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The Hsp70 Machines

Members of the Hsp70 family are strongly upregulated by heat stress and toxic chemicals, particularly heavy metals such as arsenic, cadmium, copper, mercury, etc. Hsp70 was originally discovered by FM Ritossa in the 1960s when the incubation temperature of Drosophila(fruit flies) was accidentally boosted. When examining the chromosomes, Ritossa found a "puffing pattern" that indicated the elevated gene transcription of an unknown protein. This was later described as the "Heat Shock Response" and the proteins were termed the "Heat Shock Proteins" (Hsps).

Most of the in vivo cellular processes are not spontaneous and require the assistance of specialized proteins called as Molecular Chaperones. Hsp70 family forms the class of 70 kD heat shock proteins that assist in a vast repertoire of folding processes, including the folding and assembly of newly synthesized proteins, refolding of kinetically trapped misfolded and aggregated proteins, membrane translocation of organellar and secretory proteins, and control of the activity of regulatory proteins (1). In detail, the Hsp70 chaperone system is involved in: (i) the import of proteins into cellular compartments such as mitochondria, the endoplasmic reticulum and the Escherichia coli periplasm. (ii) Folding of proteins in mitochondria and of a subset of proteins in the endoplasmic reticulum and the Cytosol. (iii) Disassembly of protein complexes such as the nucleoprotein complex which initiates replication of bacteriophage λ DNA and the clathrin baskets of clathrin coated vesicles. (iv) Degradation of unstable proteins and the bacterial heat shock transcription factor. (v) Control of the activity of regulatory proteins such as heat shock transcription factors and the initiator protein of plasmid replication (2,3).

The members of Hsp70 family include, DnaK, HscA (Hsc66) and HscC (Hsc62) in prokaryotes; the eukaryotic organisms express Hsc70 in Cytosol, Hsp70 and its paralogs HSPA1A, HSPA1B, and HSPA1L, Binding immunoglobulin protein (BiP or Grp78) in endoplasmic reticulum and mtHsp70 or Grp75 in mitochondria. All these proteins share similar domain architecture and have a alike mode of action. Other than the core molecule Hsp70, the Hsp70 chaperone system constitutes other essential factors; co-chaperones such as Hsp40/J-proteins and Hip which aid in Hsp70 function and nucleotide exchange factors.

Structure and Domain Organization

hsp90
Structure and Domain organization of Hsp70

Hsp70 class of chaperone contains two major domains; N-terminal ATPase domain that binds to ATP and hydrolyzes it to ADP (3). The exchange of ATP drives conformational changes in the other two domains. The second domain is the substrate binding domain (SBD) that can interact and transiently associate with short linear peptide segments of folding intermediates. The SBD can be further subdivided into substrate binding subdomain and a C-terminal subdomain. The substrate binding subdomain contains a substrat binding pocket that has a high affinity for neutral, hydrophobic amino acid residues. The C-terminal subdomain is rich in alpha helical structure acts as a 'lid' for the substrate binding pocket (6). When an Hsp70 protein is ATP bound, the lid is open and peptides bind and release relatively rapidly. When Hsp70 proteins are ADP bound, the lid is closed, and peptides are tightly bound to the substrate binding domain (2).

The ATPase domain has a conventional nucleotide-binding fold viz. the ‘actin fold’ which is found in diverse proteins (6). In this fold, amino acid residues contributed by residues from domains on both sides form a cleft into which ATP is bound. The binding promotes a conformational change that closes the cleft. The substrate binding domain (SBD) has a β-sandwich structure comprising of two antiparallel β-sheets of four strands each. The peptide-binding site, which lies between the two β-sheets, is hydrophobic in nature and contains a deep pocket capped by a helical lid. The helical lid structure is composed of five helices namely, A, B, C, D and E and governs substrate affinity of the chaperone. It shows specificity for hydrophobic residues, particularly leucine. The two domains are connected by a short linker segment (~6 residues) which is highly conserved (2).

Mode of Action: The Hsp70 chaperone cycle

70cycle
Model of Hsp70:J-protein chaperone cycle

The functioning of all Hsp70s is modulated by two core activities: peptide binding and ATP hydrolysis. These two activities are localized to different domains: the ATPase activity to the N-terminal domain and substrate binding to the C-terminal domain (9). All the cellular functions of the Hsp70 family are attributed to the ability of these proteins to bind to exposed hydrophobic segments in the substrate proteins or partially folded polypeptide chains, preventing non-productive association of the hydrophobic regions and thus facilitating proper folding. This transient interaction with substrates is regulated by the ATPase activity of the other domain, while the ATPase activity is in turn stimulated by substrate binding (4).

