Chapter 8 1

Molecular Chaperones: Structure-Function 2

Relationship and their Role in Protein Folding 3

Bhaskar K. Chatterjee, Sarita Puri, Ashima Sharma, Ashutosh Pastor, 4 and Tapan K. Chaudhuri 5

Abstract During heat shock conditions a plethora of proteins are found to play a 6 role in maintaining cellular homeostasis. They play diverse roles from folding of 7 non-native proteins to the proteasomal degradation of harmful aggregates. A few 8 out of these heat shock proteins (Hsp) help in the folding of non-native substrate 9 proteins and are termed as molecular chaperones. Various structural and functional 10 adaptations make them work efficiently under both normal and stress conditions. 11 These adaptations involve transitions to oligomeric structures, thermal stability, 12 efficient binding affinity for substrates and co-chaperones, elevated synthesis during 13 shock conditions, switching between ‘holding’ and ‘folding’ functions etc. Their 14 ability to function under various kinds of stress conditions like heat shock, cancers, 15 neurodegenerative diseases, and in burdened cells due to recombinant protein pro- 16 duction makes them therapeutically and industrially important biomolecules. 17

Keywords Chaperone assisted folding · Heat shock · Molecular chaperones · 18 Protein folding · Structure-function of chaperones 19

Abbreviations 20

ACD α-crystallin domain 21 ADP Adenosine di-phosphate 22 ATP Adenosine tri-phosphate 23 CCT Chaperonin containing TCP-1 24 CIRCE Controlling inverted repeat of chaperone expression 25

Bhaskar K. Chatterjee, Sarita Puri, Ashima Sharma, and Ashutosh Pastor authors are equally contributed. B. K. Chatterjee · S. Puri · A. Sharma · A. Pastor · T. K. Chaudhuri (*) Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, HauzKhas, New Delhi, India e-mail: [email protected]

© Springer International Publishing AG 2018 181 A. A. A. Asea, P. Kaur (eds.), Regulation of Heat Shock Protein Responses, Heat Shock Proteins 14, https://doi.org/10.1007/978-3-319-74715-6_8 182 B. K. Chatterjee et al.

26 CNX Calnexin 27 CRT Calreticulin 28 CS Citrate synthase 29 ER Endoplasmic reticulum 30 ERAD Endoplasmic reticulum associated degradation 31 FRET Fluorescence energy resonance transfer 32 HOP HSP90/HSP70 organizing protein 33 HSC70 Heat shock cognate 34 HSEs Heat shock elements 35 HSFs Heat shock response and specific transcription factors 36 Hsp Heat shock proteins 37 HSP Heat shock protein family 38 HSR Heat shock response 39 MalZ Maltodextrin glucosidase 40 NAC Nascent chain associated complex 41 NEF Nucleotide-exchange factors 42 NTD n-terminal domain 43 PBD Peptide binding domain 44 PPIase Peptidyl-prolyl isomerases 45 PTP Permeability transition pore complex 46 RAC Ribosome associated complex 47 RuBisCO Ribulose-1,5-bisphosphate oxygenase-carboxylase 48 SHR Steroid hormone receptors 49 sHsp Small heat shock proteins 50 sHSP Small heat shock protein family 51 TF Trigger factor 52 TPR Tetratricopeptide 53 TRiC TCP-1 ring complex 54 UPR Unfolded protein response pathway

55 8.1 Introduction

56 Living systems respond to threatening conditions at multiple levels in their quest for 57 survival. It may in the form of a fight or flight response, which is a result of any 58 imminent physical threat either to an organism or their inner homeostasis. For 59 example the temperature, ionic and sugar balance are regulated within a fixed range 60 in our bodies and are probably optimized by evolutionary mechanisms. Similarly, 61 homeostasis is also maintained at the cellular level and maintaining such a balance 62 is imperative for the survival and efficient functioning of the cell. One of the major 63 homeostasis mechanisms operating at the cellular level is the protein homeostasis, 64 commonly referred to as proteostasis (Balch et al. 2008). Starting with maintaining 65 the structural organization of a cell to catalysing various metabolic reactions; from 66 the transport of macromolecules within and across cells to various recognition and 8 Molecular Chaperones in Cellular Stress Response 183 immune functions, proteins play vital roles in our bodies and are regarded as the 67 actual workhorses of the cells. Proteins undergo various post-translational modifica- 68 tions and move through trafficking pathways before they are ready to take up their 69 function. However, acquiring their specific three-dimensional structure supersedes 70 all this because only in their specific structural forms can they undertake the func- 71 tion they are meant to carry out. In this chapter, we shall discuss the cellular machin- 72 ery responsible for maintaining proteins in their functional state during stress 73 conditions; specifically focusing on the Hsp that assist in the folding and refolding 74 of misfolded and aggregation prone proteins. 75

8.2 What Is Stress Response? 76

The stress response can be defined as our involuntary defense reaction to threaten- 77 ing conditions. This may occur at multiple levels as a response to a different range 78 of conditions. At a cellular level, the response to any alarming condition, like a 79 chronic change in the environment away from normal conditions (which may inter- 80 fere with the physiological functioning of the cells and cause damage to nucleic 81 acids and proteins) can be identified as a stress response (Kültz2004 ). While at the 82 organism level our adaptive responses are driven by hormonal changes (Charmandari 83 et al. 2005), the cellular response to damages occurring at a molecular level involves 84 a cascade of pathways, and molecules that work in cohort to bring the cells back to 85 their normal functional state. Various kinds of stress at the cellular level include 86 oxidative, heat, radiation and nutrient deprivation. The major consequences of these 87 stress are DNA damage, loss of cellular signalling, protein unfolding, misfolding, 88 aggregation, proteolysis, cellular necrosis and apoptosis. The cellular stress response 89 may be of a protective nature where the cell can defend and restore its normal func- 90 tioning, or of a destructive nature where the conditions are beyond the cell’s ability 91 to repair. The type, level, and duration of stressful conditions may ultimately deter- 92 mine the fate a cell. During stress conditions, proteins are misfolded due to changes 93 in the overall energy landscape. This causes loss of protein function and the accu- 94 mulation of misfolded proteins in the form of toxic aggregates. The protective stress 95 response for proteins includes pathways of the heat shock response and the unfolded 96 protein response (UPR); the destructive response pathways include apoptosis, 97 necrosis or autophagy (Fulda et al. 2010). The DNA damage response consists of 98 multiple, complex pathways that restore genomic integrity. These include the base 99 excision repair, nucleotide excision repair, and non-homologous end joining 100 (Kourtis and Tavernarakis 2011). The oxidative stress response helps the cell cope 101 with the reactive oxygen species, maintain redox homeostasis; and a number of 102 enzymes like superoxide dismutase and non-enzymatic antioxidants are involved 103 (Trachootham et al. 2008). In this chapter, we shall mainly focus on the diverse 104 mechanisms governing heat shock response and the various factors that are involved 105 in mediating such a response. 106 184 B. K. Chatterjee et al.

107 8.3 Heat Shock Response

108 The heat shock response was one of the earliest explored stress response mecha- 109 nisms that was initially observed in Drosophila as changes in puffing patterns of 110 salivary gland chromosomes (Ritossa 1996), followed by changes in gene expres- 111 sion patterns after heat treatment (Tissiéres et al. 1974; Hightower 1991). The heat 112 shock response imparts thermo-tolerance to the cells and protects them when they 113 are stressed due to prolonged exposure to heat. This response is activated by the rise 114 of a few degrees in temperature from the normal dwelling temperature of the organ- 115 ism. The regular transcription and translation processes in the cells are halted during 116 heat shock response, and specific transcription factors (HSFs) which selectively 117 enhance expression of a set of proteins having protective functions are activated. 118 However, even during normal conditions these HSFs play important roles in differ- 119 entiation and development of the organisms (Morimoto et al. 1996). The HSF1 120 predominantly regulates the heat shock responses, and is itself regulated by its 121 interactions with heat shock proteins HSP70 and HSP90 (Pirkkala et al. 2001). The 122 ability to activate transcription and bind to DNA are uncoupled in HSF1 imparting 123 a higher degree of regulation. The HSF1 exists as a monomer or in complex with 124 Hsp under normal conditions. During heat shock, HSF1 homotrimerizes and under- 125 goes hyperphosphorylation which leads to its activation (Cotto et al. 1996). These 126 HSFs facilitate the overexpression of Hsp by binding to cis acting sequences on the 127 genome known as heat shock elements (HSEs). HSP are commonly known as 128 molecular chaperones for their role in assisting proteins to acquire their native struc- 129 tures. Hsp prevent heat induced denaturation and aggregation of proteins, facilitate 130 the proteins to fold, and assist in the refolding of already denatured proteins 131 (Lindquist 1986). The Hsp play an active role in facilitating the degradation of 132 proteins that are unable to fold in order to maintain protein homeostasis and thus 133 promote cell survival.

