptides between different folded states. During stress, the HSP70s can bind (hold) and prevent the aggregation of misfolding proteins and thereafter act alone or in collaboration with other unfolding chaperones to solubilize protein aggregates. Here, we discuss the common ATP-dependent mechanisms of holding, unfolding-by-clamping and unfolding-by-entropic pulling, by which the HSP70s can apparently convert various alternatively folded and misfolded polypeptides into differently active conformers. Understanding how HSP70s can prevent the formation of cytotoxic protein aggregates, pull, unfold, and solubilize them into harmless species is central to the design of therapies against protein conformational diseases.

Keywords: entropic pulling, HSP40 heat-shock proteins, chaperone proteins, misfolded proteins, conformational diseases, DnaK, DNAJ homologs

## HSP70/HSP110 Evolution and Cellular Functions

Together with the HSP90s, members of the conserved HSP70/HSP110 family (HSP70s) are prevailing ATP-hydrolyzing chaperones that control all aspects of cellular proteostasis. The HSP70s and co-chaperones may constitute up to 3% of the total protein mass of unstressed human cells (Finka and Goloubinoff, 2013; Finka et al., 2015). In the test tube, proper mixtures of purified HSP70s and co-chaperones can prevent the aggregation of artificially unfolded proteins. This is, however, a rather ineffective process in the absence of ATP, which necessitates a large molar excess of chaperones over their protein substrates (Sharma et al., 2011). In stressed cells, owing to the presence of ATP and J-domain co-chaperones, the HSP70s are much more effective "holding" chaperones. Under regular physiological conditions, they may further use ATP-hydrolysis to warrant the proper de novo folding of nascent polypeptides, drive the post-translational translocation of cytosolic polypeptides across membranes and promote their assembly into active oligomers, or drive their disassembly into reversibly inactive conformers (Schuermann et al., 2008; Mattoo et al., 2013). Importantly, HSP70s can forcefully disaggregate stable stressor mutation-induced misfolded proteins, which may be toxic to animal cells and cause the apoptosis of old neurons in particular, leading to neural tissue degeneration (Bellotti and Chiti, 2008). Moreover, the HSP70s can mediate the refolding of solubilized misfolded polypeptides into harmless native proteins, or control their degradation by chaperone-gated proteases (Mattoo and Goloubinoff, 2014; Cho et al., 2015).

With the exception of exotic thermophilic and hyperthermophilic archaea, genes encoding for HSP70s (DnaK in bacteria) are present in all living organisms (Macario and De Macario, 1999). In over 1200 sequenced bacterial genomes, only two members of the order Aquificales lack HSP70 genes (Warnecke, 2012). Likewise, all eukaryotic genomes encode for at least half a dozen HSP70 genes and three or four related HSP110 (HSPHs) genes, predicted to be expressed in all the ATPcontaining compartments of the cell: cytosol, nucleus, lumen of the endoplasmic reticulum (ER), mitochondrial matrix and in plants, the chloroplast stroma and the glyoxisome (Schlicher and Soll, 1996; Wimmer et al., 1997). Moreover, HSP70s can be secreted and exposed on the extra-cellular surface, were they carry a yet unclear ATP-independent immunogenic function, particularly important for cancer detection and therapy (Tytell, 2005).

A bioinformatic search in the human genome can identify 13 HSP70 and 4 HSP110 genes, which are also actively transcribed. In addition, there are 50 J-domain co-chaperones, known to target the various HSP70s onto their substrates, and at least six nucleotide exchange factors (NEFs) called BAG and GrpE (Kampinga et al., 2009; Finka et al., 2011; Mayer, 2013; Clerico et al., 2015). Some genes are poorly expressed and may remain below detection thresholds of current mass spectrometry methods, or they may be conditionally expressed only in particular tissues, or under various stresses (Finka et al., 2011) such as heat shock (Finka et al., 2015). In Hela cells, high throughput mass spectrometry proteomic using stable isotope labeling of amino acids in cell cultures (SILAC) identified and precisely quantified ten different HSP70/HSP110 proteins, eighteen J-domain cochaperones and five NEFs (Geiger et al., 2012; Finka and Goloubinoff, 2013) (**Figure 1A**) that summed into 3.2% of the total protein mass. In comparison, a medium resolution mass spectrometry proteomic analysis of human Jurkat cells, identified and quantified ten different HSP70/HSP110 proteins, fifteen J-domain cochaperones and five NEFs (Finka et al., 2015) that summed into 2.7% of the total protein mass.

When exposed to various abiotic or chemical stresses, such as treatments with heat, the HSP90 inhibitor geldanamycin, or various non-steroidal anti-inflammatory drugs, the mRNA and protein levels of cytosolic HSP70 (mainly HSPA1A, HSPA8, and HSPA4) may increase many folds, whereas levels of ER HSP70 (also named HSPA5, BIP) remain unchanged (Saidi et al., 2007; Finka et al., 2011, 2015). Reciprocally, under a ER stress, such as treatments with deoxyglucose or tunicamycin causing a typical Unfolded Protein Response (Ryno et al., 2013), the mRNA levels predominantly of ER BIP may increase many folds, whereas the mRNA levels of cytosolic HSP70 and HSC70 remains unchanged (Finka et al., 2011). The quantitative observation that without stress, HSC70 and BIP are very abundant chaperones, implies that they both carry central house-keeping physiological functions (Finka et al., 2015), beyond their well-characterized defensive role during and following stress (Ben-Zvi et al., 2004; Shorter, 2011; Rampelt et al., 2012; Mattoo et al., 2013).

### HSP70s Can Assist Translation by Pulling Polypeptides out of Ribosomes

At the exit of eukaryotic ribosomes, the L31 protein can bind a special J-domain cochaperone called zuotin (DNAJC2), which in turn can anchor a special HSP70 (SSZ1) and cytosolic HSC70. The presence of several consecutive bulky hydrophobic residues in the sequence of some nascent polypeptides might slow down their translocation through the narrow channel of the ribosome. Remarkably, ATP-hydrolysis by SSZ1 and HSC70 and their consequent clamping onto the emerging polypeptides, alongside their dissociation from their zuotin anchor, can exert a pulling force to relieve the translocation stalling (Otto et al., 2005; Fiaux et al., 2010; Koplin et al., 2010; Shalgi et al., 2013, 2014; Zhang et al., 2014).

### ER HSP70s Can Pull Polypeptides into the ER Lumen

Not all ER proteins are synthetized and co-translocated on the SRP-bound ribosomes. Some pre-polypeptides are first synthetized on cytosolic ribosomes. For thermodynamic reasons they may have to reach a near-native fold before being presented to the ER surface to be further translocated across narrow import channels into the ER lumen. On the ER lumen side, BIP (HSPA5) in the ATP-bound state can spontaneously associate to a specific anchoring J-domain cochaperone (SEC63) and thus become poised in the opened state to bind incoming polypeptides from the cytosol. Upon ATP hydrolysis, luminal BIP may "lock" on a threaded polypeptide emerging from the pore and, owing to the

**B**

FIGURE 1 | Amounts, stoichiometries and phylogenetic relationships among HSP70s and their main cochaperones in human cells. (A) List of the significantly detected HSP70 and HSP110 cognate proteins (light green), J-domain cochaperones (orange) and NEFs (blue) in Hela and Jurkat cells and their presumed sub-cellular compartments. (B) Phylogenetic tree (neighbor joining method) from the protein sequences of the most abundant

HSP70 and HSP110 proteins in Jurkat cells, using E. coli DnaK and HSCA as out-groups. Presumed subcellular localization: CYT; cytoplasmic (cyan), ER; Endoplasmic reticulum (yellow). MIT; mitochondria (magenta). The copy number per micron cube are from human (Jurkat) cells (Finka and Goloubinoff, 2013; Finka et al., 2015) and from E. coli (gray) (Arike et al., 2012).

concomitant dissociation of ADP-bound BIP from its J-anchor SEC63, the chaperone can exert an entropic pulling force on the locked-upon incoming polypeptide. Pulling of the translocating polypeptide from inside the lumen may compel bulky structured domains that are still on the cytosolic side to collide with the pore entry, leading to their unfolding and unidirectional translocation into the lumen. There, the protein can reach the native state, assisted by soluble DNAJB11 and the ATP-fuelled unfoldases BIP (HSPA5) and HYOU (Matlack et al., 1997; Goloubinoff and De Los Rios, 2007; Griesemer et al., 2014; Melnyk et al., 2015).

