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Rhodes Centenary South African Journal of Science 100, November/December 2004 665

Molecular chaperones in biology, medicine and protein biotechnology

A. Boshoff*, W.S. Nicoll*, F. Hennessy*, M. Ludewig*, S. Daniel*, K.W. Modisakeng*, A. Shonhai*, C. McNamara*, G. Bradley* and G.L. Blatch*†

uration. Molecular chaperones do not contain any steric information for the folding process of a substrate protein, but Molecular chaperones consist of several highly conserved fami- play a vital role by guiding proteins along productive protein lies of proteins, many of which consist of heat shock proteins. The folding pathways. Chaperones also play central roles in the primary function of molecular chaperones is to facilitate the folding translocation of proteins across intracellular membranes into or refolding of proteins, and therefore they play an important role in organelles, and facilitate the proteolytic degradation of unstable diverse cellular processes including protein synthesis, protein proteins.5 In general, the main molecular chaperones function in translocation, and the refolding or degradation of proteins after cell a nucleotide-dependent manner, and the release of the substrate stress. Cells are often exposed to different stressors, resulting in protein is usually associated with an ATP-coupled conforma- protein misfolding and aggregation. It is now well established that tional change by the chaperone.6 the levels of certain molecular chaperones are elevated during Many chaperones are constitutively expressed (Hsc proteins), stress to provide protection to the cell. The focus of this review is on while the synthesis of other chaperones (Hsp proteins) increases the impact of molecular chaperones in biology, medicine and after a period of stress or the so-called ‘heat shock response’. protein biotechnology, and thus covers both fundamental and According to Finkelstein and Strausberg,7 the heat shock applied aspects of chaperone biology. Attention is paid to the func- response was first described in Drosophila by Ritossa in 1962. It is tions and applications of molecular chaperones from bacterial and now generally accepted that this response is similar for different eukaryotic cells, focusing on the heat shock proteins 90 (Hsp90), 70 types of stress, and is conserved in most organisms. One of (Hsp70) and 40 (Hsp40) classes of chaperones, respectively. The the common features of the response is an increased rate of role of these classes of chaperones in human diseases is dis- transcription of the genes that encode the inducible heat shock cussed, as well as the parts played by chaperones produced by the proteins, followed by enhanced synthesis of these proteins.8 causative agents of malaria and trypanosomiasis. Recent advances Stressful circumstances induce the synthesis of more than 20 have seen the application of chaperones in improving the yields of a heat shock proteins that enable cells to adapt to environmental particular target protein in recombinant protein production. The and metabolic changes and to survive stress conditions.9 Hsps prospects for the targeted use of molecular chaperones for the are highly conserved and play similar roles in organisms from over-production of recombinant proteins is critically reviewed, and bacteria to humans. In eukaryotic cells, heat shock genes include current research on these chaperones at Rhodes University is also a conserved sequence referred to as the heat shock element discussed. (HSE) within the promoter region; the genes are activated by a protein called the heat shock factor (HSF).10 The heat shock response in Escherichia coli is positively controlled at the level of Introduction transcription by the heat shock promoter-specific F32 subunit of The functions of molecular chaperones in living cells under RNA polymerase, that is a product of the rpoH gene.9,11 both normal and stress conditions have received intensive There are several families of heat shock or stress proteins, research interest. This has led to advances in fundamental defined on the basis of molecular weight. This review focuses on research as well as in medical and biotechnological applications the functions of the 90-kDa heat shock protein (Hsp90), 70-kDa of chaperones. Molecular chaperones consist of several highly heat shock protein (Hsp70) and 40-kDa heat shock protein conserved families of proteins, with some chaperones also being (Hsp40) classes of chaperones, highlighting the cooperation called heat shock proteins (Hsps) or stress proteins. The primary between different chaperones to carry out specific functions. functions of molecular chaperones are to bind selectively to The diverse roles of these particular classes of proteins in the life nascent polypeptides and partially folded intermediates, cycle of a protein are outlined in Fig. 1. The terms Hsp40 and preventing their irreversible aggregation and misfolding and to Hsp70 are used for the heat shock proteins in eukaryotes, facilitate protein refolding.1 Molecular chaperones are able to whereas these proteins in bacteria are called DnaJ and DnaK, distinguish between unfolded and folded proteins as they respectively. In this review we use Hsp40 and Hsp70 as general recognize the hydrophobic features of unfolded proteins. terms. A number of diseases, such as Alzheimer’s, have been Anfinsen’s early work established that the amino acid sequence attributed to the formation of aggregates of protein referred to as of a polypeptide contains all the information needed for the amyloid, and the role of molecular chaperones in the develop- polypeptide to reach its native structure.2 However,owing to the ment of these diseases is beginning to be understood.12 The crowded (300 mg/ml protein) and viscous nature of the implications of heat shock proteins in the control of disease intracellular environment, newly translated proteins are prone pathology and in the survival of pathogens are examined in this to entering non-productive folding pathways, resulting in pro- tein misfolding and aggregation.3,4 Numerous proteins therefore review. Recombinant protein production can result in the require the assistance of chaperones to fold into an active config- formation of insoluble inactive aggregates that decrease the yield of active protein.13 Therefore, we also review the targeted *Chaperone Research Group, Department of Biochemistry, Microbiology and Biotechnol- ogy, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa. use of molecular chaperones for the over-production of recombi- †Author for correspondence. E-mail: [email protected] nant proteins. 666 South African Journal of Science 100, November/December 2004 Rhodes Centenary

Fig. 1. Diagrammatic representation of the diverse functions of molecular chaperones in the life cycle of a protein, highlighting the roles of Hsp40, Hsp70 and Hsp90. (A) Nascent polypeptides are bound by molecular chaperones to facilitate correct protein folding during and after ribosomal translation. Translocation of proteins across membranes into cellular organelles, such as (B) the endoplasmic reticulum (ER) and (C) the mitochondrion, is aided by organelle-specific chaperones. (D) Denatured proteins are refolded or are targeted to (E), the lysosome for proteolytic degradation. (F) The maturation of certain specialized signalling molecules, such as the steroid receptor, is mediated by a suite of molecular chaperones. Although not shown, Hsp40 also plays a role in processes (A), (B) and (C).