134 8.4 Cellular Components Providing HS Response

135 Heat shock response in cells is mediated by concerted actions of heat shock factors 136 (HSF), heat shock elements (HSE) and Hsp. The heat shock factors as described 137 above are transcription factors activated during a heat shock. Gram negative bacte- 138 ria E.coli has a specific sigma factor 32 σ( 32), coded by the rpoH gene, which is a 139 heat shock promoter specific subunit of RNA polymerase. Theσ 32 is a positive 140 regulator and is suppressed by DnaJ during normal conditions (Bukau 1993). The 141 gram-positive bacteria B. subtilis has a negative regulator HrcA which binds to neg- 142 atively acting CIRCE elements (controlling inverted repeat of chaperone expression). 143 Folding of HrcA is mediated by GroE chaperones. During heat shock response, 144 HrcA doesn’t fold due to the unavailability of free GroE chaperones and hence 145 the negative regulation of Hsp is switched off (Hietakangas and Sistonen 2006). 8 Molecular Chaperones in Cellular Stress Response 185

A majority of the eukaryotes have either one or multiple of the four heat shock fac- 146 tors HSF1-HSF4; the condition being different in plants. Arabidopsis has 21 genes 147 encoding various heat shock factors, divided among three classes and 14 different 148 groups. The plant HSFs are induced and expressed during heat shock (Hietakangas 149 and Sistonen 2006). Unlike plants, the transcription factors in animals are like 150 HSF1. They are constitutively expressed but functionally repressed during normal 151 conditions by HSP70 and HSP90 (Shi et al. 1998). The DNA binding domain of 152 HSF recognizes the HSE in a major grove of the DNA double helix. HSEs are highly 153 conserved and contain inverted repeats of nGAAn (e.g. nTTCnnGAAnnTTCn) 154 (Amin et al. 1988; Akerfelt et al. 2010). There might be multiple HSEs in the pro- 155 moter region of a heat shock gene and hence multiple HSFs can bind simultane- 156 ously in a cooperative manner. 157 Expression of Hsp is invariably upregulated during heat shock irrespective of the 158 difference in their mechanism of action, as discussed above. Overall, Hsp or molec- 159 ular chaperones, are one of the seven different classes of proteins to be overex- 160 pressed during stress response and were the earliest discovered components of the 161 heat shock response. The second class comprises components of the proteolytic 162 system which helps in the clearance of misfolded and aggregated proteins. The pro- 163 teolytic machinery of the proteasome has a similar structural organization amongst 164 different organisms differing only in their subunit compositions. The protein degra- 165 dation machinery requires the translocation of misfolded or partially unstruc- 166 tured intermediates between cytosol, ER and Golgi in eukaryotes and multiple HSP 167 members coordinating protein folding and degradation in different cellular com- 168 partments. The third class of proteins help in DNA and RNA repair, like counteract- 169 ing the heat induced methylation of RNA. The fourth category comprises enzymes 170 of the metabolic pathways which reorganize the energy supply of the cell. The fifth 171 category includes kinases and transcription factors that further initiate or inhibit 172 expression cascades to support the stress response. The sixth class of proteins main- 173 tain the integrity of the cytoskeleton. Transport proteins and membrane-modulating 174 proteins make up the seventh class. All these proteins function together to respond 175 to the heat stress conditions (Richter et al. 2010). However, we will be discussing 176 the molecular chaperones in detail in the following sections. 177 The Hsp are divided into multiple families based primarily on their size. These 178 different classes of molecular chaperones and their localization in different cellular 179 compartments provide a great degree of organizational control and distribution of 180 roles to execute particular functions within the overall cascade of stress response. 181 HSP60, HSP70, HSP90, HSP100, and small HSP are found across all organisms 182 and are known to have high similarities within their classes among different organ- 183 isms. One similarity among all chaperones is that they recognize the hydrophobic 184 surfaces of proteins which are increasingly exposed among misfolded and unfolded 185 proteins, and function to either fold the protein (foldases) or mask the hydrophobic 186 regions to prevent aggregation (holdases). Despite the name suggesting a partic- 187 ular function, Hsp play significant roles in multiple stress response pathways in all 188 organisms, including oxidative stress in all organisms and drought and osmotic 189 stress in plants. The roles of molecular chaperones have been observed in the correct 190 186 B. K. Chatterjee et al.

191 folding of a newly translated protein, refolding of misfolded proteins, disaggrega- 192 tion, translocation, and in the degradation of proteins. HSP60, HSP70, and HSP90 193 are ATP dependent chaperones which actively support protein folding. The small 194 HSP and other chaperones simply prevent the misfolding of proteins. The heat 195 shock factors are regulated by molecular chaperones like HSP70 and HSP90, 196 thus proving their importance in the overall heat shock response of the cell. HSP70 197 helps in the translocation of proteins to cellular compartments like ER and also facil- 198 itates retrotranslocation (The reverse movement of the protein from ER to the cyto- 199 sol, where it can be degraded by the proteasomal machinery) during ER associated 200 degradation. HSP90 clients include many kinases and steroid receptors, which help 201 in regulating a multitude of functions through signaling pathways. HSP104 is 202 known to disaggregate proteins and redirect them to correct refolding pathways. 203 The widespread presence of chaperones in almost all cellular components where 204 protein folding occurs, proves that they are key players in the heat shock response 205 machinery. Almost all cellular proteins might have to interact with molecular chap- 206 erones at least once in their lifetimes, be it during synthesis, folding, targeting, or 207 degradation.

208 8.5 Role of Chaperones in Mediating Cellular Heat Shock 209 Response

210 Cells grow optimally within a narrow range of temperature, pH, and other physio- 211 logical conditions, but adapt to moderate deviation from such conditions. One of the 212 most well studied cellular adaptations is the heat shock response (HSR) (Guisbert 213 et al. 2004). During heat shock conditions, many cellular proteins work to either 214 rescue the cells from dying, or trigger apoptosis when the damage incurred is irre- 215 versible. These proteins are referred to as heat shock proteins (Hsp) (Herman and 216 Gross 2000). Few out of several such Hsp protect proteins from undergoing aggre- 217 gation, unfold aggregated proteins to make them folding compatible and refold 218 damaged proteins. These proteins are termed as chaperones (Morimoto et al. 1994). 219 Molecular chaperone is a major class of protein found at all levels of cellular 220 organizations ranging from bacteria to humans. They have variable organization 221 and function depending on the cellular location and complexity of the organism. 222 Bacterial chaperone proteins are found only in the cytosol as they are not 223 compartmentalized, but in case of higher organisms, these are also localized in 224 mitochondria, endoplasmic reticulum, and chloroplasts. Structural and functional 225 organizations of chaperones are evolutionally conserved within the same kingdom 226 but vary between them. On the basis of gross molecular weight, the major chaper- 227 one are classified as: HSP60 (GroEL/GroES, Cpn60/Cpn10, HSP60/HSP10, Tric/ 228 CCT, Thermosome), HSP70 (DnaK, DnaJ, GrpE, HSP70), HSP90 (HSP90, TRAP, 229 HtpG), HSP100 (ClpA, ClpB, ClpP), sHSP and Trigger Factor (Georgopoulous 230 et al. 1994; Gross 1996). The other important chaperone proteins playing a role 8 Molecular Chaperones in Cellular Stress Response 187 in heat shock are Prefoldin, Calnexin / Calreticulin, GRP94, GRP170, AAA 231 ATPasesPPIases, PDIases, NAC (Nascent polypeptide Associated Complex), CasA 232 and HtpX (Rani et al. 2016). 233

8.5.1 Small Heat Shock Proteins (sHsp) 234

Most organisms have a well-developed sHSP system, which help in their protection 235 from the thermal, osmotic and salt stresses (Jakob et al. 1993). sHsp have subunit 236 molecular masses of 12–43 kDa. The common feature of all sHSP is the presence of 237 a highly conserved stretch of 80–100 amino acids in their C-terminus termed as the 238 “α-crystallin domain” (ACD). It is flanked on both side by less conserved N-terminal 239 domain (NTD) and a C-terminal extension (Kappé et al. 2003; Franck et al. 2004; 240 Kriehuber et al. 2010). In E.coli major sHsp are IbpA and IbpB. Under normal cel- 241 lular conditions they help in aggregation prevention and folding of substrate pro- 242 teins in an ATP independent manner. During stress conditions, along with 243 an increased expression, these proteins undergo drastic conformational rearrange- 244 ments in order to bind to the misfolded proteins and prevent cellular aggregation 245 (Mani et al. 2016). HSP31 of E.coli functions as a holding chaperone. It cooperates 246 with the DnaK-DnaJ-GrpE system in managing protein misfolding during stress 247 conditions (Mujacic et al. 2004). In the pathogenic Halobacterium sp., sHSP1, 248 HSP-5 and sHSP2 impart protection from thermal stress, solar radiation and high 249 salt concentration (Vanghele and Ganea 2010). HSP20 of Mycobacterium tubercu- 250 losis protects them from macrophage induced stress response and helps in solubili- 251 zation of heat induced aggregates (Vanghele and Ganea 2010). 252 In yeast, sHsp HSP26 and HSP42 together, add an additional layer of protection 253 against a cellular assault like heat shock. Both HSP26 and HSP42 are poorly 254 expressed during exponential growth, but their expression increases 10-fold under 255 heat stress suggesting the dominant role they play in a thermally stressed cell 256 (Haslbeck et al. 2004a). HSP26 exists as a 24-mer under normal conditions, acts 257 like a holdase for damaged or misfolded proteins and transfers client proteins to the 258 HSP70 chaperone machinery during heat shock (Haslbeck et al. 2004a, b; 1999). 259 During heat shock, the 24-mer of HSP26 gets reversibly dissociated into dimers. 260 This dimeric form then interacts with the unfolded polypeptides and eventually 261 forms a larger complex, to be presented to chaperones capable of folding the sub- 262 strate (Stromer et al. 2004). HSP26 also interacts with aggregated proteins, making 263 them accessible to the HSP104 chaperone (Glover and Lindquist 1998). HSP42 264 oligomer is a symmetric assembly of dimers organized into two hexameric rings. 265 HSP42 binds with 30% of total yeast cytosolic proteins (Haslbeck et al. 2004a). It 266 is a more effective chaperone than HSP26, as a higher HSP26 to substrate ratio is 267 needed to prevent aggregation (Haslbeck et al. 2004a). HSP12 exhibits low sequence 268 homology to the sHSP superfamily and is structurally and functionally distinct, as 269 it exists exclusively as a monomer (Welker et al. 2010). Like the other sHsp, HSP12 270 188 B. K. Chatterjee et al.