### HSP70s can Pull Polypeptides into the Mitochondria

Similarly, in the mitochondrial matrix, a special HSP70 (mortalin, HSPA9) can associate in the ATP-bound state to two anchoring J-domain cochaperones named PAM16/18, and also to a special anchor named TIM44 (Slutsky-Leiderman et al., 2007) and thus await forincoming polypeptides from the cytosol. Given that a steep proton gradient needs to be maintained across the inner mitochondrial membrane, import pores must be as narrow as possible. When a typical hydrophobic polypeptide segment emerges, HSPA9 is triggered to clam upon it, while hydrolyzing ATP and concomitantly dissociating from its membrane anchors. This exerts an entropic pulling force that can unfold the polypeptide on the cytosolic side and unidirectionally translocate it to into the mitochondrial stroma (Goloubinoff and De Los Rios, 2007).

### HSP70s can Control Protein Degradation

HSP70s also control protein degradation by chaperoning various native polypeptides toward specific proteases. Thus, without heat stress, bacterial DnaK, together with DnaJ can specifically bind the native σ32 transcription factor and present it to the protease FtsH for rapid degradation (Arsene et al., 2000). In eukaryotes, HSP70s can direct ubiquitin E3 ligases onto a polypeptide target to be subsequently degraded by the proteasome (Demand et al., 2001; Cyr et al., 2002; Kettern et al., 2010). In addition to chaperone-assisted proteasomal degradation, HSP70s are involved in selective chaperone-assisted autophagy and chaperone-mediated autophagy (Kettern et al., 2010).

### The HSP70s Substrates

HSP70s can preferentially bind and process misfolded or alternatively-folded polypeptides, as opposed to (natively-)unfolded and natively-folded polypeptides for which they may have a lower affinity (Priya et al., 2013). By alternatively folded proteins, we intend to emphasize that such substrates can be natively folded and active proteins that also expose specific hydrophobic surfaces with a high affinity for J-domain co-chaperones and HSP70s. Under different biological circumstances, the alternatively folded proteins may acquire differently active, native states without exposed hydrophobic surfaces, thereby having low affinity for the chaperones. Examples of alternatively folded active proteins that can bind chaperones: clathrin cages (Schuermann et al., 2008; Mattoo et al., 2013), IκB oligomers (Weiss et al., 2007), nuclear heat-shock factor-1 trimers (Voellmy and Boellmann, 2007b), nuclear steroid hormone receptors (Echeverria and Picard, 2010), bacterial transcription factor σ32 (Rodriguez et al., 2008); their corresponding, differently active native conformers being, respectively, the clathrin triskelions, IkB monomers, inactive cytosolic heat-shock factor-1, cytosolic steroid hormone receptor and unfolded bacterial transcription factor σ32.

Given the general unfolding activity of HSP70s, it is unclear whether the so called intrinsically unfolded proteins are HSP70s substrates. The intrinsically unfolded α-synuclein protein does not bind DnaK or DnaJ, although it optimally exposes an ideal putative DnaK binding site (Rudiger et al., 1997). In contrast, once α-synuclein is stably aggregated into small oligomers, it strongly and specifically binds DnaJ, which in turn recruits DnaK on the stable misfolded α-synuclein oligomers. Upon ATP-hydrolysis and locking, the oligomers become partially unfolded and disaggregated (Hinault et al., 2010). DnaJ thus guides DnaK specifically onto the misfolded substrate, but not onto the unfolded polypeptide, suggesting that in general, the unfolded conformers might behave as the low-affinity products of the chaperone unfolding reactions.

### HSP70s Can Control Biological Switches

HSP70s are key regulators of protein complexes involved in vesicular trafficking. Thus, HSC70 and HSP110, both assisted by the particular J-domain cochaperone auxilin, mediate the ATP-dependent dismantling of clathrin cages for recycling (Augustine et al., 2006; Morgan et al., 2013). Together with the HSP90s, the HSP70s can also control the activation of the steroid hormone receptors (Dittmar et al., 1998) and their own expression under heat stress, by regulating the activity, the oligomeric and phosphorylation state of heat-shock transcription factor-1 (Voellmy and Boellmann, 2007a). It has been reported that the pathological form of the cystic fibrosis transmembrane conductance regulator (CFTR) is stalled with a HSC70/HSP90 complex during protein maturation, suggesting that the HSP70/HSP90 chaperone complexes act transiently more on the early folding steps of the mutant CFTR than in the case of wild type CFTR (Coppinger et al., 2012). HSP70 over-expression in TNF-treated mammalian cells may cause the deoligomerization and thus the inactivation of large active IκB complexes, thereby inhibiting apoptosis (Aschkenasy et al., 2011). In parallel, HSP70s, especially HSPA1A can also inhibit caspases (Sabirzhanov et al., 2012) and are thus attractive drug targets for pro-inflammatory diseases suffering from unchecked apoptosis, such as Parkinson and Alzheimer's diseases. Therapies based on the over-expression of HSP70s, HSPA1A and HSP110 (HSPH1, Apg-1) in particular (Mattoo et al., 2013), might arrest pro-inflammatory degenerative diseases caused by toxic protein aggregates (Hinault et al., 2006). Yet, HSP70s are also prooncogenic factors against, which specific inhibitors need to be developed for cancer therapies (Schilling et al., 2015).

HSP70s appear to use a similar mechanism to process both randomly structured aggregates and alternatively folded oligomers: A J-domain cochaperone is first observed to target a HSP70 molecule onto the substrate. This may then trigger ATP-hydrolysis by the HSP70 and its "locking" onto the target polypeptide, thereby causing the local unfolding of the clampedupon secondary structures and leading to the disaggregation and/or deoligomerization of the substrate. Upon NEF-mediated release, the unfolded substrate may then refold into a differently active polypeptide or be further degraded by an ATP-consuming protease (Goloubinoff and De Los Rios, 2007).

The large diversity of the above-described cellular functions, raises the question of how the HSP70s, which are simple ∼640 residue molecules sharing a simple common mechanism, may carry out so diverse cellular tasks? HSP70s are relatively simple two-domain individual 70 kDa polypeptides, which in order to act as effective polypeptide unfolding nanomachines, do not need to assemble into large cage-like oligomers, as in the case of the GroEL/CCT chaperonins, or ring-shaped hexamers in the case of ClpB/HSP104 (Priya et al., 2013; Mattoo and Goloubinoff, 2014). Here, we discuss various non-exclusive molecular mechanisms by which the HSP70s may carry so many diverse housekeeping and stress-damage control tasks in the cells.

### HSP70s Nomenclature and Phylogeny

As in the case of gene families that have been studied for four decades, the nomenclature of the HSP70s is very complex and confusing with historical layers of terms. For example, the most abundant HSP70 is called DnaK in bacteria, SSA in yeast, HSC70 in humans, whereas they should be called HSPA8 according to the last agreed-upon nomenclature (Kampinga et al., 2009). Similarly, the main ER HSP70, which was traditionally called BIP or Grp78, recently became HSPA5. ER HSP110, which was traditionally called Grp170, is now called HYOU1, although sequence-wise it clearly belongs to the same clade as the cytoplasmic HSPH1, which was traditionally called HSP105 or Apg1 (**Figure 1A**).

A phylogenetic tree generated from the amino acid sequences of the most abundant HSP70s in Hela cells, HSPA8 (cytosolic HSC70), HSPA1A (cytosolic HSP70), HSPA9 (mitochondrial mortalin) HSPA5 (ER BIP), HSPH1, HSPH2 (cytosolic HSP110s) and HYOU (ER HSP110), shows two large subclasses: The DnaKlike HSP70s, that are both present in bacteria and eukaryotes and the HSP110s (Mattoo et al., 2013), which are only in the cytosol and the ER lumen of eukaryotes (**Figure 1B**).

### HSP70s Structures and Functions

HSP70 and HSP110 are composed of a highly conserved twolobbed 40-kDa nucleotide-binding domain (NBD) (**Figure 2A**, light beige) and of a more variable 30–50 kDa protein-binding domain (PBD) (**Figure 2A**, orange). The highest degree of evolutionary conservation is in the 40 kDa N-terminal NBD, which functions as ATP-fuelled molecular motor regulating dramatic structural changes in the 30 kDa C-terminal PDB (Mayer, 2013). The PDB resembles a crocodile jaw composed of a beta-strand subdomain, acting as a rigid base and a flexible α-helical subdomain acting as flexible lid, across which, bulky misfolded polypeptide substrates can bind and thus become "prays" to be chewed upon. In the apo- and ATP-bound states, the lid is widely open, with its back rear sticking to the NBD, allowing bulky misfolded, or structured alternatively folded polypeptides (such as the transcription factor σ32) with exposed hydrophobic surfaces, to freely bind the hydrophobic surfaces in the wide-open chaperone jaws (Schlecht et al., 2011; Clerico et al., 2015).