Interaction of Hsp70 with non-native protein substrates and client proteins is highly dynamic and coupled to cycles of ATP binding and ATP hydrolysis (9) . Hsp70s exist in a two-state conformation: The ATP-bound form which has low affinity for substrates and the ADP-bound form with relatively higher substrate affinity. The low affinity of the ATP bound state is attributed to the fact that the SBD has an ‘open’ conformation with high rates for both association and dissociation of substrate. Hence, the peptide has a high tendency to be released even before a stable peptide-chaperone complex has been formed (2). The intrinsic ATPase activity of Hsp70s is very weak for stoichiometric coupling to the substrate binding-release cycle and co-chaperones like J-proteins and ‘nucleotide exchange factors’ function as accessory proteins, required for facilitating this process. Association with such as co-chaperones also contributes to the functional versatility of the chaperone. In vivo, Hsp70s and their J-domain partners function as ‘chaperone machines’ (5).

Hsp70 activity is characterized by multiple cycles of substrate binding and release coupled to cycles of ATP binding and hydrolysis. These two coordinated activities, attributed to the two domains of the protein, together constitute the ‘chaperone cycle’ (6). Nucleotide hydrolysis is the rate-limiting step of the ‘chaperone cycle’ and is accountable for the conformational changes in the domains that convert Hsp70 from its low substrate affinity state to its higher affinity state. Interaction with J-proteins which catalyze the ATP to ADP conversion causes the conformational change, is responsible for stabilization of the substrate-bound form. Furthermore, J-proteins are believed to bind protein substrates and recruit them to Hsp70 at this stage (5). The release of ADP and hence also of substrate, is then carried out by the nucleotide exchange factor, permitting the continuation of the cycle. In this way, repeated cycles of binding to exposed hydrophobic patches, ensures prevention of misaggregation during folding and translocation across membranes (4).


Physiological functions

Maintenance of Protein Quality Control

The broad spectrum of cellular functions of Hsp70 proteins is achieved through (i) the amplification and diversification of hsp70 genes in evolution, which has generated specialized Hsp70 chaperones, (ii) co-chaperones, which are selectively recruited by Hsp70 chaperones to fulfill specific cellular functions and (iii) cooperation of Hsp70s with other chaperone systems to broaden their activity spectrum. Hsp70 proteins with their co-chaperones and cooperating chaperones thus constitute a complex network of folding machines. Further, the reliance on Hsp70 chaperones increases even more for unstable mutated proteins. Interestingly, mutated proteins such as mutant p53, cystis fibrosis transmembrane regulator (CFTR) variant ΔF508, mutant superoxide dismutase seem to require more attention by the Hsp70 chaperones than the corresponding wild-type protein. As a consequence of this interaction the function of the mutant protein can be preserved. Thereby Hsp70 functions as a capacitor, buffering destabilizing mutations, a function demonstrated earlier for Hsp90 (4).

Other than its intrinsic protein folding/refolding function, Hsp70 prevents them from aggregation of partially synthesized proteins by binding tightly to the exposed peptide sequences and hence prevents them from being rendered nonfunctional (12). Once the entire protein is synthesized, nucleotide exchange factors (such as BAG-1 and HspBP1) cause the release of ADP and binding of fresh ATP and thus opening the peptide binding pocket. The protein either reaches to its native conformation on its own or is transferred to other chaperones such as Hsp90 for further processing. HOP (the Hsp70/Hsp90 Organizing Protein) can bind to both Hsp70 and Hsp90 at the same time and can mediate the transfer of such proteins from Hsp70 to Hsp90. Hsp70 also participates in disposal of terminally misfolded and inactive proteins (10). It interacts with an E3 ubiquitin ligase CHIP (Carboxyl-terminus of Hsp70 Interacting Protein) which allows Hsp70 to transfer the inactive proteins to the cell's ubiquitination and proteolysis pathways (3).