271 is weakly expressed in exponentially growing cells but overexpressed (100-fold) 272 during heat shock (Welker et al. 2010). 273 In plants, there are distinct gene families for sHSP found in different organelles 274 and a total of 6 families have been classified. The HSP17.6 and HSP17.9 reside in 275 the cytoplasm, HSP21 in the chloroplast, HSP22 in the ER, HSP23 in the mitochon- 276 dria and HSP22.3 in the cell membrane (Wang et al. 2004). They have been sug- 277 gested to be involved either in maintaining the structure of a heat stressed cell 278 (Lindquist and Craig 1988) or to protect the photosynthesis machinery during heat 279 shock (Schuster et al. 1988; Chen et al. 1990). Genetically modified plants with 280 higher thermo-tolerance have been designed by constitutive upregulation of 281 sHSP. For example, the HSF3 gene of Arabidopsis thaliana was modified to express 282 it in non-heat shock conditions and was shown to increase the basal thermotolerance 283 of the plant (Prändl et al. 1998). Overexpression of HSP17.6 results in a higher 284 tolerance to drought and salinity in Arabidopsis thaliana, while the natural elevated 285 gene expression is observed in case of heat shock (Sun et al. 2001). 286 In humans, Group I sHsp consists of HSP27, HSP20, HSP22, and αB-crystallin. 287 They are found in various tissues and are heat inducible. Group II sHsp consists of 288 HSPB9, HSPB10, HSPB3, HSPB2 and α-crystallin, and they are involved in cell 289 differentiation and are restricted to certain tissues (Taylor and Benjamin 2005). 290 HSP27 forms oligomeric species when subjected to higher temperatures and this 291 results in increased chaperone activity (Bakthisaran et al. 2015). They predomi- 292 nantly function as ‘holdases’, keeping the substrates in a folding competent globular 293 state that can later be presented to ‘foldases’ like HSP60/10 or HSP70 to make them 294 functional (Eyles and Gierasch 2010). Under conditions of heat stress, they also 295 prevent aggregation by binding to late unfolding intermediates and keep them in a 296 stable, soluble complex. sHSP may also be involved in the transient/reversible reac- 297 tivation of early unfolded intermediates and this process may be ATP dependent, 298 although no ATP hydrolysis has been observed (Rajaraman et al. 2001). ATP bind- 299 ing is thought to trigger a conformational change that aides Hsp to release their 300 refolding-competent substrates (Muchowski et al. 1999).

301 8.5.2 The Chaperonin System (HSP60)

302 Chaperonins are double ring complexes of 800–900 kDa which help in the folding 303 of many cellular proteins under normal and stress conditions (Spiess et al. 2004; 304 Hemmingsen et al. 1988; Vabulas et al. 2010). These are further classified as group 305 I and group II chaperonins (Horwich et al. 2007). 306 In bacteria and symbiotic organelles like mitochondria and chloroplasts, Group I 307 chaperonins (cpn60) are found. These chaperonins are termed as GroEL in E.coli, 308 mtHsp60 in mitochondria and Rubisco binding protein in chloroplast (Figueiredo 309 et al. 2004). These require co-chaperonin GroES or HSP10 to function in prokary- 310 otic and eukaryotic cells respectively. Under normal cellular conditions, GroEL/ES 311 is constitutively expressed in bacterial cytoplasm and helps in the folding of 8 Molecular Chaperones in Cellular Stress Response 189

­substrate proteins in an ATP dependent fashion. Under environmental stress condi- 312 tions, the expression of these proteins increase by 15–20% (Georgopoulos et al. 313 1994; Gross 1996) and also leads to a few structural and functional modifications of 314 the chaperone system, which enable them to fold or hold the aggregation prone 315 proteins. One such modification is the phosphorylation of the double ring which 316 mediates substrate folding in an ATP independent manner (Sherman and Goldberg 317 1994). GroEL also acts as a holding chamber for substrate proteins during thermal 318 stress and helps them regain their folding function once normality is restored 319 (Carrascosa et al. 1998). Mitochondrial Hsp60, with the help of its co-chaperonin 320 HSP10 helps in the folding and assembly of imported proteins inside the matrix of 321 mitochondria. In HSP60 conditional mutants, aggregates accumulate and are unable 322 to assemble into functional complexes. The plastid HSP60 chaperonin found in 323 chloroplasts consists of two different subunits which make them different from 324 other members of the HSP60 family (Levy-Rimler et al. 2002). These two distinct 325 subunits known as CPN60α and CPN60β share about 50% sequence identity 326 (Boston et al. 1996). The tetradecameric structure consists of α7β7oligomers where 327 the assembly of α7 is dependent on the β7 homo-oligomer, thus forming two homo- 328 geneous rings, and the oligomers having a seven fold symmetry (Waldmann et al. 329 1995). The chloroplast co-chaperonin can bind to both GroEL and ch-CPN60 and 330 assists in the folding of proteins in both cases. Its structure however, is markedly 331 different from GroES and contains two GroES like domains connected by a short 332 linker region. Some reports have confirmed the presence of 10 kDa CPN10 and 333 20 kDa dimer like CPN20 existing simultaneously in the chloroplasts (Hill and 334 Hemmingsen 2001; Levy-Rimler et al. 2002). 335 Group II chaperonins are found in archaea (Thermosome) and the cytoplasm of 336 eukaryotes (TCP/CCT) (Ditzel et al. 1998; Leitner et al. 2012). They do not require 337 the co-factor HSP10 as they have specialized α-helical extensions in their apical 338 domain that function as a built-in lid (Vabulaset al. 2010). Archaeal Group II chap- 339 eronins (Thermosome) consists of two stacked octameric rings with two different 340 kinds of subunits α & β (Klumpp et al. 1997). The mechanism of folding of non-­ 341 native protein substrates is similar to GroEL and involves binding of the substrate at 342 the apical domain, followed by ATP hydrolysis and release of the folded substrate 343 from the cavity (Lopez et al. 2016). During stress conditions, Group II chaperonins 344 form a large octadecameric β complex which is more efficient in substrate binding. 345 It also helps in membrane stabilization of archaeal cells during stress conditions 346 (Chaston et al. 2016). The group II chaperonins in the eukaryotic cytosol are known 347 as TRiC (TCP-1 ring complex) or CCT (chaperonin containing TCP-1) and like 348 their archaeal counterparts have eight or nine rings, each containing eight paralo- 349 gous subunits (Frydman 2001). The general domain structure of the group II chap- 350 eronins is akin to GroEL (Ditzel et al. 1998). The closing and opening of these 351 segments to encapsulate the substrate in the TriC/CCT cavity is ATP dependent 352 (Meyer et al. 2003a). TriCs interact functionally with the co-chaperone prefoldin 353 (Vainberg et al. 1998; Siegert et al. 2000) and HSP70 (Langer et al. 1992), which 354 serve to transfer substrates to this chaperonin. 355 190 B. K. Chatterjee et al.

356 8.5.3 HSP70

357 HSP70 family is involved in a multitude of functions in all organisms and are found 358 in various cellular compartments. HSP70 facilitates translocation, protein import, 359 and signal transduction along with assisting in the refolding of substrate proteins 360 and preventing their aggregation (Frydman 2001; Miemyk 2017). HSP70 consists 361 of two domains, a highly conserved 44 kDa N-terminal ATP binding domain, and a 362 15 kDa C-terminal peptide binding domain (PBD) which consists of a β-sandwich 363 motif and an α-helical lid segment (Vabulas et al. 2010; Yu et al. 2015). In eukary- 364 otes, under normal conditions, HSP70 exist as complexes with either HSP90 or 365 co-chaperones like HSP40 and others, and is known as Heat Shock Cognate 70 366 (HSC70). The constitutively expressed HSC70 assist in the folding and transloca- 367 tion of newly synthesized proteins during normal conditions (Hartl et al. 2011). 368 Under heat stress conditions, the stress-inducible HSP70 isoforms are over-­ 369 expressed. This is achieved by its interaction with HSF, which transcriptionally 370 activates several heat shock genes including that of HSP70. HSP70s function is 371 generally conserved in all organisms (Akerfelt et al. 2010). Nascent polypeptides 372 form the bulk of its clients and undergo chaperoning via the ATP-dependent reac- 373 tion cycle of HSP70 which is regulated by HSP40 and nucleotide-exchange factors 374 (NEF) (Kampinga and Craig 2010; Mayer 2010). The β-sandwich motif of the PBD 375 recognizes an extended, seven-residue hydrophobic patch of an aggregation prone 376 protein, especially when they are locally surrounded by positively charged residues 377 (Rudiger et al. 1997). The binding of the peptide is ATP-dependent and is regulated 378 by a conformational change in the β-sandwich motif (Mayer 2010). In the ATP-­ 379 bound state, the lid adopts an open conformation resulting in a high binding affinity 380 for the peptide. Hydrolysis of ATP facilitates lid closure and is accelerated by 381 HSP40, leading to the peptide getting locked inside. Following the hydrolysis of 382 ATP, NEF binds to the ATPase domain and catalyses ADP-ATP exchange that result 383 in the opening of the lid and release of the substrate, presumably in their non-native 384 conformation. These intermediates might then undergo multiple rounds of binding 385 and release till they acquire their native conformation (Hartl et al. 2011). Under 386 stress conditions (apart from folding nascent polypeptides) HSP70 prevents aggre- 387 gation of non-native substrates by transiently shielding exposed hydrophobic seg- 388 ments and keeping them in a folding competent state, which may subsequently be 389 transferred to the chaperonin cage for completion of the folding process (Vabulas 390 et al. 2010). BiP, the HSP70 paralog in the ER, binds to unfolded regions of the 391 protein manifesting exposed hydrophobic residues and has an ATP dependent 392 mechanism of substrate binding and release, similar to its cytosolic counterpart. BiP 393 plays an active role in the Unfolded Protein Response pathway (UPR) and in ER 394 Associated Degradation (ERAD), both of which occur when misfolded proteins 395 start accumulating in ER. It does so by interacting with several co-chaperones that 396 assist in protein folding and quality control. One of them is Erdj3, an HSP40 397 paralog in the ER, which interacts with partially folded intermediates and presents 398 them to BiP. Another cohort is BAP, a NEF that plays a similar role as its cytosolic 8 Molecular Chaperones in Cellular Stress Response 191 counterparts, promoting ADP release and facilitating BiPs transition to the open 399 state (Ma and Hendershot 2004). 400 In yeast, SSA and SSB are subclasses of cytosolic Hsp70 that are constitutively 401 expressed, while SSA3 and SSA4 are known to be induced upon exposure to heat 402 shock (Santoro et al. 1998). Interaction with the chaperone Ydj1 (Hsp40), a homo- 403 logue of the bacterial DnaJ protein, accelerates the ATPase activity of Hsp70. KAR2 404 (ER Hsp70) is a yeast homolog of BiP protein which gets induced within 10 minutes 405 of heat shock (Normington et al. 1989). Kar2 is responsible for the translocation of 406 proteins across the ER membrane through co-translational as well as post-transla- 407 tional pathways (Brodsky et al. 1995). It is also involved in the retrograde transport 408 of defective non-functional substrates from the ER to the cytosol for proteosomal 409 degradation via ERAD, and thereby contributes to ER proteostasis (McCracken and 410 Brodsky 1996). The promoter region of the Kar2 gene contain unfolded protein 411 response elements (UPREs), and it gets induced when unfolded polypeptides start 412 accumulating in the ER lumen (Cox and Walter 1996). As is the case for human 413 cytosolic Hsp70, Kar 2 interacts with co-chaperones Hsp40/J proteins (Sec63, Scj1, 414 and Jem1) and nucleotide exchange factors (Sil1) (Nishikawa and Endo 1997; 415 Sadler et al. 1989; Schlenstedt et al. 1995). Sec63 acts as a Kar2 ATPase activator 416 (Lyman and Schekman 1995), while Scj1 functions to counter the misfolding of 417 proteins occurring due to lack of a carbohydrate modification (Silberstein et al.1998 ). 418 SSC1 and SSC 3 are the two mitochondrial Hsp70 chaperones which are involved 419 in the heat shock response (Wagner et al. 1994). Binding of the SSC1 to the unfolded 420 protein is critical for post-stress (Baumann et al. 2000). Mdj1 (J protein), the yeast 421 mitochondrial HSP70 cofactor, helps in preventing heat induced protein aggrega- 422 tion in mitochondria (Rowley et al. 1994). It is also involved in protein folding. 423 Furthermore, in addition to folding, its interaction with SSC1 also facilitates the 424 clearance of misfolded mitochondrial proteins. MGE1 is the only mitochondrial 425 NEF known to interact with, and therefore assist the Ssc1 chaperone action by pro- 426 moting the release of bound nucleotide (Wagner et al. 1994). 427 The HSP70 family in plants has been subdivided into 4 subgroups based on 428 C-terminal sequence motifs and their cellular localization. The cytosolic HSP70 429 contains the EEVD motif; the ER HSP70 contains the HDEL motif, the HSP70 430 molecules found in plastids have the PEGDVIDADFTDSK motif and those in mito- 431 chondria have a conserved PEAEYEEAKK motif (Guy and Li 1998). These motifs, 432 known as anchors, are identified by co-chaperones and are involved in substrate 433 binding through the assistance of co-chaperones (Freeman et al. 1995). Plant cells 434 are different in that they contain multiple (2-5) HSP70 family members within the 435 ER (Ray et al. 2016). In Arabidopsis, the total HSP70 family members are encoded 436 by about 18 genes, (Lin et al. 2001) and about 12 genes of the HSP70 family have 437 been identified in the spinach genome (Guy and Li 1998). This gives an idea about 438 the functional diversity of this chaperone family (Wang et al. 2004). Mechanisms by 439 which HSP70 mediates its cellular function have been difficult to study in plants 440 due to the poor survival of deletion mutants and the unavailability of efficient 441 inhibitors (Sarkar et al. 2013). In prokaryotes, the DnaK/DnaJ system belongs to 442 HSP70/HSP40 family of heat shock proteins. They function with the prokaryotic 443 192 B. K. Chatterjee et al.