Hsp70 and Hsp40 are functionally linked Structural features of Hsp70- and Hsp40-like proteins The most investigated Hsp70 chaperone system is the E. coli All Hsp70s are composed of three regions: a highly conserved Hsp70 chaperone, DnaK, and its Hsp40 co-chaperone, DnaJ. ~44-kDa ATPase domain, a conserved ~15-kDa substrate-bind- DnaJ and DnaK were initially identified as being necessary for ing region, and a less conserved ~10-kDa C-terminal region bacteriophage 8 DNA replication, hence the Dna nomenclature.3 (Fig. 2A). The structures of the ATPase domain and the Hsp70s are found in all prokaryotes and eukaryotes, as well as in substrate-binding region have been determined. The structures some, but not all, of the archaea.14,15 Some methanogens, extreme have been determined of ATPase domains of bovine Hsc70,29,32 halophiles and extreme thermophiles do not appear to synthe- E. coli DnaK (in conjunction with the nucleotide exchange factor size, or even to contain, a genomic copy of an Hsp70 gene.15,16 GrpE),33 bovine Hsc70 (in conjunction with the nucleotide There appear to be minimal differences between Hsc7017 exchange factor Bag-1)34 and human Hsp70.35 The ATPase (also called Hsp73) and Hsp70 (also called Hsp72) in terms of domain consists of two lobes with a nucleotide-binding site function. The cognate form acts as a molecular chaperone under between them. The two lobes are similar in size, and each lobe normal cellular conditions, whereas the heat shock protein will can be further subdivided into two subdomains, giving function under stress conditions. Hsp70s recognize unfolded subdomains IA, IB, IIA and IIB (Fig. 2B).29 It bears structural proteins, binding and stabilizing them, thereby giving them similarity to the ATPase domains of hexokinase and actin, enough time to fold correctly. They are also involved in the stabi- despite low sequence similarity between these proteins.36,37 The lization of precursor proteins, aiding in the translocation of presence of ATP or ADP in the nucleotide-binding cleft affects newly synthesized proteins, the rearrangement of protein Hsp70s chaperone activity. oligomers and the protection of the cell against the effects of The structures of substrate-binding domains have been cellular stress.18 Hsp70s have also been implicated in protein determined from E. coli DnaK30,38–39 and Hsc70.40 The domain degradation,19 including the specific degradation of the heat comprises two subdomains, with the first subdomain a predomi- shock transcription factor F32 in E. coli,20 and polyglutamine nately $-strand-rich region involved in the binding of amino repeat containing proteins via the ubiquitination pathway in acids in a substrate protein, followed by an "-helical region eukaryotes.21 These functions are all dependent on Hsp70 keep- proposed to act as a lid that locks the substrate in the domain ing polypeptides in an extended conformation, and stabilizing (Fig. 2C). A number of studies have been conducted on the bind- the exposed hydrophobic regions of the target polypeptide.22 ing of Hsp70 to peptides, with the aim of defining what amino Hsp40-like proteins are involved in facilitating the folding of acid types and combinations are recognized by the sub- nascent polypeptides by regulating partner Hsp70 proteins,23 strate-binding domain. The results of these studies have sug- and have also been implicated in protein translocation,24 protein gested that the substrate specificity of different Hsp70s is degradation,25 clathrin uncoating26,27 and viral infection.28 different, but overlapping. E. coli DnaK has been shown to inter- Rhodes Centenary South African Journal of Science 100, November/December 2004 667

Fig. 2. Diagrammatic representation of the structural features of Hsp70. (A) Schematic representation of the domain organization of Hsp70: The N-terminus consists of an ATPase domain linked to a central substrate-binding domain, and the C-terminus contains a lid region. (B) Ribbon representation of the structural features of bovine Hsc70 ATPase domain with a bound molecule of ATP/ADP: The ATPase domain consists of two domains I and II, which are further divided into subdomains IA, IIA, IB and IIB (PDB code: 3HSC29). (C) Ribbon representation of the structural features of the DnaK substrate-binding domain with a bound substrate molecule. N, N-terminus; C, C-terminus (PDB: code 1DKX30). Figures B and C were drawn using MOLSCRIPT.31 act strongly with peptides enriched in phenylalanine, (HPD motif) located between helices II and III53 (Fig. 3B). The isoleucine, leucine and valine.41,42 In addition, leucine is found in HPD motif is present in all known J-domains, with the exception approximately 90% of all regions specifically recognized by of the ring-infected erythrocyte-surface antigen (RESA) proteins DnaK, with sites on unfolded polypeptide chains occurring sta- of Plasmodium falciparum,60 and the recently identified DjlB/DjlC tistically every 36 residues,43 so that at least one site is likely to be family of proteins described in E. coli.61 The minimal interacting present in all proteins. The optimal length of the substrate pep- region in the J-domain is between amino acids 2 and 35, which tide has been shown to be at least seven or eight amino acids.44 includes helices I and II, and the HPD motif.62,63 The J-domain The subtle variations in the peptides that can be bound by DnaK also appears to be flexible in structure, with some workers pro- suggest that there may be distinct but overlapping pools of sub- posing an induced-fit mechanism, with the HPD motif aiding in strates in prokaryotes and in eukaryotic organelles where multi- the alteration of the orientation of the charged residues in helix II ple Hsp70s are present. The E. coli protein DnaK has been shown to allow them to interact correctly with the ATPase domain of a to bind exposed hydrophobic residues, regardless of whether partner Hsp70.64 Structural features in addition to the J-domain they were in unfolded or native proteins.45 This may indicate a present in E. coli DnaJ include: a glycine-phenyalanine (G/F)- conserved mechanism for all Hsp70s, as some Hsp70 functions rich region, proposed to act as a linker between the J-domain and require an ability to bind folded proteins, e.g. during clathrin the C-terminal portion of the protein; cysteine repeats of the uncoating.46,47 The nature of the substrate proteins recognized by form Cys-X-X-Cys-X-Gly-X-Gly proposed to play a role in pro- Hsp70s may be regulated by the partner Hsp40-like protein.48 tein binding; and a poorly characterized C-terminus (Fig. 3A and The J-domain is the specific feature that defines a protein as an C). The presence and absence of these domains of E. coli DnaJ Hsp40-like protein.49 However, the presence of a J-domain does have been used to sub-divide Hsp40 proteins into three classes not imply that the protein is a true homologue