## The Multilayered Molecular Mechanisms of the HSP70s

### Mechanism of Passive Prevention of Aggregation

Once a protein is artificially denatured in the presence of a molar excess of HSP70 (and HSP40) but without ATP, it can weakly bind the chaperones and thus become partially prevented from forming large aggregates that scatter light. Hence, many in vitro chaperone assays measure the decrease in the net light scattering signal of an aggregating polypeptide in the presence of increasing concentrations of chaperones (Haslbeck and Buchner, 2015). But light scattering intensities are not quantitative and moreover don't reflect the ability of HSP70 chaperone molecules to use the energy of ATP-hydrolysis to change the conformational fate and thus the biological activity of hundreds different polypeptides in the cell.

### Mechanisms of Active Unfolding by Clamping

Upon ATP hydrolysis, the rear of the lid detaches from the NBD and undergoes a closing movement toward the base in a wide rotation motion around the hinge (**Figure 2A**, step 3). There is experimental evidence that this mere clamping motion may already apply a destabilizing pressure on the misfolded structures of a bound substrate (Sharma et al., 2010). This motion may be futile, either when it is blocked by an overresistant bulky polypeptide (Schlecht et al., 2011), or when the substrate has escaped the jaws before complete closure has occurred (Sousa, 2012). Importantly, however, this motion may be productive when it reaches complete closure, or "clamping," allowing the entrapment of a small, fully extended segment of the substrate, typically composed of five-six non-bulky hydrophobic residues preferably flanked by positive charges (Rudiger et al., 2000). Indicative of the complete unfolding of the clamped-upon segment, the ATP-dependent closure of DnaK's PDB has been shown to cause the peptide bonds of the substrate to undergo cis-trans isomerization of their secondary amides, reminiscent of peptidyl prolyl cis/trans isomerase activity (Schiene-Fischer et al., 2002). In the clamped state, the substrate is tightly bound to the ADP-containing HSP70 with an affinity estimated to be about 1000 fold higher than in the ATP bound or apo states, which both display wide-opened PBDs (Goloubinoff and De Los Rios, 2007).

### Mechanisms of Active Holding

When clamping occurs under heat-shock conditions, substrate dissociation from HSP70s may come to a halt. This is the classic "holdase" activity of the chaperone (Slepenkov and Witt, 2002; Haslbeck and Buchner, 2015). The holding mode of HSP70s may be mediated by the NEFs, in particular bacterial GrpE, which can undergo a reversible thermal denaturation and thus reversibly loose its physiological ability to accelerate ADP- and substrate release from the HSP70s (Diamant and Goloubinoff, 1998; Grimshaw et al., 2001). This "holding" activity of the HSP70s under an ongoing heat-shock makes biological sense, as the untimely release of stress-labile polypeptides would obligatorily lead to their misfolding and aggregation, ultimately into species becoming increasingly resistant to the HSP70s unfolding activity (Sharma et al., 2011) (**Figure 2A**, step 2). The

differences between HSP70-mediated passive and active holding activities were shown in online light scattering measurements during the gradual denaturation and aggregation of a heat-labile luciferase in the presence of prokaryotic DnaK + DnaJ (Sharma et al., 2011), or human HSP70 + HSP40 and HSP110 + HSP40 (Mattoo et al., 2013), in the absence or presence of ATP. Although both chaperone and co-chaperone have specific hydrophobic surfaces that can bind misfolding polypeptides, without ATP, they were poorly effective at preventing the aggregation of the thermolabile luciferase, or in other words, much more of them was needed to prevent luciferase aggregation. In contrast, with ATP, both displayed a strong synergic holding activity at low concentrations, implying that when the conditions are unfavorable, DnaK, Hsp70 or HSP110 can use the energy of ATP hydrolysis to tightly clamp upon the heat-labile polypeptides to actively prevent aggregation and halt their release as long as heat-shock persists. Exactly the same ATP-dependent "holding" behavior was observed with HSP70 (HSPA1A) and HSP40 (Mattoo et al., 2013). The need under a prolonged heat-stress for very large amounts of non-catalytic holding HSP70s to prevent the aggregation of as many polypeptide substrates, could explain why the HSP70s are among the most abundant proteins in human cells (Finka and Goloubinoff, 2013). ATPfuelled HSP70 clamping may not only cause the local unfolding of the clamped-upon segment but also maintain unfolded also some distal segments that are protruding away from the chaperone jaws.

Remarkably, at physiological temperatures, the clamping by a single DnaK molecule embracing at most 8–10 residues, at any of the dozen or so putative DnaK binding sites (Rudiger et al., 1997) along the 572 residue-long luciferase polypeptide, sufficed to maintain the latter nearly completely unfolded. This was revealed by partial protease treatments: The clamped-upon luciferase species were significantly more sensitive to a limited amount of trypsin, compared to the much more compact resistant misfolded substrates and natively folded products of the chaperone reaction (Sharma et al., 2010). The particular ability of the firefly luciferase to be maintained nearly completely unfolded by a single HSP70 molecule might be attributed to the fact that it folds mostly as a large single domain. In a multi-domain polypeptide, except for the clamped-upon domain that would be maintained unfolded by the chaperone, the other independent domains could be either misfolding or natively folded (Fitter, 2009). Thus, at variance with monomeric misfolded luciferase, aggregationprone misfolded multi-domain polypeptides, are expected to necessitate the cooperative action of several HSP70 bound at different hydrophobic clusters along the same polypeptide, in order for it to become sufficiently unfolded, to reach after release the native state (Ben-Zvi and Goloubinoff, 2002).

### Mechanism of Active Catalytic Unfolding/Refolding

In contrast to heat shock conditions, where the chaperone can be acting as a holding protein platform to prevent aggregation, upon returning to physiological temperatures and GrpE renaturation, the chaperone can switch back into a more effective catalytic mode of action: It can turn into an unfolding enzyme capable to iteratively bind, unfold and release polypeptides, until many substrates become converted into many native products.

Strong evidence that mere ATP-fuelled clamping by a single DnaK molecule may suffice to cause extensive unfolding, leading thereafter to native refolding of the 63 kDa luciferase, was presented by Sharma et al. (2010). Even in the presence of an unprecedented 15 fold molar excess of misfolded substrates over the chaperone, DnaK could effectively regenerate several native luciferase molecules in several consecutive cycles of DnaJmediated binding, ATP-fuelled unfolding and GrpE-mediated release (Sharma et al., 2010). At such stoichiometric ratios, chances of concomitant binding of two DnaK molecules to the same polypeptide are excessively low, excluding an obligate cooperative action between them. Given that each stable misfolded luciferase polypeptide that was successfully converted into a native luciferase monomer must have been produced by the action of a single DnaK molecule, ATP-fuelled unfolding by clamping was likely the central mechanism at work in this particular case.

This reaction did set a low consumption record of merely five ATP hydrolyzed per refolded luciferase protein. This strategy, whereby an inactive misfolded polypeptide could be "rehabilitated" by an ATP-consuming unfoldase into a functional one, was estimated to be about a thousand times more economic than the alternative, consisting of degrading it first by an ATPconsuming unfolding protease and then synthetizing a new polypeptide at the cost of three ATPs per peptide bond.