444 NEF GrpE. DnaK is an ATP-dependent chaperone which requires DnaJ and GrpE 445 for substrate binding through its ATPase cycle. During stress conditions, DnaJ 446 undergoes certain functional modifications which stimulates the ATPase activity of 447 DnaK (~500 fold). During stress conditions, DnaK/DnaJproteins also interact with 448 ClpB and reactivate the inactivated proteins (Liberek et al. 1992). DnaK/DnaJ 449 system is absent in archaeal species except for a few halophiles, hence may not have 450 any particular role in alleviating archaeal stress. The functional analogs of DnaK/ 451 DnaJ system in archaea are prefoldin and GimC protein (Rani et al. 2016).

452 8.5.4 HSP90

453 HSP90 is another major player that functions downstream of HSP70 in the confor- 454 mational maturation and functional activation of several important classes of pro- 455 teins that actively take part in various cell signaling pathways. While the basal 456 HSP90 content is about 1% of the total cellular protein content, it may increase to 457 4–6% during the stress response (Young et al. 2001; Wegele et al. 2004). HSP90 is 458 usually present in the cytosol, but they may also be found in the ER, mitochondria 459 and plastids (Krishna and Gloor 2001). HSP90 functions as a dimer; the monomer 460 units covalently joined at their C-terminal domains via the dimerization domain. 461 The N-terminal domain binds and hydrolyzes ATP and is joined to the C-terminal 462 domain by a middle domain, which participates in substrate and co-chaperone bind- 463 ing. In all organisms HSP90 acts as a scaffold for several if not all protein folding 464 pathways and components. HSP90 dimer undergoes an ATP driven reaction cycle of 465 substrate binding and release achieved by the transition from an open nucleotide-­ 466 free to a closed ATP-bound state ‘committed to hydrolysis. HSP90 works in cohort 467 with multiple co-chaperones like HSP70, HOP, the J-domain proteins (HSP40) and 468 p23. These co-chaperones mediate the interactions between HSP90 and the sub- 469 strates in most of the cases (Li et al. 2012). 470 In humans, clients of this ~ 90Kda chaperone number over 200, which includes 471 kinases, nuclear receptors, transcription factors, telomerase and many other proteins 472 (Zhao et al. 2005; McClellan et al. 2007). Under stress conditions HSP90α is over- 473 expressed and shows marked increase in interactions with certain clients. HSP90α 474 has a higher potential for oligomerization and undergoes temperature-dependent 475 oligomerization above 45 °C, and denatured substrates like DHFR bind specifically 476 with these higher order oligomers (4-mer, 6-mer, and 8-mer). Oligomeric forms of 477 HSP90 display manifold higher affinity for denatured substrates as they themselves 478 undergo ‘unfolding', where their hydrophobic patches are exposed (Csermely et al. 479 1998). Like HSP70, HSP90 prevents irreversible denaturation of substrates like 480 luciferase and can act as ‘holdases’. When cells go back to the resting phase, these 481 substrates can be captured by ‘foldases’ and reactivated (Minami and Minami 482 1999). Tumour Necrosis Factor Receptor-Associated protein 1 is the mitochondrial 483 homologue of Hsp90 (Felts et al. 2000). It is a ~75 kDa protein (Chen et al. 1996) 484 which is structurally similar to its cytosolic counterpart, except the absence of an 8 Molecular Chaperones in Cellular Stress Response 193

MEEVD motif in the C-terminus that binds to co-chaperones like p23, indicating 485 that the regulation of TRAP-1 might be orchestrated by a different set of co-­ 486 chaperones in a yet unknown fashion (Felts et al. 2000). TRAP-1 functionally dif- 487 fers from its ER counterpart, GRP94 in that it adopts a closed conformation upon 488 ATP binding, but this alone is insufficient for commitment towards ATP hydrolysis 489 as the propensity to adopt an open confirmation even before ATP hydrolysis is 490 greater than its ATP hydrolysis rate. This kinetic partitioning essentially reduces the 491 turnover number of ATP and signifies that the dissociation of ATP is favoured to its 492 ATPase activity (Leskovar et al. 2008). Under heat shock conditions, its ATPase 493 activity increases by ~ 200 fold (Altieri et al. 2012). Although downstream targets 494 involved in this process is not fully understood, it is probably the chaperone 495 Cyclophilin D (a mitochondrial matrix PPIase), a key component of the Permeability 496 Transition Pore complex (PTP). Opening of the PTP is known to induce mitochon- 497 drial apoptosis, and by refolding Cyclophilin D in a closed PTP configuration 498 through its ATPase activity, TRAP-1 is perhaps able to mediate cell survival (Green 499 and Kroemer 2004; Kang et al. 2007). The proteins classified in HSP90 family 500 range from 80–94 kDa in size and about 70% sequence identity has been observed 501 between the plant and other eukaryotic counterparts of HSP90 (Lindquist and Craig 502 1988). The HSP90 family in Arabidopsis has seven members where AtHSP90–1 to 503 AtHSP90–4 are present in the cytoplasm, AtHSP90–5 is present in the plastids, 504 AtHSP90–6 in the mitochondria and AtHSP90–7 in the ER (Krishna and Gloor 505 2001; Wang et al. 2004).It has been observed in Arabidopsis that HSP90 is involved 506 in mediating the stress response pathways through its interactions with the heat 507 shock factor (HSF) (Yamada et al. 2007). Overall, studies carried out in plants sug- 508 gest important roles of HSP90 in many aspects of plant development and stress 509 response. HtpG is the Escherichia coli homolog of HSP90. It normally acts as a 510 holder chaperone that transiently maintains the nascent protein in a conformation 511 accessible to DnaK. During stress conditions, its expression increases 5–10-fold 512 and it binds to aggregated proteins with the help of ClpB and re-presents them to the 513 DnaK/DnaJ system (Thomas and Baneyx 2000). In yeast, HSP90 chaperone is 514 encoded by the Hsp82 gene, which is overexpressed under heat shock conditions 515 (Smith et al. 1991). Yeast HSP90 chaperone system is also involved in overcoming 516 the deleterious impact of heat shock on the cell surface (Imahi & Yahara 2000). 517

8.5.5 HSP100/Clp 518

The HSP100 family is primarily known for its unique function of remodeling pro- 519 tein complexes and disassembling protein aggregates, facilitating either refolding or 520 degradation of the aggregated proteins (Goloubinoff et al. 1999). The HSP100 are 521 hexameric ring structures belonging to AAA+ ATPase family (Burton and Baker 522 2005) with two defined classes based on distinct N and C-domains. The class I pro- 523 teins are ClpA, B, C, D, E having two AAA modules, whereas the class II proteins 524 ClpM, N, X, and Y have only one AAA module (Lee et al. 2007). In E.coli the major 525 194 B. K. Chatterjee et al.