of Hsp40. Many of Hsp40-like proteins: type I (J-domain, G/F region and cysteine proteins do not contain all the typical domains found in a true repeats), type II (J-domain and G/F regions), and type III Hsp40 homologue such as in E. coli DnaJ,50 and the presence of (J-domain).50 other domains may allow a specific Hsp40-like protein to fulfil a specific function. The J-domain has a similarity with the initial The chaperone function of Hsp70 is dependent on its 73 amino acids of DnaJ, and is normally found at the amino ATPase activity terminus of proteins, although this is not always so.23 This region In general, the ATPase cycle of Hsp70 is stoichiometrically is believed to be the site of the interaction between Hsp40-like coupled to the cycle of substrate binding and release. The type proteins and their partner Hsp70s. It also appears to be the of nucleotide bound to the ATPase domain determines the minimal region required for interaction between Hsp40-like efficiency of the Hsp70’s chaperone activity. When ATP is proteins and partner Hsp70s.51 present, there is a low affinity for unfolded proteins by the The structures of the J-domain in E. coli DnaJ,49,52 human substrate-binding region. By contrast, when ADP is present HDJ1,53 E. coli Hsc20,54,55 the large T-antigen from murine there is a high affinity for unfolded proteins. This higher affinity polyomavirus,56 the large T-antigen from SV40 in conjunction allows a longer period of time for stabilization of the unfolded with the retinoblastoma tumour suppressor,57 and bovine protein, and hence gives it a greater chance of folding correctly. auxilin58 have been determined using nuclear magnetic reso- The Hsp70 ATPase cycle involves ATP hydrolysis and nucleotide nance (NMR) and X-ray crystallography. The structures com- exchange. ATP hydrolysis is important for converting Hsp70 to prise four "-helices, with a loop region containing a highly the high-affinity substrate-binding state. Nucleotide exchange is conserved tripeptide of histidine, proline and aspartic acid important for ensuring that Hsp70s are not permanently in the 668 South African Journal of Science 100, November/December 2004 Rhodes Centenary

Fig. 3. Diagrammatic representation of the structural features of E. coli DnaJ. (A) Schematic representation of the domains present in E. coli DnaJ. The J-domain is followed by a G/F-rich region (glycine–phenylalanine region) and the cysteine repeats. (B) A ribbon representation of the J-domain (1XBL49). The conserved HPD motif is depicted and the four helices are shown. (C) A ribbon representation of the cysteine repeats (1EXK59) that represent four zinc finger-like motifs. Figures B and C were 31 generated using MOLSCRIPT. high-affinity ADP state and can release substrate and return to cellular proteins under physiological conditions.72 The Hsp90 the low-affinity state. Hsp70s typically have a low basal ATPase protein family includes the endoplasmic 94-kDa glucose-regu- activity,65 which implies that the Hsp70 will be predominately in lated protein, grp94,73 the mitochondrial matrix proteins the ATP-bound form, and hence will manifest relatively poor Hsp75/TRAP1,74 and the prokaryotic HtpG protein (high chaperone activity. temperature protein G).75 The prokaryotic homologue HtpG is Close inter-domain communication between the substrate- not as well characterized as its eukaryotic counterparts.76 binding domain and ATPase domain is critical for effective Cytosolic Hsp90 occurs in two isoforms (" and $) that are chaperone activity and substitutions in the linker region differentially regulated, and it is primarily the Hsp90-a gene that between these two domains affected this activity.66 Stimulation is inducible, while the Hsp90-b gene is constitutive.76 The genes of either ATP hydrolysis or nucleotide exchange will enhance encoding both isoforms of Hsp90 contain introns,76 which is the ATPase activity and therefore the chaperone activity of unusual for Hsps in general. Another isoform, Hsp90N, has been Hsp70. Hsp40 proteins are critical in that they stimulate the added to the Hsp90 family, which is associated with cellular ATPase activity of partner Hsp70s by stimulating ATP hydroly- transformation.77 The rest of this section will be devoted to sis.65 The enhancement of the basal ATPase activity has been eukaryotic cytosolic Hsp90. reported at greater than 5 times the basal rate,67 and also as much Hsp90 is a hydrophobic,78 phosphorylated dimer79 containing as 1000-fold, but only at high DnaJ concentrations.68 Binding 2–3 covalently bound phosphate molecules per monomer80 that of peptide substrate has been found to stimulate the ATP undergoes conformational change after binding to ATP.81 Stress hydrolysis activity of DnaK.69 GrpE plays an important role in produces an increase in oligomerization of the molecule,82 as the ATPase cycle of DnaK in E. coli, and was found to stimulate well as an increase in hydrophobicity.83 Hsp90 contains an N-ter- the basal ATPase activity up to 50-fold by enhancing nucleotide minal and a C-terminal domain, connected together by a hinge exchange.67 region, with both domains having an ATP-binding site.84 The While no GrpE homologue has been identified in the cytosol of hinge domain and the C-terminal domain are highly charged eukaryotic cells, various nucleotide exchange factors have been areas76 that bind preferentially to hydrophobic or positively characterized.70 In eukaryotic systems, the Hsp70-substrate charged proteins.85 Hsp90 is involved in the folding and matura- conformation can be stabilized by the Hsp70-interacting protein, tion of various specialized signalling molecules including ste- Hip, which has been shown to be involved in the stabilization of roid receptors,86,87 phosphatases,88 protein kinases86,87 and various the ADP-bound form of Hsp70.71 This allows further time for other signalling intermediates of the mitogenic signal the unfolded polypeptide to attain a native-like conformation transduction pathway that regulate cell-cycle progression and before being released to fold to the native state, or facilitates apoptosis (Fig. 4). passage of the substrate on to the Hsp90 chaperone machinery The N-terminal domain of Hsp90 is a highly twisted, 8-stranded as occurs during the maturation of certain specialized signalling $-sheet covered on one side by "-helices and contains an molecules. ATP/ADP binding site,91 which is also the binding site for the Hsp90-specific anti-tumour drug geldanamycin92 and the The Hsp90 family interacts with a diverse range of antibiotic radicicol.93 The N-terminal Hsp90 domain is also signalling proteins involved in binding substrate proteins.94 Harris et al.95 recently Members of the Hsp90 family are highly conserved and resolved the crystal structure of the C-terminal domain of HtpG; present in most prokaryotic and eukaryotic cells. Hsp90 is an the structure revealed a potential substrate-binding site. The abundant eukaryotic cytosolic protein contributing 1–2% of C-terminal domain of Hsp90 binds calmodulin96 and is also the Rhodes Centenary South African Journal of Science 100, November/December 2004 669