This notwithstanding, the hydrolysis of five ATPs implied that the chaperone may have undergone five productive clamping/unfolding events, but upon HSP70 dissociation, four luciferases spontaneously misfolded again and only one succeeded to reach the native state (Sharma et al., 2010). Yet, there was an alternative scenario, in which the chaperone could have undergone five ATP-hydrolysis events accompanied by five attempted clamping events, but only one would have succeed to reach the native state, whereas the other four would have caused only partial and ineffective unfolding (Sharma et al., 2010). This second scenario was confirmed by an order of addition experiment in which the misfolded luciferase monomers were first interacted with an excess of DnaK, DnaJ and alphalabeled radioactive ATP for 2 min, to allow initial binding and clamping. Then, apyrase was added to readily convert all the ATP into AMP. Following the separation of the proteins from the nucleotides by a quick spin column chromatography, an inactive chaperone-luciferase complex was isolated that was found to contain 0.6µM of apyrase-resistant ADP. Remarkably, subsequent addition of GrpE to this inactive complex, readily yield up to 590 nM of native luciferase, although this happened in the total absence of ATP. This experiment produced two important clues about HSP70s mechanism. (1) Whereas the unfolding of a stable misfolded substrate by HSP70s necessitates ATP-hydrolysis, the refolding of the chaperone-released unfolded products does not necessitate ATP and is thus spontaneous. This confirmed Anfinsen's seminal observations (Anfinsen, 1973) that unfolded polypeptides should, in principle, be able the reach their native state unassisted by other proteins. (2) The excellent match between the amounts of entrapped ADP in the isolated inactive chaperone-luciferase complexes, and the subsequently refolded luciferase, implies that upon release, the native refolding was nearly 95% efficient, excluding scenario one, in which only 20% of the released unfolded species would have reached the native state while the other would have misfolded again. Rather, this experiment indicated that four out of five clamping events by the chaperone ended up being unproductive. The possibility that upon ATP-hydrolysis, the ADP-bound HSP70 might undergo only a partial but incomplete clamping and unfolding motion was shown by Mayer and collaborators, who used fluorescence spectroscopy to demonstrate that following ATP hydrolysis, some ADP-bound DnaK molecules need not obligatorily end up being with tightly closed SBDs that clamp around fully extended polypeptides (Schlecht et al., 2011).

Relevant to this chaperone mechanism, quantitative proteomics of human Jurkat cells (**Figure 1A**) showed copy numbers of 18500:1350:1180, representing an overall stoichiometry of about 16:1:1 for HSP70s, J-domain cochaperones and NEFs (Bags and GrpE), respectively. Assuming an equal distribution of Hsp70s through the cytosol of Jurkat cells, this suggests that neither J-domain nor NEF cochaperones can be persistent structural constituents of the core unfoldase machineries. Rather, they may both need to act as substoichiometric catalysts (Pierpaoli et al., 1998; Laufen et al., 1999; Hinault et al., 2010), which in iterative cycles of binding and dissociation, can accelerate respectively, the ATP-fuelled unfolding of the misfolded substrates by the chaperone and the release of the unfolded products from the chaperone (Sharma et al., 2010).

### Mechanism of Active Unfolding by Entropic Pulling

In contrast to chaperone-amenable substrates, such as monomeric misfolded luciferase, which could be productively unfolded by the solitary clamping action of individual DnaK molecules, larger soluble aggregates of the same misfolded luciferase (Mattoo et al., 2013), or of heat-denatured G6PDH (Diamant et al., 2000; Ben-Zvi et al., 2004), or of freezethawed denatured rhodanese monomers (Natalello et al., 2013), stringently required the presence of a large molar excess of chaperone (DnaK), in order to become productively unfolded and renatured. This suggested that the productive unfolding of severely damaged misfolded polypeptides may require the concomitant binding of several DnaKs at different hydrophobic binding sites along same misfolded polypeptide (Rudiger et al., 1997, 2000). Together with the observed unfolding by pulling effect of mitochondrial HSP70, which is distally applied on translocating polypeptides from within the mitochondria, a common mechanism of unfolding by entropic pulling was proposed that described the forces applied on a misfolded substrate by two or more clamped ADP-DnaK molecules on a misfolded segment situated in between two chaperone-bound segments in an aggregate. Due to the excluded volumes of the two clamped chaperones and their bumping into each other and into the aggregate during a very long time in molecular terms (0.1-1 minute), they were found to apply unfolding-pulling forces, which can pull away and unfold vicinal misfolded segments (De Los Rios et al., 2006; Goloubinoff and De Los Rios, 2007). Thus, in the case of more damaged protein aggregates, the active local clamping of the HSP70s may not suffice to productively unfold a multidomain polypeptide and an additional cooperative mechanism of unfolding by entropic pulling may be necessary (**Figure 2A**, step 4).

One major limitation of unfolding by entropic pulling is that in order to be effective, more than one HSP70 molecule has to be concomitantly bound to the same misfolded polypeptide. Although as many as 12,000 HSC70 molecules may be present in a micron cube of human cytoplasm (**Figure 1**) (Finka et al., 2015), some of the remaining 1,200,000 polypeptides may misfold. Once these would exceed 6000, none of them would be reverted to the native state by the 12,000 HSC70s, even at the futile cost of several times 12,000 hydrolyzed ATP.

One possible way for HSP70s to overcome dilution by their own substrate would be to assume that they don't necessarily have to be equally distributed in the cytosol. Moreover they may have a natural tendency to form, at rest, low affinity oligomers (Motohashi et al., 1994). When challenged by misfolded polypeptides, these would readily dissociate and, because of their slow diffusion rate, readily bind nearby each other, increasing the chances of productive multiple binding on the same misfolded polypeptide, despite the unfavorable stoichiometry.

A second way for HSP70s to overcome this limitation would be to function with dimeric J-cochaperone. Indeed all DNAJA/B and HSP40 are dimers associated by a small dimerization domain through their C-terminal ends (Mattoo et al., 2014). Thus, a dimeric DnaJ may optimally present its two N-terminal Jdomains for the binding of the NBDs of two ATP-bound HSP70s, while still keeping the two far apart enough so that, after binding to the substrate, they might optimally exclude each other and apply a cooperative unfolding force by entropic pulling. Given that cells have a large excess of ATP over ADP and that the cytosolic HSP70s are about 10 times more abundant than their J-domain cochaperones, it is tempting to speculate that without a stress, most DNAJA/B dimers in the cell will be associated to two ATP-bound HSP70s. Once challenged by a stress-induced misfolded polypeptide, these loose heterotetramers could dissociate and the two HSP70s would be thus able to lock onto nearby regions on the same misfolded polypeptide substrate. Simple order of addition kinetics of DnaK-DnaJ-GrpEmediated refolding of inactive FT-luciferase, illustrates this. When DnaK and DnaJ was first pre-incubated (for 5 min) with ATP and at T = 0', supplemented by FTluc and GrpE (**Figure 2B**, DnaKJ->no), luciferase refolding readily started without a delay and at a rapid rate of 140 nM per 5 min. When DnaJ alone was first preincubated with TFluc and ATP at T = 0', DnaK and GrpE were added (**Figure 2B**, DnaJ->DnaK), refolding was slightly delayed and the refolding rate was slightly lower (90 nM in the first 5 min). In contrast, when DnaK alone was first preincubated with ATP and FTluc, and at T = 0' incubated with DnaJ and GrpE (**Figure 2B**, DnaK->DnaJ), luciferase refolding was strongly delayed and reached less than a net 15 nM active Luciferase in the first 5 min of the reaction. These results indicate that, as initially shown by Bukau and colleagues (Gamer et al., 1992), the DnaJ dimer may indeed be the first to bind the substrate and thereafter effectively recruit two DnaKs onto the substrate. Noticeably, when DnaK was compelled to pre-interact first with the substrate, the strong delay in the refolding rate suggests that some time was needed for the wrongly associated DnaKs to first dissociate from the substrate and for DnaJ to properly bind and only then, optimally recruit DnaK on the substrate. Moreover, the fastest rate that we observed with preincubated DnaK + DnaJ + ATP prior they were presented to the substrate and GrpE, suggest that in the presence of ATP, DnaK and DnaJ may form a loose hetero-oligomer, likely a DnaJ2DnaK2, optimally poised to bind cooperatively to the same misfolded substrate and engage in the HSP70-unfoldase cycle (**Figure 2A**, step 1).

A third way to overcome the limitation for unfolding by entropic pulling caused upon dilution of the HSP70s by their own substrate, would be the spontaneous formation of loose hsp70dimers. Thus, HSP110:HSP70 heterodimers associated by their N-terminal domains can apparently form and act reciprocally as NEFs on each other. Somehow, together they can efficiently disaggregate large stable aggregates (Schuermann et al., 2008; Mattoo et al., 2013). Similarly, bacterial DnaKs might also form loose homodimers, which would favor their ability to act upon resistant substrates by entropic pulling (Sarbeng et al., 2015).

Finally, one elegant way in which evolution may have chosen to overcome HSP70 limitation by substrate dilution, was recruitment of powerful disaggregating nanomachines, such as ClpB in E. coli or HSP104 in yeast. These are AAA+ type hexameric cylinders capable of applying ATP-fuelled powerstokes on inserted misfolded polypeptide loops from the aggregates in the hexamer cavity and unfold them by stretching strokes (Goloubinoff et al., 1999; Winkler et al., 2012). Once activated by HSP70, ClpB or HSP104 can convert large protein aggregates into lower oligomeric species amenable to HSP70 mediated unfolding by clamping.