526 HSP100 are ClpA, ClpB, ClpC, ClpX and ClpY. They help in the disassembly of 527 oligomers and aggregates during stress (Smith et al. 1999). HSP104 is the yeast 528 homolog of ClpB. It recognizes misfolded proteins within an aggregate, unfolds 529 them and ultimately delivers the substrates into various refolding pathways 530 (Schirmer et al. 1996). Together with HSP70 and HSP40, it resolubilizes and refolds 531 the substrates. HSP78 (a yeast mitochondrial aggregase), plays a role in the reacti- 532 vation of proteins damaged due to stress, thus imaprting thermotolerance (Janowsky 533 et al. 2006). It binds to misfolded polypeptides in the matrix and stabilizes them, 534 thereby preventing their aggregation (Schmitt et al. 1995). One of the major roles of 535 HSP78 is resolubilization of the Ssc1 (mtHsp70) chaperone which itself tends to 536 misfold during stress (Sichting et al. 2005). HEP1 (mtHsp70 escort protein) and 537 Pim1 (involved in mitochondrial proteolysis) are some of the other proteins in mito- 538 chondria which are involved in HSR (Sichting et al. 2005; Wagner et al. 1994). 539 HEP1 plays a complementary role to Hsp78 in maintaining functional SSC1 540 (Sichting et al. 2005). It assists Ssc1 to maintain its solubility and function during 541 stress (Sichting et al. 2005). Multiple HSP100 members exist in Arabidopsis; four AU1542 ClpB, two ClpC, and one ClpD. Although they are present at basal levels in cells 543 under normal conditions, an increased expression is observed during heat stress. 544 The cytosolic ClpB1 of Arabidopsis, known as AtHSP101 is present in high levels 545 during heat stress and imparts thermotolerance to plants (Glover and Lindquist 546 1998; Lee et al. 2007).

547 8.5.6 Other Chaperones

548 Many other chaperones also play a role in stress tolerance; e.g., PPIases, AAA 549 ATPases and so on. PPIase proteins include cyclophilins, FKBPs and parvulins 550 (Maruyama et al. 2004). They help in cis-trans proline isomerization in a polypep- 551 tide chain and help in the fast folding of kinetically trapped proteins. AAA ATPases 552 are mainly found in archaea and eukaryotes. The major AAA ATPase of archaea is 553 CDC48 and AMA which function as major proteasomal ATPases. These proteins 554 regulate proteasomal protein degradation in archaea (Forouzan et al. 2012). In yeast 555 and humans, most proteins that are translocated into the ER are N-glycosylated with 556 a branched glucose-3-mannose-9-N-acetylglucosamine-2 (Glc3Man9) glycan chain 557 (Helenius and Aebi 2004). This oligosaccharide moiety serves as a recognition sig- 558 nal for lectin-like chaperones calnexin (CNX) and calreticulin (CRT) (Daniels et al. 559 2003). CRT prevents thermal aggregation and promotes recovery of nonglycosyl- 560 ated substrates. This happens due to enhanced polypeptide binding property of CRT 561 under heat stress. CRT also forms oligomers under heat stress via certain conforma- 562 tional changes that occur in its C-terminal acidic domain. Oligomerization of CRT 563 and enhanced polypeptide binding are concomitant and explain how CRT acts as a 564 chaperone. CNX may act to recruit other chaperones like the PDI family member 565 Erp57 that catalyses disulphide bond formation in a highly oxidized ER lumen or it 566 may even act as a chaperone, binding to exposed polypeptide stretches of misfolded 8 Molecular Chaperones in Cellular Stress Response 195

Table 8.1 Major Chaperones described herein and their subcellular localization (Modified from t1.1 (Graner et al. 2014)) t1.2

Subcellular localization t1.3 Major chaperones ER Mitochondria Cytosol Nucleus Cell Surfacea t1.4 HSP27 ✓ ✓ ✓ t1.5 HSP60 ✓ ✓ ✓ t1.6 HSP70/HSC70 ✓ ✓ ✓ t1.7 GRP78 (BiP) ✓ ✓ ✓ ✓ ✓ t1.8 HSP90 ✓ ✓ ✓ t1.9 HSP110 ✓ ✓ ✓ t1.10 GRP94 (gp96) ✓ ✓ ✓ t1.11 CNX/CALRb ✓ ✓ ✓ t1.12 PDIc ✓ ✓ t1.13 aCell surface localization is mostly associated with tumour cell surfaces t1.14 bCalnexin/Calreticulin t1.15 cProlyl Disulphide Isomerase t1.16

glycoproteins. Both these functions of CNX are enhanced under heat stress condi- 567 tions. However, these chaperones may also ‘generally’ bind to glycoproteins inde- 568 pendent of their folding state, suggesting that there is no specific recognition of the 569 attached polypeptide chain (Buchberger et al. 2010). Other chaperones like GRP94, 570 Peptidyl-Prolyl Isomerases (PPIase) and GRP170 along with the ones mentioned 571 above form a large ER-localized multi-protein complex that functions as a network 572 and bind to unfolded proteins in the ER rather than existing as free pools that get 573 individually assembled onto protein clients. Grp94 most likely acts as a scaffold like 574 its cytosolic HSP90 counterpart and forms an important part of this multi-chaperone 575 complex (Ma and Hendershot 2004). Table 8.1 lists the major chaperones involved 576 in attenuating the adverse effects of heat shock and their subcellular localization. 577

8.6 Detailed Mechanism of Chaperone Assisted 578 Protein Folding 579

Out of the many chaperone systems described briefly in the previous section, our 580 group has mainly focussed on a couple of chaperone systems namely GroEL/ES (E. 581 coli) and HSP90 (human). Based on the various structural and functional studies 582 carried out by our group since the last decade on the chaperonin GroEL/ES system, 583 we have better understood the importance of this system in aggregation prevention 584 of denatured substrates, refolding of substrates and survival of E. coli. While we 585 have only recently started working on the human HSP90 system, we have obtained 586 novel information regarding the contribution of HSP90s structure in modulating its 587 chaperone properties using certain HSP90 inhibitors. The following section 588 describes in detail the structure and function of GroEL/ES and HSP90 during nor- 589 mal and under stress conditions. 590 196 B. K. Chatterjee et al.

591 8.7 GroEL/ES Mediated Protein Folding

592 8.7.1 GroEL/ES Structure

593 GroEL in its normal state is a porous cylindrical protein made of 14 subunits 594 arranged in nearly 7- fold rotationally symmetrical rings stacked back to back, and 595 forms a cage like structure with a central cavity (Braig et al. 1994). Each GroEL 596 subunit further folds into three domains (Braig et al. 1994; 1995). The apical domain 597 (residues 190–345) which is rich in hydrophobic residues and acts as a binding site 598 for the non-native substrates and co-chaperonin GroES (Fenton et al. 1994). The 599 intermediate domain (residues 134–190, 377–408) acts like a hinge between the 600 apical and equatorial domains. This also helps in allosteric communication between 601 the two domains. The equatorial domain consists of sub-domains E1 (residues 602 4–133) and E2 (residues 409–523) that form all the intra and inter-subunit interac- 603 tions required for the proper folding of the monomeric subunit and their assembly 604 into the tetra-decameric form (Braig et al. 1994, Hayer-Hartl et al. 2016). It also 605 houses the nucleotide binding pocket since GroEL works in ATP-dependent manner 606 (Braig et al. 1994). GroES is a single seven membered ring with identical subunits 607 of 10 kDa that binds to one or both ends of the GroEL cylinder in presence of a 608 nucleotide. Each subunit consists of a β-barrel body with an extended hydrophobic 609 mobile loop that interacts with the apical domain of GroEL and provides an enclosed 610 cavity for the folding of substrate proteins (Braig et al. 1994, Fenton et al. 1994; 611 Hendrick and Hartl 1993). Binding of GroES to the GroEL cylinder doubles the 612 volume of central cavity to provide sufficient space for the folding of substrate 613 proteins. Under normal conditions, GroEL interacts with non-native substrates 614 post-­translationally (Fenton et al. 1997), while native proteins under stress are sub- 615 jected to unfolding that leads to the formation of intermediate ‘aggregation prone’ 616 states that remain bound to GroEL. These can then be refolded back to their native 617 form, presented to other chaperones, or taken up by the proteolysis machinery 618 (Hartl et al. 2011).

619 8.7.2 GroEL Mechanism of Substrate Folding under Normal 620 Conditions

621 GroEL helps in the folding of about 5–15% of total cellular protein under normal 622 conditions (Ewalt et al. 1997). Depending upon the size of the substrate protein, it 623 assists in folding in two different ways: (I) Cis- mechanism of folding and (II) 624 Trans- mechanism of folding. The GroEL cavity can encapsulate substrate proteins 625 ranging between 54–57 kDa (Sakikawa et al. 1999) and help in their folding by 626 providing an Anfinsen cage inside the cavity. This is termed as the cis-mechanism 627 of substrate folding. GroEL is not able to encapsulate large proteins (> 60 kDa) inside 628 its cavity and they can undergo multiple rounds of binding and release at the apical 8 Molecular Chaperones in Cellular Stress Response 197 domain of GroEL before reaching their final folded or ‘committed to folding’ state. 629 Both GroES and ATP are required to release the folded/partially folded substrate 630 from the cis-ring (Fenton and Horwich 1997; Dahiya and Chaudhuri 2014). 631