Fig. 4. Mitogenic signal transduction pathways regulated by Hsp90 that are disrupted in cancers.89,90 Hsp90 substrate proteins include growth factor receptors, kinases, steroid receptors and cell cycle regulators. Hsp90 substrate proteins are indicated in bold. Abbreviations: ER, estrogen receptor; AP, androgen receptor; HER, human epidermal growth factor receptor; IGF-1R, insulin-like growth factor-1 receptor; PDGR-R, platelet-derived growth factor receptor; EGF-R, epidermal growth factor receptor; PI-3K, phosphoinositide 3-kinases; PDK-1, 3-phosphoinositide-dependent kinase 1; PKB, protein kinase B; BAD, Bcl-2-associated death protein; MEK-1, mitogen-activated protein kinase kinase-1; ERK, extracellular signal-regulated kinase; PKC, protein kinase C; MEKK, mitogen-activated protein kinase kinase kinase; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; ROS, reactive oxygen species; HSF1, heat-shock transcription factor-1. site for dimerization.97 The C-terminal domain is reported to to facilitate the folding process and reduce the number of contain chaperone activity as it binds steroid receptors and actin non-productive folding interactions that occur within the cell.101 filaments.98,99 It is therefore proposed that polypeptide binding Not surprisingly, recent evidence indicates that altered protein by Hsp90 involves all its domains either through several folding is the basis of a growing list of human diseases (Table 1). different sites or working together in a concerted action.76 These protein-folding-related diseases generally take two forms: Hsp90 associates with a wide range of co-chaperones, where most commonly a substrate protein contains mutations that the type of co-chaperone appears to depend on the protein result in a misfolded protein that chaperones are unable to substrate of interest.72 The elucidation of the cellular functions of correct, and less commonly a defect in the chaperone itself Hsp90 is a complicated task and has received the attention of results in target proteins not being folded correctly.125 Table 1 many researchers during the last decade. Hsp90 has been impli- indicates that diseases caused by protein misfolding are many cated in protein folding, signalling, cytoplasmic organization, and varied, and this list will probably grow as further roles for nuclear transport, DNA rearrangements and DNA-protein chaperones in other diseases are uncovered. Molecular pheno- interactions, the cell cycle and apoptosis and protein presenta- types of misfolding-related diseases generally fall into three tion to the proteasome.76 After cellular stress, Hsp90 binds to categories — misfolding and subsequent loss of function, partially denatured proteins in order to prevent total denatur- misfolding that leads to incorrect localization, and misfolding ation and maintain them in a ‘folding-competent state,’100 that results in the formation of toxic aggregates.126 thereby indicating a role for Hsp90 in salvaging proteins that have undergone partial denaturation due to cell stress. Hsp90 is Involvement of Hsp70 and Hsp40 in disease thought to play a major role in preserving the structural integ- As discussed above, Hsp70 and its co-chaperone Hsp40 are rity of both the cytoplasm and the nucleus, thus accounting for integrally involved in a wide range of processes within the cell, the high cellular concentration of Hsp90. The elucidation of the from prevention of aggregation and disassembly of multimeric actual cellular functions of Hsp90, however, requires further protein complexes to protein trafficking and regulation of the investigation.76 heat-shock response.127 With this almost ubiquitous involvement in biochemical processes, it is not surprising that these Importance of molecular chaperones in human disease chaperones are turning out to be involved in an increasing From an understanding of the biology of chaperones, we can number of diseases. Interestingly, the wide-ranging roles of consider their role in human disease. Chaperones have evolved Hsp70 often mean that it is involved in both positive and 670 South African Journal of Science 100, November/December 2004 Rhodes Centenary

negative regulation at different stages of a single Table 1. Human diseases that are a consequence of protein misfolding and the formation of toxic folds. process. Disease Protein involved Molecular phenotype One of the prominent roles of Hsp70 in disease is in cancer and the associated apoptosis. How- Misfolding ever, while Hsp70 seems to perform a multitude "-Antitrypsin deficiency102 "-Antitrypsin Mislocalization of apparently conflicting functions, its exact role Amyotrophic lateral sclerosis103 Superoxide dismutase Misfolding 104 is unclear. Tumour cells can be characterized by Bardet-Biedel syndrome MKKS Loss of chaperone activity Cancer105 P53 Misfolding their ability to shut down the apoptotic path- Charlevoix-Saguenay106 Sacsin Loss of chaperone activity way, resulting in runaway cell multiplication. Cystic fibrosis107 CFTR Misfolding Hsp70 has the ability both to enhance and retard Ethylmalonic aciduria108 SCAD Misfolding the apoptosis pathway. In caspase-dependent Familial hypercholesterilemia109 LDL receptor Mislocalization Leprechaunism110 Insulin receptor Mislocalization apoptosis, over-expression of Hsp70 has been 111 128 Marfan syndrome Fibrillin Misfolding How- 112 shown to reduce Fas-induced apoptosis. McKusick-Kaufman syndrome MKKS Loss of chaperone activity ever, contrary to this, Hsp70 over-expression Osteogenesis Imperfecta113 Procollagen pro aaa Misfolding 129,130 enhances apoptosis in Jurkat cells. Over- Phenylketonuria114 PAH Misfolding expression of Hsp70 also inhibits apoptosis Retinosa pigmentosa115 Rhodopsin Mislocalization 116 through the inhibition of caspase activation in Tay-Sachs disease $-Hexosaminidase Mislocalization U-937 cell cultures131 and in mitochondrial-con- Toxic folding Alzheimer’s disease117 $-Amyloid Aggregation trolled apoptosis through proapoptotic fac- 118 132 Cataracts "-Crystallins Aggregation tors. Conversely, over-expression of the Hsp70 119 Creutzfeldt-Jakob disease Prions Aggregation homologue Hsp105a results in the induction of Familial amyloidosis120 Transthyretin Aggregation 133 apoptosis through caspase activation. Huntington’s disease121 HD Aggregation Hsp70s are also found within the lysosome, Parkinson’s disease122 PARK2 Aggregation Spinocerebellar atrophy123 Ataxin-3 Aggregation suggesting a major role for this protein in the 124 proteolytic pathways that are integrally associ- Spinal and bulbar muscular atrophy Androgen receptor Aggregation ated with apoptosis.134,135 Additionally, Hsp70s