The multilayer mechanisms of HSP70 as holding and unfolding chaperones can be summarized in four steps (**Figure 2A**). Without ATP, the wide opened protein-binding domain may weakly bind to hydrophobic patches on the surface of misfolding polypeptides, thereby passively and rather inefficiently preventing aggregation (**Figure 2A**, step 1). But passive binding is probably irrelevant to the physiological situation in cells. ATP-hydrolysis, by driving the closure of the PBD in the substrate and causing local unfolding, can dramatically increase the affinity of the misfolded polypeptides for the chaperone, thereby actively and efficiently prevent aggregation (**Figure 2A**, step 2). Owing to the transient thermal inactivation of the NEF (GrpE), dissociation during heat-shock is prevented. Upon returning to the physiological conditions, GrpE may become functional again and trigger the release of the unfolded substrate, leading to efficient spontaneous refolding. Catalytic cycles of misfolded substrate binding, ATP-fuelled unfolding by clamping, release and spontaneous refolding may thus convert non-aggregated misfolded species (**Figure 2A**, step 3), such as FT-luciferase into native proteins (Sharma et al., 2010). In case the misfolded species are tightly associated into large insoluble aggregates, the binding and local unfolding by clamping of a single Hsp70 molecule may not suffice to unfold distal misfolded segments in the same polypeptide. The binding and locking of additional HSP70 molecules elsewhere on the same polypeptide would add another unfolding effect by way of the Brownian movements of the locked chaperones and their volume exclusion from each other, applying unfolding forces on the misfolded segments located in between the HSP70-bound sites (**Figure 2A**, step 4).

This additional mode of ATP-dependent unfolding by distal entropic pulling of the HSP70, can predominate in the case of polypeptide precursors that are partially folded in the cytosol and need to be imported across the narrow pores of the mitochondria (Elsner et al., 2009), the ER (Matlack et al., 1997) and chloroplasts (Shi and Theg, 2011). Here, the organellar ATP-bound HSP70s are uniquely targeted to the import pores by specific poreassociated J-domain cochaperones to clamp upon the entering polypeptides segments, which are unstructured. In these cases, the unfolding action of the organellar HSP70s is not local but distal, by pulling on the bulky structures that are still on the cytoplasmic side of the precursor protein and are forced to collide onto the narrow pore entry and thereby to unfold prior translocation. Moreover, ATP-dependent unfolding by entropic pulling is likely to be the mechanism by which HSC70 and HSP110, targeted by their specific J-domain cochaperone auxilin, actively deoligomerize clathrin cages into triskelions (Zhuo et al., 2010).

## Conclusion

HSP70s are a central hub of the chaperone network carrying very diverse physiological functions in proteostasis, such as polypeptide folding/unfolding, protein assembly/disassembly, protein activation/inactivation, polypeptide translocation, and degradation. Moreover, HSP70s act as polypeptideunfolding nanomachines that serve as defenses against stress- and mutation-induced formation and accumulation of cytotoxic misfolded protein conformers. Assisted by J-domain cochaperones, the HSP70s can specifically detect among a large excess of low-affinity native proteins a minority of high-affinity misfolding intermediates on their way to form toxic aggregates. HSP70s may further collaborate with each other (DnaK-DnaK, HSP70-HSP110) and with AAA+ unfoldases (HSP100/ClpB), at disentangling already formed aggregates and convert them into natively refoldable, or protease-degradable polypeptides. The pharmacological upregulation, or adenoviral-mediated over-expression of specific HSP70s, HSP110s and their specific J-domain and NEF cochaperones, are attractive avenues for therapies against degenerative protein conformational diseases and aging (Ebrahimi-Fakhari et al., 2013). Moreover, the development of specific inhibitors of HSP70s and J-domain co-chaperones, is a promising approach to develop therapies against aggressive cancers (Patury et al., 2009; Chang et al., 2011).

## Acknowledgments

Research was financed by Swiss National Science Foundation Grants 125502/1 and 140512/1.

### References

transgenic Physcomitrella patens as a sensitive biosensor for organic pollutants. Plant Cell Environ. 30, 753–763. doi: 10.1111/j.1365-3040.2007.01664.x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Finka, Sharma and Goloubinoff. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# DNAJs: more than substrate delivery to HSPA

### Suzanne L. Dekker, Harm H. Kampinga and Steven Bergink \*

*Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, Netherlands*

Proteins are essential components of cellular life, as building blocks, but also to guide and execute all cellular processes. Proteins require a three-dimensional folding, which is constantly being challenged by their environment. Challenges including elevated temperatures or redox changes can alter this fold and result in misfolding of proteins or even aggregation. Cells are equipped with several pathways that can deal with protein stress. Together, these pathways are referred to as the protein quality control network. The network comprises degradation and (re)folding pathways that are intertwined due to the sharing of components and by the overlap in affinity for substrates. Here, we will give examples of this sharing and intertwinement of protein degradation and protein folding and discuss how the fate of a substrate is determined. We will focus on the ubiquitylation of substrates and the role of Hsp70 co-chaperones of the DNAJ class in this process.

#### Edited by:

*Pierre Goloubinoff, University of Lausanne, Switzerland*

#### Reviewed by:

*Nico P. Dantuma, Karolinska Institutet, Sweden Krzysztof Liberek, University of Gdansk, Poland Pierre Genevaux, Centre National de la Recherche Scientifique, France*

#### \*Correspondence:

*Steven Bergink, Department of Cell Biology, FB30, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan 1, Groningen 9700AD, Netherlands<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Protein Folding, Misfolding and Degradation, a section of the journal Frontiers in Molecular Biosciences*

> Received: *30 April 2015* Accepted: *12 June 2015* Published: *30 June 2015*

#### Citation:

*Dekker SL, Kampinga HH and Bergink S (2015) DNAJs: more than substrate delivery to HSPA. Front. Mol. Biosci. 2:35. doi: 10.3389/fmolb.2015.00035* Keywords: DNAJ, ubiquitin E3 ligases, HSP70 heat-shock proteins, degradation, protein folding

### Protein Stress

The capacity of the protein quality control network is limited and collapse occurs when the system is overwhelmed. This can occur after sudden massive stress including temperature shifts, nutrient deprivation or pathogenic infection (Lee, 1992; Scheuner et al., 2001; Kaufman et al., 2002; Schelhaas et al., 2007; Morimoto, 2008). These types of stresses are usually sensed and the organism responds with the activation of stress pathways, such as the heat shock response after temperature elevation (Morimoto, 2008).

Also "normal" conditions in the cell, for example the increased synthesis of proteins during cell cycle progression (Morimoto, 2008) can be considered as physiological protein stress requiring increased folding capacity and adaptations in the quality control network. The difference between these two types of protein stress is that the latter is enlisted and that substrate recognition and fate are pre-determined, for example cyclins are degraded upon orchestrated phosphorylation events (Murray, 2004). In contrast, sudden or accidental protein stress is unpredictable and requires a system that can recognize the client(s), which lack(s) a common motif that is usually present in the regular enlisted clients. Typically, these accidental clients become unfolded after stress or are intrinsically misfolded (i.e., genetically encoded).

### Protein Stress and Its Impact on Human Pathology

Protein stress can accumulate over time as well, as is the case in many diseases that are associated with an accumulation of misfolded proteins. Often this type of slowly accumulating protein damage is—initially—not triggering a strong stress signaling, but remains undetected. Yet, this type of stress may also overwhelm the protein quality control network and cause a similar collapse, ultimately resulting in the associated pathology. Diseases driven by protein stress range from cataract, type II diabetes, atrial amyloidosis to neurodegenerative diseases (Chiti and Dobson, 2006).

Moreover, it is known that hypomorphic mutations, a partial loss of gene function, can lead to an accelerated phenotype in the background of a compromised protein quality control network (Ben-Zvi et al., 2009). Suggesting that genome alterations can lead to metastable or aberrant polypeptides that stochastically accumulate in time. If true, the source of protein stress would expand to single nucleotide polymorphisms (SNPs) and somatic mutations that slowly accumulate as we age.