8.7.3 Cis- Mechanism of GroEL Action 632

The asymmetric complex of GroEL-GroES is the most common form found in the 633 cellular milieu, along with a few symmetric complexes of GroEL (Weissman et al. 634 1995). A few other reports however, show that both types of complexes can exist in 635 equimolar concentration in cellular milieu and that the ratio of symmetric to the 636 asymmetric complex is an ADP dependent phenomenon (Yang et al. 2013; Lizuka 637 and Funatsu 2016). The asymmetric complex has one ring of GroEL occupied by 638 GroES, while another ring remains ready to accept a non-native substrate protein. 639 Due to the presence of hydrophobic residues at the apical domain, newly synthe- 640 sised polypeptides or partially folded substrates bind to the empty GroEL ring. The 641 binding of substrate protein leads to a conformational change in the same ring which 642 promotes subsequent binding of GroES and ATP. This leads to the formation of an 643 isolated cage for the folding of the substrate, known as the cis-ring. The conforma- 644 tional changes that occur during the binding of GroES and ATP make the GroEL 645 cavity hydrophilic, which in turn provides a proper environment for the folding of 646 substrate protein. ATP hydrolysis is a slow process with one molecule of ATP 647 hydrolysed in 8–10 seconds. The hydrolysis of ATP lowers the binding affinity of 648 GroES to the apical domain. This releases GroES and the folded substrate from the 649 GroEL cis ring; simultaneously ATP binds to the opposite ring along with another 650 substrate and a new cycle is initiated. (Fenton and Horwich 1997; Weissman et al. 651 1995). Recent reports show that both rings of GroEL act in a concomitant fashion 652 during the folding process (Yang et al. 2013). The following schematic explains the 653 mechanism of cis- folding (Fig. 8.1). 654

8.7.4 Trans Mechanism of GroEL 655

Large proteins with molecular weight (>60 kDa) are too big to fit inside the GroEL 656 cavity. This does not involve cis-encapsulation, but requires GroES binding to the 657 trans ring to release either folded or partially folded substrates (Chaudhuri et al. 658 2001). As the cis-ring binds with the substrate protein, the trans ring acts as a bind- 659 ing site for GroES, so this mechanism is termed as folding in-trans. There are many 660 substrate proteins, e.g., 70 kDa tail spike protein of phage p22, 86 kDa α/β heterodi- 661 mer, 82 kDa mitochondrial aconitase, dimeric citrate synthase, etc. that fold via this 662 mechanism (Weissman et al. 1995, Chaudhuri et al. 2001). In this process, the non-­ 663 native substrate binds to the apical domain of the asymmetric GroEL-GroES com- 664 plex, and undergoes folding with domain rearrangements or prevents aggregation 665 198 B. K. Chatterjee et al.

Fig. 8.1 Proposed model for GroEL/ES assisted folding of small proteins via cis-­mechanism): (I) Open ring of GroEL–GroES–ADP complex acts as an acceptor state for the non-native poly- peptide. (II) Binding of GroES to the GroEL–ATP complex leads to conformational changes in the apical domain of GroEL; consequently, polypeptide enters in the central cavity. (III) Folding occurs in the cis cavity before the ATP gets hydrolysed, this weakens the interaction between GroEL and GroES. (IV) Binding of ATP to the trans ring promotes the release of (N – Native, Ic – partially folded, U – misfolded) from the cis ring. (V) At the same time, binding of GroES to GroEL allows GroEL to alternate its rings between binding-active and folding-active states. (Copyright clearance order number 4177600370026.

666 (Chaudhuri et al. 2001). The apical domain of GroEL can also bind with the ‘burst 667 phase intermediate’ state of substrate proteins. To prove this hypothesis, experi- 668 ments were carried out using slow folding Malate Synthase G (MSG), a 89 kDa 669 multi-domain monomeric protein. Our observations suggest that binding of MSG to 670 GroEL accelerates the slowest kinetic phase of the spontaneous protein folding 671 pathway. Due to the large size of the substrate, GroES is not able to bind to this ring, 672 but ATP hydrolysis continues, which helps in the release of the folded substrate 673 from the ring. Sometimes the release of the substrate depends on the binding of both 674 GroES and ATP to the opposite ring. The binding of GroES and ATP to the opposite 675 ring also accelerates the release of folded/partially folded substrate from the GroEL 676 ring (Paul et al. 2007; Dahiya and Chaudhuri 2014). The trans mechanism of sub- 677 strate folding occurs under normal cellular conditions and also during thermal 678 stress. It helps in preventing the irreversible aggregation of thermally denatured 679 proteins. Study of the thermal unfolding pathway of citrate synthase (CS) show that 680 CS unfolds via an inactive dimeric intermediate. Further unfolding of these interme- 681 diates led to their irreversible aggregation. GroEL interacts with this dimeric unfold- 682 ing intermediate, dissociating them into monomers which stably associate with 683 GroEL (Grallert et al. 1998). The following schematic explains the mechanism of 684 Trans-folding (Fig. 8.2). 8 Molecular Chaperones in Cellular Stress Response 199

Fig. 8.2 Proposed model for GroEL/ES assisted folding of large proteins via trans-­ mechanism: The burst phase intermediate of MSG is captured by GroEL (orange colored) to form GroEL-MSG complex. This binding induces minor structural rearrangements to give rise to a more folding-compatible state. Further addition of GroES/ATP or ATP releases the GroEL-bound form of MSG, which folds to the native state via formation of a compact intermediate, that is structurally quite close to the native MSG. GroES (shown in blue) binds in Trans to the folding polypeptide and doubles the ATP-dependent reactivation rate

8.7.5 Passive Models of GroEL-Mediated Folding 685

GroEL passively suppresses protein aggregation (Pelham 1986; Ellis and van der 686 Vies 1991; Agard 1993) by binding to the exposed hydrophobic regions of aggrega- 687 tion prone proteins. GroEL specifically recognises and binds with the on-pathway 688 intermediate states, which shifts the overall folding equilibrium away from aggrega- 689 tion and provides a pool of folding competent monomers that could reach their 690 native state by further folding or assembly. In a purely passive folding model, high 691 concentrations of an aggregating molecule cause non-linear increase in the rate of 692 aggregation (Van den Berg 1999; Ellis 2001). GroEL prevents the formation of 693 unfavourable intermediates that could lead to aggregation and hence aid in the 694 proper folding of its substrates. 695 200 B. K. Chatterjee et al.

696 8.8 GroEL in Stress

697 8.8.1 Synthesis of GroEL during Stress

698 During heat shock, activation of σ32 transcription factor leads to an increased 699 production of Hsp inside E.coli. GroEL population increases from a basal level of 700 1–2% to 10–15% (of the total cellular protein content). Non-native substrates bind- 701 ing to GroEL results in a 2-fold increase (30% of the total cytoplasmic protein 702 content) in the clientele of GroEL, as compared to normal conditions (10–15%) 703 (Bukau 1993).

704 8.8.2 Structural and Functional Modifications 705 during Heat Shock

706 Foldase During heat shock, GroEL undergoes phosphorylation allowing it to 707 function without GroES. Enhanced ATPase activity in the phosphorylated form 708 ensures relatively fast release of the folded substrate from GroEL. The folding effi- 709 ciency of phosphorylated GroEL is 50–100 fold higher than its native counterpart. 710 Phosphorylation is a reversible process and once the cell is relieved of stress, GroEL 711 undergoes de-phosphorylation and resumes its normal function inside the cell 712 (Sherman and Goldberg 1994).

713 Holdase Our studies on monomeric GroEL show that the apical domain is the most 714 stable region in the GroEL subunit as it requires 4.0 M urea and 70°C to undergo 715 complete unfolding (Golbik et al. 1998; Puri and Chaudhuri 2017). This is a kind of 716 adaptation in the GroEL structure that can possibly hold aggregating substrates under 717 stress conditions. It is also observed that during thermal stress, GroELs protein fold- 718 ing activity is reduced and it starts acting as holdase by binding to the aggregation 719 prone proteins, thus behaving as a storehouse of proteins. The molecular basis for 720 such a functional transition is the loss of inter-ring signalling and negative co-­ 721 operativity, which slows down the release of GroES and the unfolded proteins from 722 the GroEL cavity. This phenomenon is also reversible with GroEL reverting to its 723 normal function after heat shock (Llorca et al. 1998).

724 Unfoldase Strong binding of substrate proteins at the GroEL apical domain can be 725 the main cause of unfolding or ‘stretching’ of bound substrates. The unfolding 726 activity occurs through inter-domain movement where stretching of the apical 727 domain helps in ‘opening up’ of the aggregating substrates. The unfolded substrate 728 remains bound to the GroEL apical domain during stress conditions, but once cel- 729 lular conditions become normal, GroEL is able to perform its folding function 730 (Grallert et al. 1998). 8 Molecular Chaperones in Cellular Stress Response 201

8.8.3 ATP Independent Aggregation Prevention 731 by GroEL Protein 732

Aggregation prevention tendency of GroEL is an ATP independent process. This 733 proves useful during stress conditions, because of increasing load of aggregating 734 proteins and relatively more ATP consumption to maintain cellular homeostasis 735 (Soini, et al. 2005). In vivo studies in bacteria and yeast demonstrated that depletion 736 of either GroEL or mitochondrial Hsp60 resulted in aggregation of a large number 737 of newly translated proteins (Cheng et al. 1989; Horwich et al. 1993). Similarly, 738 many in vitro studies demonstrated that GroEL can rapidly and efficiently bind 739 to non-native states of several proteins and arrest their aggregation e.g., malate 740 dehydrogenase (MDH; Ranson et al. 1995), ribulose-1,5-bisphosphate oxygenase-­ 741 carboxylase (RuBisCO; Goloubinoff et al. 1989), etc. Our current study on aggrega- 742 tion prevention of Maltodextrin glucosidase (MalZ) demonstrate that GroEL can 743 prevent aggregation of this protein without the involvement of GroES and ATP (Puri 744 and Chaudhuri 2017). The passive binding of non-native intermediates to GroEL 745 can prevent their aggregation by disallowing random hydrophobic interactions. 746 Once captured proteins reach the folding competent state, they must be released 747 back into the free solution, to undergo completion of the final steps of folding or 748 oligomer assembly without ATP (Lin and Rye 2006). 749

8.9 HSP90 Mediated Protein Folding 750

Structure 751 HSP90 functions as a flexible dimer inside the cell; the dimers consist of two mono- 752 mers joined at their C-terminus via the dimerization domain. The N-terminus consists 753 of a conserved Bergerat ATP/ADP binding fold that also binds to Geldanamycin and 754 Radicicol, competitive inhibitors of ATP binding (Pearl 2016; Bergerat et al. 1997; 755 Stebbins et al. 1997; Roe et al. 1999). An N-terminus ‘lid’ segment that responds to 756 ATP binding has been found, consisting of two highly conserved glycine clusters 757 (Prodromou et al. 2000). The N-terminus is connected to the Middle (M) domain by 758 a flexible linker that has been shown to convey allosteric modulations from the 759 M-domain and the C-terminus to the N-terminus, resulting in global conforma- 760 tional changes. The M-domain binds to co-chaperones that “present” client pro- 761 teins to HSP90. The C-terminus contains a unique, conserved MEEVD domain that 762 binds to tetratricopeptide (TPR) domain containing co-chaperones. The nucleotide 763 binding site lies in a deep pocket on the helical face of the N-domain. The adenine 764 base, sugar, and the α- phosphate group make extensive contacts within this pocket, 765 whereas the β- and the γ- phosphate groups display weak and no contacts respec- 766 tively. A hydrogen bond connects the adenine base with Asp79, while all other 767 contacts observed are polar in nature with water molecules interacting with the 768 202 B. K. Chatterjee et al.