have been shown to protect cells from ceramide-induced cell line suppressed aggregation of the mutant ataxin protein.143 apoptosis136 and AIF (for apoptosis-inducing factor)-induced It is postulated that neurodegenerative diseases such as those apoptosis.137 Furthermore, the chaperone activity of cytosolic affecting elderly people are due to mutations occurring in the Hsp70 is required for inhibition of apoptosis, as non-functional body as it ages, and the chaperone system gradually becomes Hsp70 mutants exhibited no inhibition.138 In tumour cells Hsp70 overrun with an ever-increasing workload.144 This type of is highly expressed and required for survival, with depletion disease could potentially be addressed through drugs that up- of Hsp70-inducing apoptosis,139 possibly through a change in regulate the overall chaperone system before the disease symp- regulation of the proapoptotic p53 protein.140 A number of toms set in. Hsp70 has also been investigated as a possible tool in compounds have been shown to alter the apoptotic pathway treatment of heart attack or stroke, as it has been shown that through induction of heat shock proteins, including stannous induction of Hsp70 chaperones can prevent cardiac muscle from chloride (used to improve the success rate of tissue transplanta- the damage induced by both ischaemia and reperfusion.145 As tions), geranyl-geranyl acetone (suppresses induced apoptosis the roles of Hsp70 in human disease are many and varied, the in rats), carbenoxolone (used in ulcer treatment) and aspirin potential for targeting the Hsp70 folding pathway as a therapeutic (used for a wide range of ailments).90 Targeting of Hsp70 in approach is great. However, further research into its particular tumour cells could be a feasible approach to tumour control. role in specific diseases is required. However, the specific roles of Hsp70 in tumour development need to be understood better, as manipulation of Hsp70 levels Role of Hsp90 in disease may result in changes in the control of apoptosis to protect Hsp90 chaperone complexes involving signalling proteins are tumour cells from cytostatic drugs and promoting tumori- found over-expressed in human cancers.89 The wide range of genesis.131 Additionally, there could be effects on the role substrate proteins involved in signal transduction suggests apoptosis plays in the body such as that of a defence mechanism that inhibition of Hsp90 may result in the simultaneous during infectious disease. destabilization of various oncoproteins involved in multiple In cystic fibrosis, Hsp70 has been shown to associate with the signalling pathways, thereby potentiating a role for Hsp90 misfolded cystic fibrosis transmembrane conductance regulator inhibitors in attacking late stage cancer cells.89,146 The diverse (CFTR) that causes the disease phenotype. This suggests a role nature of its substrate proteins makes way for targeting of for Hsp70 in maintaining the correct form of CFTR. It is postu- various cancers in which the particular Hsp90 substrate protein lated that this association is responsible for retention of mutant is involved. proteins in the endoplasmic reticulum and their ultimate degra- Geldanamycin interacts with the nucleotide-binding site on dation.141 Many neurodegenerative diseases such as Alzheimer’s Hsp90, which is the N-terminal site, thus blocking the conver- and Parkinson’s involve aberrant protein folding that causes a sion of Hsp90 from its ADP conformation to its ATP conforma- conformational rearrangement and results in protein aggrega- tion.147 Although geldanamycin displays potent anti-tumour tion, subsequent tissue deposition and further cell degenera- activity in vitro, its hepatotoxic properties do not warrant its clin- tion.142 While the causes of many neurodegenerative diseases is ical use.148 A less toxic derivative of geldenamycin, 17-allyl- unclear, general housekeeping chaperones such as Hsp70 are aminogeldanamycin (17-AAG), has been shown to exert potent implicated as modulators of disease pathology due to their anti-tumour activity in pre-clinical models and enhanced ability to modulate protein aggregation. Both Hsp40 and Hsp70 tumour cell susceptibility to the cytotoxicity of ionizing radia- are found in ataxin aggregates that are common in spino- tion.149 Geldanamycin is also known to cause an upregulation of cerebellar atrophy (SCA). Over-expression of Hsp40 in an SCA heat shock proteins including Hsp27, Hsp70 and Hsp90 via the Rhodes Centenary South African Journal of Science 100, November/December 2004 671

HSF-1 pathway. These have a protective function during identified in P. falciparum. The remainder are type III Hsp40s.161 stressed conditions such as ischaemia, which accompanies most The most well-characterized Hsp40-like protein is the type III cardiovascular diseased conditions. Protection by these heat ring-infected erythrocyte-surface antigen (RESA), that is shock proteins is exerted through the inhibition of the assembly translocated to the inner face of the erythrocyte membrane of the caspase 9-Apaf-1-cytochrome C apoptosome.150 A role for during the erythrocytic stages.163 This is a highly immunogenic Hsp90 as a diagnostic tool has also been proposed in diseased antigen and has been the focus of a number of vaccine develop- conditions of the central nervous and cardiovascular systems.89 ment efforts, although its functional role is still unknown.164,165 Since diseased cells may be dependent on the upregulation of RESA could be a novel drug target as the highly conserved Hsp90 activity, this could allow for the use of Hsp90 as a diagnos- J-domain HPD motif found in human Hsp40 has been found to tic tool through its selective high-affinity binding of Hsp90 be substituted with a YPY motif.166 The YPY motif has been inhibitors within the diseased tissue. shown still to interact with Hsp70 during protein folding.167 Heat shock proteins, including Hsp90, have been shown to be While it is expected that the malarial agents produce heat a principal target for the immune response to various bacterial, shock proteins as a way of facilitating pathogenesis in the fungal and parasitic infections.151 Viral infections are known to human host, it is interesting to note that the human host cause upregulation of Hsp90 activity in the cell. Many viruses chaperones Hsp70 and Hsp90 have been implicated in the ‘steal’ the host Hsp90 for their own survival, and Hsp90 is an externalization of parasitic proteins to the surface of the red essential host factor for the replication of hepatitis B virus and blood cells, forming knob-like sub-units.168 This suggests that the hepatitis C virus. Inhibitors of Hsp90 like geldanamycin have parasite commandeers the erythrocytic chaperone machinery demonstrated broad-spectrum activity against a range of for its own use. Sera from humans, mice and squirrel monkeys viruses, with HIV-1 and