### Molecular Chaperones

Heat Shock Proteins (HSPs) play a central role in protein homeostasis and are upregulated by the diverse protein stress signaling pathways in cells under many conditions, most prominently acute stress, that challenge protein homeostasis (Morimoto, 2008). For various HSPs, it has been established that they have so-called molecular chaperone activity. Usually, this is associated with folding nascent chains into their native state or refolding of stress-unfolded proteins. However, their activities are also needed for degradation of misfolded or mutant proteins and even for remodeling of active protein complexes. As a matter of fact, the most ancient members of the molecular chaperones, DnaJ, and DnaK (Hsp70/HSPA), were originally identified as essential components controlling (dis)assembly of phage lambda replication complexes (Konieczny and Zylicz, 1999).

A wide network of molecular chaperones exists that encompasses the chaperonins, the HSPAs (Hsp70s and Hsc70s, eleven in humans) with its co-chaperones: the DNAJs (50 members in humans) and the Nucleotide Exchange factors (NEF, 13 members in humans), the HSPBs (small HSPs, 10 members in humans) and the HSPCs (Hsp90s, five members in humans) (Kampinga and Craig, 2010). Here we will focus on the DNAJs in the HSPA system (**Figure 1A**), as these are involved in substrate recognition and seem to function at the crossroad of folding and degradation.

### The DNAJs: A Crucial Role in Protein Quality Control

The HSPAs function with the help of several co-chaperones and collaborate with the other chaperones. The most important co-chaperones are the DNAJs and the NEFs. DNAJs recognize substrates/clients and subsequently bind to the ATP-bound form of HSPA (for the HSPA cycle see **Figure 1A**).

DNAJs are defined by the presence of a J-domain, an approximately 70 amino acid domain that consist of four alpha helices and an accessible loop to which the (ATP-bound) ATPase domain of HSPA binds (Jiang et al., 2007). In this loop lies a Histidine Proline Aspartate (HPD) motif, which is crucial for binding to HSPAs. DNAJs are divided based on their domain structure into three classes, DNAJAs, DNAJBs and DNAJCs (Kampinga and Craig, 2010). Briefly, in both DNAJAs and DNAJBs, the N-terminal J domain is followed by a Glycine/Phenylalanine-rich stretch. In the DNAJA class proteins, two domains (CTDI and CTDII) are located at the Cterminus that are involved in substrate recognition by forming a hydrophobic pocket that binds to the substrate (Li et al., 2003). The first of these CTDs contains a zinc finger. The C terminus in the DNAJB lacks the organization into two CTDs or and more specifically contains no zinc finger and specific information of substrate binding regions is still lacking. The DNAJC class comprises any J-domain containing protein that does not fit in the A or B class.

A wide variety of domains can be found in the different J proteins. Many of these are known or thought to be involved in substrate interactions, thus a division based on substrate recognition has been proposed as well (Kampinga and Craig, 2010). Many J-proteins are believed to have a promiscuous client binding (mostly from class A and B and a few from class C) while others seem to display high substrate specificity (mostly class C).

### Interplay between Degradation and Folding

The notion that there are around 50 DNAJs, 11 HSPAs, and 13 NEFs at least suggest a high potential of many possible combinations, theoretically resulting in flexibility and a broad substrate range. Some DNAJs and HSPAs are localized to specific organelles and seem to form combinations with the compartment specific Hsp70 (HSPA) members, but for other (cytosolic) HSPAs it has been shown that they can indeed operate with different DNAJs (Kampinga and Craig, 2010). The interplay between the HSPA cycle and the degradation machineries makes matters even more complex. The idea that the fate of a protein is determined by this interplay of the chaperone systems and the degradation systems is often referred to as the triage decision (Connell et al., 2001; Houck et al., 2012). The classical view is that the cell first attempts to (re)fold the damaged or metastable protein. If folding fails, the cell will then attempt to degrade it (**Figure 1B**). Many proteins that are part of the folding machinery interact with the cellular degradation machinery to make this transition smoothly. For example, DNAJB2 interacts with ubiquitylated proteins via its ubiquitin interacting motifs (UIM) (Westhoff et al., 2005) and Bag1 (a NEF) contains a ubiquitin-like (UBL) domain which are known to interact with subunits of the proteasome (Lüders et al., 2000). An alternative view would be that degradation and (re)folding act in parallel and that chaperones are an integral part of the degradation machinery and have a different function besides folding. These scenarios are not mutually exclusive and, depending on the substrate, both might happen.

How the HSPA machinery selects and recognizes the different types of substrates and which parameters determine the fate of the clients (folding, degradation or aggregation) are still open questions. Here we will give examples of clients that are dealt with by different combinations of DNAJs and HSPAs. Moreover, we will discuss the recent insights in the interplay between the HSPA cycle and the ubiquitin proteasome system (UPS) (**Box 1**).

## De Novo Protein Folding

The folding of newly synthesized proteins is different from refolding reactions, partly due to the vectorial nature of translation. Indeed, early studies clearly demonstrated that nascent chain folding and refolding require different sets of chaperones (Frydman et al., 1994, 1999). Folding of newly synthesized proteins can occur co-translationally and sometimes also post-translationally. In prokaryotes, folding involves many different partners including trigger factor which prevents misfolding of nascent chains by delaying protein folding (Agashe et al., 2004). Although far from completely resolved, in cotranslational folding, the different domains fold separately. As often, the fundamental studies in yeast have shed light on this process (Albanèse et al., 2006). Two complexes are associated with the ribosome and the emerging polypeptide. First, the nascent polypeptide associated complex (NAC), a heterodimer that consists of an alpha and a beta subunit, binds to all nascent chains. Second, a set of Hsp70s (Ssb1 and 2) is bound to translating ribosomes, which function together with the DNAJ Zuo1 and the NEF Sse1 and bind to the polypeptide that extrudes from the ribosomal exit tunnel. Zuo1 together with Ssz1 (another Hsp70) forms the Ribosome Associated Complex (RAC), and is thought to recruit Ssb1 and 2 to the ribosome. Global identification of Ssb1/2 substrates revealed that especially the more difficult substrates (e.g., longer, several domains, beta sheet enriched, and aggregation prone) are clients (Willmund et al., 2013). Beside Zuo1, two other DNAJ proteins have been identified as being important for nascent chain folding namely Ydj1 and Sis1. These DNAJs function with the "soluble" or cytoplasmic Hsp70s (the Ssa1-4 proteins) and the chaperonin system to guide the final (posttranslational) folding of newly synthesized polypeptides (Kim et al., 2013).

Most of these yeast proteins have mammalian counterparts: Ssz1 is homologous to HSPA14, Zuo1 is homologous to DNAJC2 [also called, Mpp11 (M-phase phosphoprotein 11) (Hundley et al., 2005) or ZRF1 (zuotin related factor 1) (Richly et al., 2010)], Ydj1 is homologous to DNAJA1 and Sis1 is homologous to DNAJB1. HSPA8 is associated with the ribosome and homologous to Ssb2 (Hundley et al., 2005).

#### BOX 1 | Protein Degradation.

The various proteases in the cell are capable of degrading targeted substrates. As a general theme, access to proteases is restricted either due to a high specificity for specific substrates, a common but inducible recognition signal (e.g., the proteasome), or spacious confinement (e.g., the lysosome). The UPS and autophagy are the systems that degrade the bulk of proteins.

#### UPS

Recognition by the 26S proteasome is done by lysine (K) 48-linked polyubiquitin chains conjugated to substrates that require degradation. Ubiquitin is a polypeptide that is conjugated to target proteins by the subsequent action of three enzymes, an E1 (activating enzyme), an E2 (conjugating enzyme) and an E3 (ligating enzyme). The ligases are responsible for substrate recognition and thus determine the specificity, whereas the E2s usually mediate the conjugation of ubiquitin to the substrate. Occasionally an E4 ligase is required to extend already existing mono- or oligo-ubiquitylation events to polyubiquitin trees. The C-terminal glycine of ubiquitin either forms a peptide bond with the N-terminus of a substrate or an isopeptide bond with the epsilon-N in internal lysines of the substrate (although conjugation to cysteine and serine have been reported as well). Due to the internal lysines in ubiquitin itself a so-called polyubiquitin tree is build. Polyubiquitylation with a K48 linkage is considered canonical for degradation. Indeed, monoubiquitylation or other types of polyubiquitin trees with different linkage are not necessarily recognized as targeted for destruction. Receptors at the 26S proteasome recognize the polyubiquitin trees, which are cleaved off and recycled. The 26S proteasome is a multimeric complex that consists of a 20S and 19S component. After deubiquitylation, the substrate is unfolded by ATPases associated with various cellular activities (AAA-ATPases) of the 19S, and funneled into the proteolytic chamber (20S). Only when a protein resides in this chamber it is cleaved into peptides.