Hsp70 in Cancer and Apoptosis

In addition to improving overall protein integrity, Hsp 70 directly inhibits apoptosis. One hallmark of apoptosis is the release of cytochrome c, which then recruits Apaf-1 and dATP/ATP into an apoptosome complex. This complex subsequently cleaves procaspase-9, activating caspase-9 and eventually inducing apoptosis via caspase-3 activation. Hsp 70 inhibits this process by blocking the recruitment of procaspase-9 to the Apaf-1/dATP/cytochrome c apoptosome complex. It does not bind directly to the procaspase-9 binding site, but likely induces a conformational change that renders procaspase-9 binding less favorable (14). Hsp70 is shown to interact with Endoplasmic reticulum stress sensor protein IRE1α and thereby, protects the cells from ER stress induced apoptosis. Other studies suggest that Hsp 70 may play an anti-apoptotic role at other steps, but is not involved in Fas-ligand-mediated apoptosis. Considering that stress-response proteins like Hsp70 evolved before apoptotic machinery, Hsp 70’s direct role in inhibiting apoptosis provides an interesting evolutionary picture of how more recent apoptotic machinery accommodated the machinery of heat shock proteins and thus aligning the integrity of a cell’s proteins with the better chances of that particular cell’s survival (8).

The upregulation of HSP90 and HSP70 has been traditionally observed in human tumors and is often associated with increased chemotherapy resistance and poor patient prognosis. It protects cells from a wide range of apoptotic and necrotic stimuli and hence confers survival advantage to tumor cells (8). Hsp70 overexpression is associated with carcinogenesis of the oral epithelium, breast, ovary, and lung carcinomas and as a marker of early hepatocellular carcinoma. Hsp70 has been involved not only with poor tumor differentiation but also with increased cell proliferation (breast, uterine cervix, lung), lymph node metastasis (breast, colon), increased tumor size (uterine cervix), presence of mutated p53 (breast, endometrium), and higher clinical stage (oral, colon, melanoma) (13). Hsp70 has been described as an important molecule in the assembly and trafficking of steroid receptors. In breast cancer, Hsp70 has been found associated with ERα where it increases ERα transcriptional activity and hence results in increased cell-proliferation breast-tumors (8). However, little information is available on how Hsp70 regulation is subverted in cancer and how Hsp70 dysregulation affects the molecular events involved with tumor growth, invasiveness, and metastasis. Further studies are required for interpreting and directing the studies aimed at targeting Hsp70 in cancer therapy.

References

  1. Bukau, B. and Horwich, A.L. (1998) The Hsp70 and Hsp60 chaperone machines. Cell, 92, 351-366
  2. Mayer, M.P., Brehmer, D., Gassler, C.S. and Bukau, B. (2001) Hsp70 chaperone machines. Adv Protein Chem, 59, 1-44.
  3. Mayer, M.P. and Bukau, B. (2005) Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci, 62, 670-684.
  4. Hartl, F.U. and Hayer-Hartl, M. (2002) Molecular chaperones in the cytosol: from nascent chain to folded protein. Science, 295, 1852-1858.
  5. Laufen, T., Mayer, M.P., Beisel, C., Klostermeier, D., Mogk, A., Reinstein, J. and Bukau, B. (1999) Mechanism of regulation of hsp70 chaperones by DnaJ cochaperones. Proc Natl Acad Sci U S A, 96, 5452-5457.
  6. Lund, P.A. (2001) Molecular chaperones in the cell. Oxford University Press, Oxford.
  7. Bukau, B., Weissman, J. and Horwich, A. (2006) Molecular chaperones and protein quality control. Cell, 125, 443-451.
  8. Sherman M, Multhoff G. (2007) Heat shock proteins in cancer. Ann N Y Acad Sci., 1113, 192-201.
  9. Young JC. (2010) Mechanisms of the Hsp70 chaperone system. Biochem Cell Biol., 88(2), 291-300.
  10. Goloubinoff P, De Los Rios P. (2007) The mechanism of Hsp70 chaperones: (entropic) pulling the models together Trends in Biochemical Sciences, 32(8), 372-380.
  11. Matouschek A, Pfanner N, Voos W. (2000) Protein unfolding by mitochondria. The Hsp70 import motor. EMBO Rep., 1(5), 404-410.
  12. Stefan Rüdiger, Alexander Buchberger and Bernd Bukau (1997) Interaction of Hsp70 chaperones with substrates Nature Structural Biology, 4, 342-349.
  13. Renu Wadhwa, Kazunari Taira, and Sunil C. Kaul (2002) An Hsp70 family chaperone, mortalin/mthsp70/PBP74/Grp75: what, when, and where? Cell Stress Chaperones., 7(3), 309-316.
  14. C Garrido and E Solary (2003) A role of HSPs in apoptosis through "protein triage"? Cell Death and Differentiation, 10, 619-620.

Database Statistics

Total Number of Genomes
277
Numbers in Bacteria
338
Numbers in Archaea
26
Numbers in Protists
239
Numbers in Fungi
236
Numbers in Algae
39
Numbers in Plants
260
Numbers in Animals
388
Total Number of Hsp70 Proteins
1526