769 ribose sugar moiety. The α- and the β- phosphate groups are bound to an octahedrally 770 co-ordinated Mg2+ ion, making an indirect coupling with the protein (Pearl 2016; 771 Panaretou et al. 1998).

772 ATPase Cycle 773 HSP90s function as a chaperone is dependent on its inherent ATPase activity. The 774 molecule undergoes a series of conformational changes upon ATP and client protein 775 binding that culminates in ATP hydrolysis and release of the partially folded client 776 protein (Li et al. 2012). Understanding the mechanism of this ATPase coupled con- 777 formational cycle has progressively advanced with several research groups using 778 sophisticated biophysical techniques like FRET and Analytical Ultracentrifugation 779 to identify the kinetic states in this cycle and the switch that occurs between these 780 states (Hessling et al. 2009; Mickler et al. 2009). The defining mechanism is the 781 ‘molecular clamp mechanism’, (Ali et al. 2006) where ATP binding to the open 782 V-shaped constitutive HSP90 dimer (apo-state) induces structural changes via inter- 783 mediates, the formation of each of which is regulated by co-chaperones that have 784 specific roles to play in the cycle. The rate limiting step is the formation of a closed 785 ‘tensed’ state, where the N-domains are dimerized and associated with the 786 M-domains. This closed conformation is committed to ATP hydrolysis with subse- 787 quent release of the substrate and ADP occurring concomitantly as the N-domains 788 dissociate and HSP90 returns to its open conformation, ready for another round of 789 the ATPase cycle (Pearl 2016; Li et al. 2012). The rate limiting step involves major 790 structural changes occurring in the N-domain in two distinct ‘switch’ regions: (a) 791 the β-strand in the N-domain of one monomer hydrogen bonds with the main β-sheet 792 in the N-domain of the other monomer. Simultaneous movement of the α-helix 793 exposes a large hydrophobic patch that dimerizes with the equivalent patch of the 794 other monomer; and (b) the ‘lid’ segment, which flips over ~180° from its apo con- 795 formation to fold over the bound nucleotide in the pocket and ‘cradle’ the γ-phosphate 796 of ATP in a series of main-chain hydrogen bonds (Pearl 2016). Additionally, the 797 flexible loop from the M-domain associates with the lid segment of the N-domain 798 via hydrophobic residues. This intra-molecular docking of the N and M-domains 799 facilitates the interaction between the γ-phosphate and the R380 residue, resulting 800 in the assembly of the two- halves of a split active site for ATP hydrolysis (Pearl 801 2016; Meyer et al. 2003a, b; Cunningham et al. 2012). The stable HSP90 C-terminal 802 dimer model was challenged by Ratzke and his team (Ratzke et al. 2010) who 803 observed through FRET experiments that apart from the transient dimerization 804 observed at the N-domains, the C-terminus can also open and close with fast kinet- 805 ics, even when ATP/ADP is bound to the N-domain. They proposed a unique mech- 806 anism where HSP90 may undergo multiple transitions from being a dimer at the 807 C-domain and open at the N-domains, to being open at the C-terminus while 808 N-domains are dimerized, thereby associating a higher degree of dynamic flexibility 809 that may influence substrate release (Ratzke et al. 2010). The following schematic 810 depicts the conformational change from the open to the closed state of HSP90 via 811 several transition intermediates (Fig. 8.3). 8 Molecular Chaperones in Cellular Stress Response 203

Fig. 8.3 An overview of the conformational cycle of HSP90: Several co-chaperones and ATP mediate the transition between the early, intermediate and late stages of substrate-bound HSP90 (Copyright clearance order number- 4177600710200)

HSP90 co-Chaperones 812 HSP90 can only keep its substrates in a folding competent state and prevent them 813 from undergoing aggregation, but cannot refold any of its clients by itself (Freeman 814 and Morimoto 1996). It requires the assistance of other proteins to carry out its 815 biological function. These proteins are also known as co-chaperones. Around 20 816 co-chaperones have been identified in eukaryotes and while some of them have been 817 well-characterized, the mechanism by which some of the other co-chaperones func- 818 tion is unclear. These co-chaperones mainly regulate the ATPase activity and bind- 819 ing of client proteins to HSP90 (Prodromou et al. 1999; 2002; Richter et al. 2004; 820 Roe et al. 2004; Chen and Smith 1998). They associate and dissociate dynamically 821 and help in the transition from one intermediate conformation to another, or stabi- 822 lize a certain conformation in the protein folding cycle. Some of these co-­chaperones 823 like HSP90/HSP70 Organizing Protein (HOP) (Johnson et al. 1998), protein 824 phosphatase PP5 (Silverstein et al. 1997) and PPIase family members FKBP51 825 204 B. K. Chatterjee et al. t2.1 Table 8.2 List of some well-studied co-chaperones of HSP90 and their role in the ATPase cycle t2.2 (Modified from (Li et al. 2012)) t2.3 Protein Name Function t2.4 TPR containing t2.5 co-chaperones t2.6 HOP Stabilizes the open conformation of HSP90; inhibits ATP hydrolysis; t2.7 simultaneously binds HSP90 and HSP70 and aids in transfer of nascent t2.8 polypeptides recognized by HSP70. t2.9 FKBP51/52 Maturation of steroid hormone receptors (SHR); chaperone. t2.10 CYP40 Maturation of Estrogen receptors specifically; chaperone (Chen et al.1998 ; t2.11 Pirkl and Buchner 2001). t2.12 PP5 Post translational modification of HSP90; dephosphorylates HSP90 and t2.13 CDC37; plays a role in processing of client proteins. t2.14 TPR2 Recognizes both HSP90 and HSP70 through its TPR domain; may act in the t2.15 client transfer from HSP70 to HSP90 (Brychzy et al. 2003). t2.16 Non-TPR t2.17 co-chaperones t2.18 AHA1 Stimulates ATPase activity; induces conformational change (Retzlaff et al. t2.19 2010; Sato et al. 2000). t2.20 P23 Binds and stabilizes closed client bound HSP90 heterocomplex; maturation t2.21 of client proteins; inhibits ATP hydrolysis; chaperone (Johnson and Toft t2.22 1994; Obermann et al. 1998; Bose et al. 1996; Freeman et al. 1996). t2.23 CDC37 Binds specifically to client kinases and ‘presents’ them to HSP90 by binding t2.24 to HSP90s N-terminal domain via its C-terminal; inhibits ATP binding and t2.25 ATPase activity of HSP90; chaperone (MacLean and Picard 2003; Gaiser t2.26 et al. 2010; Siligardi et al. 2002; Ali et al. 2006).

826 (Nair et al. 1997) and FKBP52 (Cox et al. 2007; Johnson and Toft 1994) have a TPR 827 domain that binds to the MEEVD sequence located in the C-terminus of the 828 chaperone (Scheufler et al.2000 ; Das et al. 1998). Table 8.2 (Modified from Li et al. 829 2012) lists some of the well-studied co-chaperones that have been identified and 830 their role in the ATPase cycle of Hsp90.

831 HSP90 co-chaperone Cycle 832 The most recent understanding of the chaperone cycle is that there are three differ- 833 ent complexes formed in a chronological fashion with different co-chaperone com- 834 position (Smith 1993). The first complex, called the early complex consists of 835 HSP70, HSP40 and a client protein (Smith et al. 1992; Patricia Hernández et al. 836 2002; Cintron and Toft 2006), which presumably is a misfolded or nascent polypep- 837 tide. This complex docks to HSP90 via HOP which acts as a scaffold. One TPR 838 domain of HOP binds to one MEEVD motif of the HSP90 dimer while another 839 HOP domain binds to the early complex (Li et al. 2011). This HOP bound HSP90 840 complex can be called the intermediate complex. Addition of ATP and a PPIase 841 results in the formation of an asymmetric complex, where the TPR domain of the 842 PPIase binds with the unoccupied MEEVD motif of the other monomer, and 8 Molecular Chaperones in Cellular Stress Response 205 concomitantly ATP binds to the N-terminus (Smith 1993). The chaperone is still in 843 its open confirmation. HSP90 adopts the closed ‘committed to ATP hydrolysis’ 844 conformation after p23 binds to the intermediate complex (Johnson et al. 1994; 845 Johnson and Toft 1995; McLaughlin et al. 2006; Freeman et al. 2000). This is called 846 the late complex where HOP and HSP70/40 dissociate from HSP90 as their binding 847 affinity is weakened due to the conformational change. After ATP hydrolysis, p23 848 and PPIase is released along with the partially folded client protein. Not much is 849 known about the ADP bound conformation that re-positions the relative orientations 850 of the N-domains just prior to the release of the client (Pearl 2016). Dynamic X-ray 851 scattering data has revealed that HSP90 can exist in a highly flexible conformational 852 ensemble (Rice et al. 2008; Zhang et al. 2004), even in the nucleotide free state and 853 that the ADP bound state is a partially closed one with the N-domains close to each 854 other, but different to the ATP bound closed complex where the N-domains dimer- 855 ize (Scherrer et al. 1990). 856