SARS coronavirus exhibiting the high- exposed to Plasmodium contained abundant amounts of anti- est sensitivity. Whether therapeutic selectivity for viral Hsp90 body reactive to the Hsp90 protein,169 suggesting that it may can be achieved remains to be determined.89,152 have an antigenic role in malaria. Geldanamycin is a specific inhibitor of Hsp90 and has been shown to suppress the growth Chaperones found in human parasites and their potential of P.falciparum in erythrocytic cultures,170 suggesting that Hsp90 role in survival function is essential for the parasite’s survival.171 The immuno- Pathogenic organisms are known to produce chaperones, genic nature of PfHsp90, PfHsp70 and RESA proteins could be which in turn play a role in the development of immune exploited for vaccine development, and the involvement of the responses during the course of infections and inflammatory Hsp40, Hsp70 and Hsp90 proteins in potentially fundamental conditions.153 The entry of a parasite into its host organism is pathways makes them possible targets for specific inhibitors. usually under high stress conditions, where it is exposed to Trypanosomes are single cellular eukaryotic organisms, many abrupt changes in temperature, pH, ionic strength, nutritional of which are parasitic. Trypanosoma cruzi is the aetiologic agent of composition and a hostile immune system. The over-expression Chagas’ disease, sometimes referred to as American trypanoso- of heat shock proteins in the parasitic system is highly likely to be miasis. Trypanosoma brucei causes African trypanosomiasis, necessary for survival.76 The roles of molecular chaperones from better known as African sleeping sickness. Both these parasites Plasmodium and Trypanosoma species in human parasitic infec- are transmitted by insect vectors. Proteins of the Hsp100, Hsp90, tions are discussed below. Hsp70, Hsp60 and Hsp40 families have been described in Plasmodium species are the causative agents of malaria, a T.cruzi,172,173 with the Hsp70 family the best characterized of the T. parasitic disease responsible for over three million deaths per cruzi Hsps.172,174 Five cytoplasmic Hsp40-like proteins have been year, with over 40% of the world’s population at risk in some 90 described in T. cruzi (designated Tcj1, Tcj2, Tcj3, Tcj4 and countries.154 The current World Health Organisation campaign Tcj6).173,175 It is likely that more of these proteins will be discov- ‘Roll Back Malaria’ aims to halve the deaths due to malaria by ered in the T. cruzi genome database, TcruziDB,176 when a 2010. Plasmodium species are protozoan parasites transmitted by completed draft sequence for T. cruzi is available. Less is known the Anopheles mosquito.155 P. falciparum is the cause of cerebral about the T. brucei Hsp70/Hsp40 system, but homologues of Tcj2 malaria, the most deadly of the malarial infections. Plasmodium and the stress-inducible Hsp70 of T. cruzi (TcHsp70) have been has three cycles of infection: the mosquito, the erythrocytic and located.173 Both the GST-tagged TcHsp70172 and the His-tagged the exoerythrocytic cycles.155 The parasite infects the erythro- TcHsp70177 indicated that TcHsp70 has a significantly higher cytes and, depending on the species, these cells are ultimately basal ATPase activity than most other Hsp70s that have been sequestered within specific organs. In the case of P. falciparum characterized. this occurs within the endothelial capillaries of the brain.156 The expression of TcHsp70 increases when the growth temper- A number of molecular chaperones have been described in ature of cultured parasites is increased from 26°C (body temper- Plasmodium, the most important of which are predicted to be the ature of the insect vector) to 37°C (temperature in the Hsp40, Hsp70 and Hsp90 proteins, which are thought to be mammalian host),172,178 indicating that the trypanosome experi- important in cytoprotection under both normal and stress ences heat stress as it moves from the insect vector to the conditions.157 Little is known about the Plasmodium Hsp70/Hsp40 mammalian host.172 It is speculated that TcHsp70 plays a chaperone system. However, at least five genes for Hsp70-like cytoprotective role in the trypanosome by counteracting proteins have been established in P. falciparum,158 and people oxidative and temperature stress associated with infection.179 living in malaria-endemic areas have been shown to produce Trypanosomal Hsp70s have been found to elicit a strong antibodies that recognize the PfHsp70-1 protein.159 In addition, immune response in host organisms180–182 and that human only PfHsp70-1 has been biochemically characterized,160 Hsp70s do not elicit the same response.180 This suggests that whereas four Hsp40s have been described from the P.falciparum inhibition of the TcHsp70/Hsp40 system to hinder the establish- parasite, namely Pfj1 – Pfj4;161 20 others have been identified ment of an infection in the mammalian host may be a feasible through the P. falciparum genome resource database project, approach. However, this will be viable only if there is at least a PlasmoDB.162 Only one type I Hsp40-like protein (Pfj1) has been significant difference between the trypanosomal stress-inducible identified and at least 9 type II Hsp40-like proteins have been Hsp70 and its partner Hsp40 protein and their human counter- 672 South African Journal of Science 100, November/December 2004 Rhodes Centenary

parts. The immunogenic nature of TcHsp70 provides potential N-carbamoyl-D-amino acid amidohydrolase (N-carbamoylase) application in vaccine development; murine protection against are used sequentially for the enzymatic synthesis of D-amino trypanosomes has been observed after DNA immunization with acids for use as important intermediates in the production of a chimeric gene involving TcHsp70.182 pharmaceuticals.192 The recombinant production of these enzymes from Agrobacterium radiobacter NRRL B11291 resulted Inclusion body formation during recombinant production in protein aggregation; however,the co-expression of the molec- decreases protein yield ular chaperones DnaJ and DnaK from E. coli led to a 4-fold There are many systems available for heterologous protein increase in enzyme activity.184 production, and E. coli is one of the most widely used as its The expression of eukaryotic proteins in E. coli has met with genetics are well characterized and it grows rapidly to high cell limited success as eukaryotic proteins are generally larger than densities.183 Thus, the over-production of recombinant proteins E. coli proteins with multiple domains, and thus have a tendency in E. coli strains is a common approach used in protein biotech- to aggregate.6 In addition, the proper folding of certain nology.However,a limitation in the over-production of recombi- eukaryotic proteins is problematic, since postranslational modi- nant proteins is the formation of insoluble