#### Autophagy

Autophagy is a process in which a double membrane-vesicle (the autophagosome) containing the cargo is targeted to, and fused with the lysosome in which the degradation (hydrolysis) takes place. The cargo consists of proteins, protein-complexes, aggregated proteins and even entire organelles. Autophagy is mediated by a set of proteins and includes two types of ubiquitin-like conjugation reactions; the most crucial one is the conjugation of LC3 or ATG8 (in yeast) to phosphoatidylethanolamine at the expanding autophagic membrane. Originally it was thought that autophagy is mostly nonspecific and just randomly engulfs cytoplasm. However, the current dogma is that most autophagy is in fact specific. Again, ubiquitylation can form the signal that is recognized by specific receptors that both bind polyubiquitylated substrates and LC3, the double membrane then encloses the cargo and translocates to the lysosome.

### Quality Control at the Ribosome

Translational errors may occur due to aberrant mRNAs, misfolding due to intrinsic properties of the nascent chain itself or to other events that lead to stalling of the translational machinery. As a general theme, these errors are sensed during translation and the emerging polypeptide is co-translationally ubiquitylated, released and degraded. Depending on the type of translational problem and substrate, different chaperones and components of the UPS are involved. For example, long stretches of basic amino acids are recognized by the NAC complex and ubiquitylated by the E3 ligase Not4 (Dimitrova et al., 2009). Other types of translational difficulties, such as the translation of the polyA sequence that leads to pausing, require another E3 ligase, namely Ltn1. The ubiquitylated polypeptide requires Cdc48Ufd1/Npl4 (p97/VCP in mammalians), a ubiquitin/ubiquitin-like-modifier-specific segregase (Jentsch and Rumpf, 2007), to remove it from the ribosome (Brandman et al., 2012; Defenouillère et al., 2013; Verma et al., 2013). Thus, translational errors usually result in immediate, cotranslational, degradation (**Figure 2A**) in which chaperones act before ubiquitylation.

Degradation of the emerging polypeptide can in principle always occur at the ribosome, even in the absence of translational errors, as shown by elegant experiments using tandem expressed degradation-reporters (Turner and Varshavsky, 2000). This implies that folding or degradation is an intrinsic fate of the client rather than it is driven by different sets of chaperone machines. In other words, the different ribosome-associated chaperone complexes (in yeast Ssb1/2, RAC, and NAC) prevent aggregation during translation which -depending on the substrate- can result in folding or maintain the substrates/clients accessible to the various E3 ligases that are either present at, or have access to the ribosomes (examples in yeast: Ltn1, Not4, Ubr1).

### Chaperones: the Prelude for Ubiquitylation of Proteasomal Clients?

The idea that unfolded or misfolded clients, similar to nascent chains, need to be kept accessible/soluble in order for a protein quality control E3 ligase to work seems appealing. The idea would be that a chaperone binds first to a potential substrate and keeps this substrate soluble or accessible for the E3 ligase to ubiquitylate this target. The requirement for a chaperone as a recognition or accessibility factor for ubiquitin E3 ligases would not be necessary for regular or enlisted clients (e.g., the cyclins), but only for those that become (partly) unfolded by a random stress event or for those clients that are intrinsically (genetically encoded) misfolded. Chaperones of the HSPB and DNAJ class are well known to be able to recognize these clients and thereby ensure their solubility. In the canonical view, these clients are next transferred to the HSPAs (Hsp70s). Release from the HSPAs occurs via NEFs and interactions with E3 ligases at which level client-fate is determined. However, as we will explain below, evidence is accumulating that other scenarios may also be operational (**Figures 1B**, **2**).

### Ubiquitylation and DNAJs in ERAD

Clear examples where a DNAJ precedes the action of an E3 can be found within ER associated degradation (ERAD). Proteins that are targeted to the ER, but are unwanted because they are intrinsically unstable, aggregation prone or unfolded, are cleared via the ERAD pathway. For ER luminal and transmembrane clients, DNAJs (DNAJB11, DNAJB9, and DNAJC10 also known as Erdj3, -4, and -5), recognize misfolded clients that subsequently are ubiquitylated by the membrane embedded E3 ligases Hrd1 or RMA1 and the E4 ligase gp78 (Buchberger et al., 2010). For those clients that expose their unstable region to the cytosol, soluble-DNAJs play a role in

conjunction with the E3 ligase Doa10 (Ravid et al., 2006). After or during ubiquitylation, all ERAD clients require retrotranslocation over, or dislocation from the lipid bilayer, a process that is mediated by the p97/Cdc48Ufd1/Npl4 complex (Tsai et al., 2002).

Also for integrated membrane proteins, DNAJs may act prior to E3 ligases in degradation. Several model substrates exist exposing their unstable region to the cytosol and for some of these the relationship between chaperone function and ubiquitylation status has been determined. For example degradation and ubiquitylation of the Doa10 substrates Ste6<sup>∗</sup> and PMA1<sup>∗</sup> depends on the cytosolic Hsp70s (Ssa1-4) and the (cytosolic) DNAJs Ydj1 and Hlj1 (Han et al., 2007; Nakatsukasa et al., 2008) (**Figure 2B**). Other DNAJs are also involved in Doa10-mediated substrate ubiquitylation as Sis1, but not the cytosolic Hsp70s (Ssa1-4), is required to ubiquitylate the degradation prone part from the kinetochore protein Ndc10. Instead, Hsp70s are required to prevent aggregation of the ubiquitylated client (Shiber et al., 2013) and clearly function after ubiquitylation. These results are in line with the idea that E3 ligases involved in clearing misfolded substrates require a molecular chaperone for their functioning (**Figure 2**).

However, for other substrates (e.g., soluble targets of the protein quality control E3 ligases Ubr1 and San1) Sis1 (or Ydj1) is not a prerequisite for ubiquitylation but is essential for efficient client degradation (Park et al., 2013; Mehnert et al., 2015; Miller et al., 2015) and thus seem to play a role downstream of ubiquitylation.

### Ubiquitylation and DNAJs of Cytoplasmic and Nucleoplasmic Clients

Whereas, translation quality control and ERAD are vectorial by nature, the organization of quality control over soluble proteins is not. If the soluble (stress induced or accidentally misfolded) clients in the cytosol and nucleoplasm also require a chaperone prior ubiquitylation is currently unclear. The notion that overexpression of certain DNAJs accelerates degradation of substrates is suggestive of a direct impact of DNAJs on degradation/ubiquitylation (Westhoff et al., 2005). However, clear evidence that DNAJs or HSPBs are required for ubiquitylation of these accidental clients is currently lacking (**Figure 2C**). Deletion of individual DNAJs or other molecular chaperones typically does not result in accumulation of their unmodified clients. It is not unlikely that there is functional redundancy between the different and numerous chaperones (e.g., DNAJs) in these compartments making the fate of substrates/clients harder to study. Moreover, to distinguish this process from translational protein quality control (Wang et al., 2013), a substrate that becomes unstable after translation vs. a constitutively unstable substrate, is required. For example a substrate that after the addition of a specific small molecule triggers unfolding of that substrate would be a great asset. This type of substrate would enable the study of posttranslational protein quality control under normal, non-stressed, conditions. This type of research is especially important as,

in many of the aforementioned diseases, protein stress occurs post-translationally.

### Nuclear Transport of Cytosolic Substrates

As mentioned above, ERAD substrates are degraded in the cytoplasm and retro-translocation is necessary as the proteasome is not present in the ER. One might suspect that degradation of cytoplasmic clients is more straightforward as the proteasome is present in the same compartment. However, recent evidence suggests that, at least for some substrates, this is not the case. Instead, cytosolic model substrates were found to shuttle to the nucleus for degradation (**Figure 2C**) (Heck et al., 2010; Prasad et al., 2010; Park et al., 2013). This transfer across the nuclear envelope depends on the DNAJ Sis1 (DNAJB1 in humans) (Park et al., 2013). Increasing the proteotoxic stress hampers this process, probably due to a sequestration of Sis1 to other clients, and results in a reduction in degradation of these substrates (Park et al., 2013). Transport of un- or misfolded substrates to a (juxta)nucleolar localization seems to be a more general feature for both nuclear (Nollen et al., 2001) and cytosolic proteins (Miller et al., 2015). Targeting of substrates to these structures depends on, again, Sis1 and the v-snare protein Btn2 (Malinovska et al., 2012; Miller et al., 2015). Whether the nucleolar accumulation per se determines the fate of the client is, however, unlikely as the accumulation in the nucleolus also occurs to nuclear clients that require refolding after heat stress (Nollen et al., 2001). Here, the accumulation of substrates in the nucleolus is reversible and accompanied by a strong sequestering of, normally cytosolic, HSPA1A (Hsp70) to the nucleolus. In line, ubiquitylation does not seem to play a role in the transfer across the nuclear envelope (Park et al., 2013; Miller et al., 2015).