Expression and Regulation of HSP90s Function under Thermal Stress 857 The upregulation of chaperones under heat shock conditions is mediated by the 858 Heat Shock Transcription Factor 1 (HSF-1) (Fiorenza et al. 1995). HSF-1 under 859 normal conditions remains in its inactive monomeric state bound to HSP90. Upon 860 heat shock, HSP90 dissociates from the complex and HSF-1 undergoes trimeriza- 861 tion (Baler et al. 1993; Sarge et al. 1993). This trimer can bind to DNA regulatory 862 elements called the Heat Shock Elements (HSEs) and upregulate the transcription 863 of HSP genes like HSP90 and HSP70 (Akerfelt et al. 2010). Elevated levels of HSP 864 lead to inactivation of HSF-1; HSP90 and its cohorts FKBP2 and p23 bind to the 865 trimeric state and attenuates its DNA binding affinity (Zou et al.1998 ; Bharadwaj 866 et al. 1999; Guo et al. 2001) while HSP70/HSP40 bind to HSF-1 and prevent its 867 transactivation (Shi et al. 1998; Abravaya et al. 1992; Baler et al. 1992). This nega- 868 tive feedback loop regulates the chaperones at the transcription level, and thereby 869 modulates the levels of misfolded nascent polypeptides inside the cell. Additionally, 870 certain post translational modifications affect the chaperone functions of HSP90 871 (Scroggins and Neckers 2007). Under normal circumstances, HSP90 remains exten- 872 sively phosphorylated, with as many as four phosphorylated residues in each iso- 873 form (Sefton et al. 1978; Kelley and Schlesinger 1982). Under heat shock conditions, 874 the general understanding is that HSP90 is rapidly dephosphorylated, leading to the 875 loss of HSP90s ability to stimulate the activity of its client proteins (Lees-Miller and 876 Anderson 1989; Morange and Bensaude 1991). Dephosphorylation is mediated by 877 Protein Phosphatase 1 (PP1), while phosphorylation is mediated by several kinases 878 like CKII, DNA-PK, and Akt (Wandinger et al. 2006; Dougherty et al. 1987; Walker 879 et al. 1985; Lees-Miller and Anderson 1989; Chalovich and Eisenberg 2012). After 880 clients that are bound to HSP90 under normal temperature get released due to 881 dephosphorylation, one of HSP90s client proteins, heme-regulated Inhibitor Kinase 882 (HRI), is activated by the chaperone upon rapid phosphorylation and this in-turn 883 down regulates protein synthesis by inactivating eukaryotic initiation Factor-2α 884 subunit (Scroggins and Neckers 2007). This cycle of dephosphorylation and phos- 885 phorylation of HSP90 reduces the load of misfolded proteins inside the cell. 886 206 B. K. Chatterjee et al.

887 8.10 Combinatorial Assistance of Various Chaperones

888 There exists various checkpoints to ensure correct folding from the beginning of 889 protein synthesis till it attains its native, biologically active conformation. Molecular 890 chaperones act as a crucial buffer providing conditions conducive for the partially 891 unfolded intermediate to fold. The chaperone machinery helps newly synthesized 892 protein to navigate the complex energy landscape in order to achieve their native 893 form. The following paragraph describes the presence of various chaperones at 894 different spatial and temporal points, coordinating with each other to regulate 895 protein folding. 896 The chaperones are present at different levels: The first chaperonic tier consists 897 of ribosome associated chaperones. These chaperones stabilize the nascent poly- 898 peptides which are being synthesized on the ribosome and initiate their folding 899 process. The second tier of components act subsequently and aid in the complete 900 folding of the proteins. Both systems cooperate to form one single folding pathway. 901 Chaperones involved in the first tier include prokaryotic chaperones Trigger Factor 902 (TF) and eukaryotic Ribosome Associated Complex (RAC) (Kramer et al. 2009). 903 These are present on the large ribosome near the exit tunnel of a polypeptide chain. 904 This RAC complex comprises HSP70 homologs Ssb1, Ssb2 and Ssz1, zuotin (in 905 yeast) and the corresponding homologs in higher eukaryotes (Hundley et al. 2005; 906 Otto et al. 2005). These chaperones primarily bind to the exposed hydrophobic 907 regions of the proteins. TF works in an ATP independent manner to fold a newly 908 synthesized polypeptide. The bound polypeptide tries to bury its hydrophobic 909 regions which facilitate the folding process. Interestingly most of the nascent chains 910 (~70%) seek the assistance of TF and are successfully folded without any further 911 assistance. Small proteins also fold spontaneously after their synthesis without any 912 further assistance (Ferbitz et al. 2004; Merz et al. 2008). 913 Another class of proteins (~ 20%) possess strong hydrophobic elements (Teter 914 et al. 1999; Thulasiraman et al. 1999) and thus TF and other ribosome associated 915 chaperones do not provide enough assistance for their folding. It has been reported 916 that TF dissociates from the ribosome while bound to the polypeptide chain (Agashe 917 et al. 2004), thereby presenting the unfolded polypeptides to the downstream chap- 918 erones like DnaJ/DnaK (Martinez-Hackert and Hendrickson 2009). The DnaJ/ 919 DnaK system further interacts with the longer polypeptide chains and helps in their 920 folding in an ATP dependent manner. In eukaryotes the second tier also consists of 921 NAC (Nascent chain associated complex) and like TF, it interacts with the newly 922 synthesized polypeptides and helps them attain their folded conformation with the 923 assistance of RAC, HSP70, and other cofactors. 924 HSP70s along with other cofactors like HSP40, J-proteins and various NEFs aid 925 in the folding of substrates in an ATP dependent manner (Zhu et al. 1996; Mayer 926 et al. 2000; Pellecchia et al. 2000). HSP40 also binds with the misfolded polypep- 927 tides and recruits HSP70 to assist in the proper folding of substrates (Young et al. 928 2003). In eukaryotes, subsequent to the HSP70 system, HSP90 with the help of 929 numerous regulators and co-chaperones acts as a finisher, helping the polypeptide 8 Molecular Chaperones in Cellular Stress Response 207 attain its biologically active structure (Pearl and Prodromou 2006; Zhao and Houry 930 2007; Scheufler et al. 2000). The remaining 10% of substrates that remain in a non-­ 931 functional, yet non-aggregated state, are shifted to the chaperonin cage for their 932 folding (Hartl 1996; Horwich et al. 2007). The environment in the nano cage of 933 chaperonin facilitates the misfolded proteins to attain a native-like structure 934 (Gromiha and Selvaraj 2004). In bacteria, GroEL/ES help in the folding of substrate 935 proteins. In eukaryotes, substrate proteins are presented to the chaperonin TRiC. The 936 reaction is mediated by HSP70 and Prefoldin which interact directly with the TRiC 937 system resulting in the release of folded, functional proteins (Hartl and Hayer-Hartl 938 2002). 939

8.11 Conclusions 940

Compilation of studies both past and recent, on chaperone mediated protein folding 941 unequivocally show that chaperones play a basal role in maintaining protein homeo- 942 stasis inside the cell. While some of them function as ‘foldases’, hydrolysing ATP 943 to carry out the folding and release of various substrates, a few others function 944 independent of ATP, primarily as ‘holdases’. Under stress conditions however, 945 chaperones undergo multiple levels of modification; from the enhanced rates of ATP 946 hydrolysis and more efficient substrate binding to various post-translational modifi- 947 cations that aid in the transcriptional upregulation of several hsp genes. Chaperones 948 can also prevent aggregation of misfolded proteins that tend to accumulate under 949 heat shock conditions. Chaperones play a dual role inside the cell; either rescuing 950 misfolded or unfolded polypeptides and preventing aggregation or triggering degra- 951 dation of substrates when cell damage is irreversible, both of which needs to be 952 tightly regulated to maintain proteostasis. Such properties of chaperones are cur- 953 rently being utilized in the industry as well as in designing therapy for certain debil- 954 itating diseases like cancer and neurodegenerative disorders. Overexpression of 955 certain proteins can be a major problem, especially when they are being expressed 956 in a different host. Several studies including some of our own have shown that 957 a chaperone or a combination of chaperones have been proven effective in increas- 958 ing the yield as well as the active fraction of total protein expressed. Some of those 959 proteins are considered therapeutically important; hence, large-scale productions of 960 such proteins are regularly uptaken by the pharmaceutical industry. Chaperones 961 thus ablate a significant clog in this giant wheel of industrial-scale protein produc- 962 tion and such strategies have come to the rescue over the years. HSP90 has been 963 used as a target to design inhibitors that have been successful in the amelioration of 964 tumor progression and development, and is considered a hot prospect for drug-­ 965 based therapy for the treatment of certain types of cancer. Although a promising 966 approach, only a few compounds have reached the clinical trials and none of them 967 have made it to the market. On the other end of the spectrum, the ability to prevent 968 aggregation of misfolded proteins and even refold partially folded ‘aggregation-­ 969 prone’ intermediates can be used to treat neurodegenerative disorders. We are 970 208 B. K. Chatterjee et al.

971 currently working on one such compound that stimulates the chaperone functions of 972 HSP90 in-vitro, and plan to carry out several studies to investigate its potential in 973 preventing certain neurodegenerative conditions. Overall, chaperones make an 974 interesting topic of study, not only because they play such important roles in regu- 975 lating protein function, stability and degradation but also because they possess tre- 976 mendous value both industrially and therapeutically.

977 Acknowledgments The authors acknowledge the financial assistance from IIT Delhi and infra- 978 structural facility from IIT Delhi, India. AS and AP acknowledge financial assistance from CSIR, 979 Government of India for providing fellowships in their doctoral course programme. SP acknowl- 980 edge financial assistance from UGC, Government of India for providing fellowships in their doc- 981 toral course programme. BKC acknowledges IIT Delhi for providing fellowship in the doctoral 982 course program.

983 References

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