aggregates called fications such as glycosylation do not occur in E. coli, and struc- inclusion bodies, which can reduce yields of active protein.184,185 tural disulphide bonds do not form in the reducing environment Bacterial inclusion bodies are aggregates of protease-resistant of the E. coli cytoplasm.183 During recombinant expression, E. coli misfolded proteins that accumulate during the over-expression chaperones were unable to fold mutant >-crystallin (a protein of a cloned gene.185 The reasons for aggregation of heterologous found in the eye lens).193 Ahn and Yun194 co-expressed HDJ-1 proteins in E. coli are not well understood. Manipulation of the (human Hsp40) and human cytochrome P450 3A4 in E. coli and growth conditions, such as decreasing the fermentation temper- observed a 15-fold increase in catalytic activity of cytochrome ature or changing the pH, can marginally improve the solubil- P450 3A4 compared to the enzyme activity in the absence of ity.186 The recovery of active protein from inclusion bodies has chaperone. typically been carried out using chemical denaturants or acids followed by dilution into a refolding buffer.187 Molecular Limitations and future prospects of molecular chaperones chaperones have been widely exploited in protein biotechnol- in recombinant protein production ogy for the minimization of inclusion body formation and the The co-expression of molecular chaperones does not always recovery of folded functional protein. improve the yields of recombinant proteins186 and the criteria needed to match the class of chaperone to a specific protein are Molecular chaperone assisted recombinant protein folding currently being investigated. Immobilization would be a useful While Hsp70 and Hsp40 have demonstrated extensive appli- tool to retain and re-use chaperones for in vitro protein refolding, cations in the improved folding or refolding of recombinant but there are few reports on immobilization of Hsp70 or Hsp40. proteins, Hsp90 has found limited application.100 Goloubinff Buchner et al.195 used E. coli DnaK immobilized on Affi-Gel beads et al.188 first reported the use of chaperone co-expression to to improve the refolding yield of recombinant immunotoxin; achieve a 10-fold increase in the yield of active ribulose bisphos- and the immobilized DnaK could be re-used several times. The phate carboxylase from E. coli. The proposed use of chaperones factors influencing the recovery of active protein from inclu- to improve the yields of protein during heterologous production sion bodies still need to be investigated, and robust inclusion was first derived from its in vivo refolding capabilities.6 However, body solubilization methods still need to be developed.196 Recent the current application of molecular chaperones in biotechnol- work by de Marco and De Marco197 has shown that recipro- ogy exploits the ability of chaperones both to prevent and cal expression tuning by cross-inhibition between the target reverse protein aggregation.6 The E. coli DnaK-DnaJ-GrpE protein and the chaperones improved the amount of soluble system was determined to be one of the most important protein. chaperone systems used to suppress protein aggregation186 and Many of the limitations arising from the expression of DnaK principally mediates protein disaggregation in E. coli with eukaryotic proteins in prokaryotes can be overcome by using the assistance of particular co-chaperones.189 eukaryotic expression systems, such as yeast, insect cell lines or The mechanisms used by molecular chaperones to improve mammalian cells.198 However, recent advances in molecular the yields of recombinant protein are currently the subject of biology, such as the formation of functionally important intensive research. Thomas and Baneyx187 used a fusion protein disulphide bridges in E. coli mutant strains, are making E. coli a as a model to study inclusion body formation and concluded more attractive option for recombinant-protein production.183 that these bodies resulted from the aggregation of unchaperoned Improved E. coli strain engineering should enhance future recombinant protein and that chaperones were unable to prospects of eukaryotic protein production.183 Ahn and Yun194 disaggregate inclusion bodies. However, previous research were responsible for one of the earliest reports on the introduc- demonstrated the active participation of the DnaK chaperone tion of mammalian chaperones into bacteria to help generate machinery in disaggregating protein aggregates.190,191 Ben-Zvi soluble mammalian proteins. This appears to be part of a trend and Goloubinoff189 proposed that the ability of molecular involving the co-expression of molecular chaperone systems chaperones to solubilize and refold protein present in aggre- that are homologous to the recombinant protein of interest. gates is in fact a novel function; they critically reviewed the roles played by molecular chaperones as well as cooperation of Hsp70 Current chaperone research at Rhodes University and with other chaperones during disaggregation. future perspectives Numerous publications have demonstrated that the co- We have been studying the mechanism by which co-chaperone expression of molecular chaperones from E. coli increased the proteins regulate the function of the major molecular solubility of particular recombinant proteins. A list of some early chaperones, Hsp70 and Hsp90, in prokaryotic, eukaryotic and examples is given in a review by Thomas et al.186 Molecular parasitic systems. We have conducted extensive work on the chaperones from E. coli have also been used to improve the Hsp70/Hsp90 organizing protein, Hop, which is responsible yields of industrially important enzymes. D-hydantoinase and for bringing Hsp90 into contact with Hsp70-client protein Rhodes Centenary South African Journal of Science 100, November/December 2004 673