What remains a question is how cytoplasmic proteins cross the nuclear barrier and whether or not posttranslational modifications play a role in this cytoplasmic-nuclear shuttling. Post-translational modifications as SUMOylation (Small Ubiquitin-like Modifier) and poly(ADP-ribosyl)ation have been implemented in protein stress responses (Haince et al., 2006; Golebiowski et al., 2009), the formation of stress bodies (Nagai et al., 2011) and also in nucleolar integrity (Finkbeiner et al., 2011; Boamah et al., 2012).

Why the nucleolus would serve as storage for proteins before folding or degradation has still remained enigmatic. Even more so, one may wonder why cytosolic substrates are translocated to the nucleus/nucleoli for degradation, given that in the cytosol proteasomes are fully active and autophagy is present. Notably, in all of these experiments the substrates used are engineered proteasomal clients. The transfer of these clients across the nuclear pore could therefore be an extension of the aforementioned ribosomal quality control mechanism, which indeed involves Sis1, and that shuttling for these substrates may occur co-translationally. However, adding an nuclear localization signal increases the degradation speed significantly (whereas adding a NES inhibits it) (Park et al., 2013), suggesting that the nuclear shuttling of cytoplasmic substrates is not that efficient and probably not coupled to translation.

One could hypothesize that substrates are normally already shuttling between the cytosol and nucleus and that degradation is simply faster in the nucleus in which the proteasomal concentration is higher (Russell et al., 1999). However, this would suggest that the capacity of the UPS in the cytoplasm is rate limiting, which seems not to be the case (Park et al., 2013). So, shuttling of un- or mis-folded clients to the nuclear/nucleolar compartment indeed seems an active, regulated process. Perhaps the cytosolic compartment, or the organelles herein, are more vulnerable to proteotoxic stress and the re-localization to the nucleus/nucleolus serves to avoid immediate cellular arrest or death. However, given the complex processes that take place in the nucleus, and the presence of the genetically vulnerable rDNA in the nucleolus, this might hamper nuclear processes such as transcription and replication.

### CFTR; Escaping Initial Quality Control Is Not Enough

One substrate that illustrates the complexity of quality control is the disease-associated form of cystic fibrosis transmembrane conductance regulator (CFTR) (Younger et al., 2006; Okiyoneda et al., 2010). For some of the CFTR mutations, in particular the deletion of phenylalanine at position 508 (CFTRdelta508) (Lukacs and Verkman, 2012), premature degradation is thought to underlay CF-pathology. This deletion destabilizes or kinetically traps CFTR, which targets this protein to the ERAD pathway. Numerous chaperones, including DNAJs and HSPAs, and E3 ligases have been implicated in the clearance of mutant CFTR. CFTRdelta508 is ubiquitylated either by the Hrd1 E3 ligase complex or the Rma1/Derlin1 E3 ligase complex and the GP78 E4 ligase in a process that is enabled by the membrane embedded DNAJB12, after which retro-translocation by p97 and degradation occurs (Younger et al., 2006; Grove et al., 2011).

Skipping this initial ERAD quality control enables part of the protein to arrive at the plasma membrane to fulfill its function (Kälin et al., 1999). Unfortunately, this rescued mutant protein is still degraded, now by a different set of chaperones and E3 ligases, involving DNAJA1, DNAJB2, HSPA8, HSPA1A, Bag1, and the E3 ligase CHIP (C-terminal Hsc70/HSPA8 interacting protein) (Meacham et al., 2001; Okiyoneda et al., 2010). CHIP has a well-known function in cytoplasmic protein quality control, it binds directly to HSPA and is involved in ubiquitylating HSPA clients that need to be degraded. The work on CFTRdelta508 nicely illustrates that each cellular compartment has a different set of protein quality control components. Moreover, it points out that in the lifetime of a protein it encounters various and different quality checks.

### Summary

The examples of substrates in the ERAD pathway and protein quality control during translation illustrated that for certain substrates a molecular chaperone (a DNAJ) is required for ubiquitylation while this is unclear for others.

The "holdase" function of DNAJs is well suited to assist E3 ligase function for those substrates that are intrinsically unstable and thus would misfold, form aggregates or fibrils in the absence of a molecular chaperone. Intriguingly, for some of these substrates HSPAs are necessary for degradation but not for ubiquitylation, indicating that they act after the E3 ligase but before proteolysis. Perhaps to prevent aggregation in the brief moment a substrate is delivered to the proteasome. Instead, the energy provided by the ATP hydroslysis of Hsp70s could also facilitate the release of the DNAJ and the E3 ligase from the ubiquitylated substrate (**Figure 1B** right panel). This un-entanglement-function of HSPA would not be required for substrates that come from the ER lumen, as both E3 ligase and DNAJ are embedded in the membrane, and retro-translocation is sufficient to clear the substrate from these factors. The view that degradation substrates are immediately marked for destruction without attempts to fold the substrate is not ruling out that futile folding can lead

### References

to degradation as well (**Figure 1A** left panel), but indicates that the degradation pathways are even more intertwined with the folding machinery as was previously thought. In that regard, it is not unlikely that the HSPA cycle, besides its canonical role in protein folding, could function to untangle the substrate-E3/E2 ligase complexes for transfer of regular enlisted clients to the proteasome as well (Mehnert et al., 2015).

### Acknowledgments

SD is supported by the Graduate School of Medical Science of the UMCG. HK is supported by a grants from Senter Novem (IOP-IGE7004), high Q foundation (project # 3.1.2), Hersenstichting (project 15F07(2)-58). SB is supported by a Hersenstichting grant (project no. F2013(1)-52).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Dekker, Kampinga and Bergink. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Metazoan Hsp70-based protein disaggregases: emergence and mechanisms

Nadinath B. Nillegoda\* and Bernd Bukau\*

Center for Molecular Biology (ZMBH) of the University of Heidelberg and German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany

Proteotoxic stresses and aging cause breakdown of cellular protein homeostasis, allowing misfolded proteins to form aggregates, which dedicated molecular machines have evolved to solubilize. In bacteria, fungi, protozoa and plants protein disaggregation involves an Hsp70•J-protein chaperone system, which loads and activates a powerful AAA+ ATPase (Hsp100) disaggregase onto protein aggregate substrates. Metazoans lack cytosolic and nuclear Hsp100 disaggregases but still eliminate protein aggregates. This longstanding puzzle of protein quality control is now resolved. Robust protein disaggregation activity recently shown for the metazoan Hsp70-based disaggregases relies instead on a crucial cooperation between two J-protein classes and interaction with the Hsp110 co-chaperone. An expanding multiplicity of Hsp70 and J-protein family members in metazoan cells facilitates different configurations of this Hsp70-based disaggregase allowing unprecedented versatility and specificity in protein disaggregation. Here we review the architecture, operation, and adaptability of the emerging metazoan disaggregation system and discuss how this evolved.

Keywords: Hsp70, J-protein, Hsp110, protein disaggregation, metazoan

### Introduction

In healthy cells, toxicities associated with protein misfolding are countered by regulated cellular processes that sequester damaged, sticky and potentially harmful proteins to intracellular protein deposit sites (Taylor et al., 2003; Arrasate et al., 2004; Miller et al., 2015) where protein quality control machineries operate to resolve aggregates (disaggregation) (Parsell et al., 1994; Mogk et al., 1999; Tyedmers et al., 2010; Doyle et al., 2013). Accumulation of protein aggregates however is a distinguishing feature of cellular stress and aging in all organisms (Morimoto, 2008; Hipp et al., 2014) and is associated with toxicities leading to pathology (Olzscha et al., 2011; Polymenidou and Cleveland, 2012; Park et al., 2013). Aggregates hallmark a plethora of human disorders ranging from neurodegeneration to diabetes and cancers (Knowles et al., 2014; de Oliveira et al., 2015; Mukherjee et al., 2015). Persistence of protein aggregates eventually also poses a threat to the integrity of the cytoskeleton and cellular signaling (Perutz et al., 1994; Kopito, 2000; Lee et al., 2004).