complexes in the cytosol of eukaryotic cells. We have shown that While these data suggest that parasitic chaperone systems the interaction of Hop with Hsp70 requires its N-terminal are different from those of their human hosts, only a complete tetratricopeptide repeat domain (TPR1),199–202 but Odunuga characterization of the parasitic system (including structural et al.203 determined that this domain is not sufficient and may data) will enable researchers to make a thorough evaluation of require other domains elsewhere on Hop for complete func- their potential as targets for anti-parasitic drug development. tional association with Hsp70. Studies by Longshaw et al.204,205 The expression of bacterial dnaK genes is typically upregulated and Odunuga et al.206 highlighted the importance of Hop as not during heat shock or starvation; however, the Myxococcus just an adaptor for Hsp70 and Hsp90, and concluded that it may xanthus dnaK homologue was not regulated by heat shock but move between the cytosol and the nucleus and play an impor- rather constitutively expressed during vegetative growth.213 The tant role in the folding and regulation of nuclear transcription characterization of further Hsp70 and Hsp40 homologues will and DNA replication machinery. improve our understanding of specialized functions, and the Further structural studies are needed to shed light on the selec- substrate specificity or substrate range of these chaperone tivity of the interactions between Hsp40 proteins and their families. Hsp70 and Hsp40 chaperone partnerships from novel Hsp70 partner proteins.207 We are particularly interested in the sources are steadily being isolated and characterized, thereby specificity determinants/features of the Hsp40 proteins. The contributing to our general understanding of the mechanism of specificity determinants and other features of the interaction action of this chaperone machine. between an Hsp40 and an Hsp70 may reside at the level of (i) dif- The Hsp70s from the African coelacanth (Latimeria chalumnae) ferential expression, (ii) compartmentalization (in eukaryotes), are examples of novel Hsps being investigated by our group.214 or (iii) structural features such as the J-domain of Hsp40. Modisakeng et al.214 have isolated and analysed the genes encod- Hennessy et al.208,209 have been investigating the levels of conser- ing both an inducible and constitutive form of Hsp70 from this vation and functional significance of J-domain residues. Our fish. The regulation of gene expression and the genomic struc- studies suggest that there are certain key residues on helix II of ture or arrangement of fish hsp70 genes have been studied in the J-domain of Hsp40 that are important for general binding af- detail for only a limited number of species.215 In addition, fish are finity to Hsp70, and that the specificity determinant appears to quickly becoming a model organism in which the expression of be located on the other helical surfaces, especially that of helix IV. Hsp70s can be investigated, due to the varying range of stresses Molecular chaperones provide cytoprotection in numerous in their natural habitat.215 pathological states, and their inhibition can be used to fight Research has shown that chaperones can be used to improve cancer.210 The inhibition of chaperone activity in the body has fo- the refolding of proteins recovered from inclusion bodies or cused on Hsp90 due to the specificity and permeability of its in- they can be expressed with the protein to limit inclusion body hibitors, and the role it plays in the folding of cell cycle kinases.210 formation. The use of specialized chaperones during the recom- We are particularly interested in elucidating the precise mecha- binant production of an aggregation-prone target protein is a nism by which Hop delivers Hsp90 to Hsp70-kinase complexes. growing area of research in protein biotechnology. Assuming This knowledge would greatly facilitate the development of that molecular chaperones are evolutionarily matched to their Hop and Hsp90 inhibitors specific for Hop-Hsp90 complexes in substrate proteins, the use of molecular chaperones that are cancer cells. One Hsp90 inhibitor that demonstrated anti-tumour from the same source as a recombinant protein could potentially activity, 17-AAG, was in phase I clinical trials in 2002.211 Drugs tar- be better suited to the refolding of such a protein within a geting other chaperone proteins have not yet been identified.211 heterologous system. We propose that the properties of the The range of Hsp90 substrates is expanding as well as the roles of particular target protein need to be critically evaluated when Hsp90 co-chaperones, suggesting that the chaperone function is choosing the chaperones to aid in the formation of functionally more complex than originally believed.212 Structural require- active protein. For example, the DnaK/DnaJ/GrpE chaperone ments for substrates to associate with Hsp90 also need to be machinery of a thermophilic bacterium could be utilized to determined. It is likely that there are human diseases still to be improve the heterologous over-production of proteins from the identified as being linked to dysfunctional chaperones.125 same thermophile.217 The agricultural and biotechnological The role of molecular chaperones in the development and importance of Agrobacterium tumefaciens prompted us to isolate survival of malarial and trypanosomal parasites is poorly under- and characterize its molecular chaperone machinery. Hennessy stood, and we have only recently made a contribution in this et al.209 and Boshoff et al.216 have isolated and characterized area.160,177 Interestingly, while the malarial and trypanosomal the DnaJ and DnaK proteins from A. tumefaciens, and we are Hsp70s (PfHsp70 and TcHsp70) show considerable sequence currently investigating the application of these proteins for the and predicted structural similarities to bacterial and mammalian improved heterologous over-production of other A. tumefaciens Hsp70s, there appear to be certain distinct functional differ- proteins of biotechnological importance. ences. In our studies by Matambo et al.,160 PfHsp70 was found to As the understanding of protein folding and the role of molec- have a slightly higher basal ATPase activity and turnover rate ular chaperones increases, the application of chaperone systems than human Hsp70, but a much lower affinity for ATP. in medicine and protein biotechnology will start to escalate. Intracellular ATP concentrations range from 2 to 4 mM, suggest- Indeed, more X-ray crystallographic and nuclear magnetic ing that PfHsp70’s Km of 616.5 µM will make it particularly sensi- resonance structures of chaperones and chaperone complexes tive to fluctuations in the level of free ATP in the cytosol, and that need to be generated before the full potential of chaperone its regulation by co-chaperones will be somewhat different from applications is realized. that of human Hsp70. TcHsp70 and Tcj2 appear to form a We are grateful for funding received from Rhodes University through the chaperone partnership, and Tcj2 is functionally equivalent to the Joint Research Committee. We also acknowledge national (National Research essential yeast Hsp40 protein, Ydj1. In our studies by Edkins Foundation and Medical Research Council) and international (Wellcome Trust and 177 et al., TcHsp70 was determined to have a high basal ATPase Volkswagen Foundation) support. activity that was only weakly stimulated by Tcj2, suggesting that 1. Fink A.L. (1999). Chaperone-mediated protein folding. Physiol. Rev. 79, ATP hydrolysis and/or nucleotide exchange may not be as rate 425–449. limiting as has been found for human Hsp70. 2. Anfinsen C.B. (1973). Principles that govern the folding of protein chains. 674 South African Journal of Science 100, November/December 2004 Rhodes Centenary

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