Molecular Chaperones

Molecular chaperones are a large group of structurally conserved and genetically diverse protein families unified by their main functions - to facilitate folding and assembly of proteins into macromolecular structures, to facilitate protein transport across membranes, to inhibit misfolding and to prevent and/or reverse aggregation of proteins (Feder and Hofmann 1999). Initially this group of proteins was discovered as heat shock proteins (Hsps) by Ritossa (1962) in his study on the gene expression in Drosophila after exposure to heat. They are thus often referred to as Hsps/chaperones. Chaperones not belonging to Hsps – some enzymes called foldases which catalyse covalent reactions involved in the protein folding at the endoplasmic reticulum are not considered here. Rather, a comprehensive overview of the families of molecular chaperones and their involvement in drought response is provided.

General Characteristics

Chaperones are essential for the proper functioning of cells and are distributed ubiquitously in all living organisms, in almost all cell compartments (Ellis, 1990). Chaperones are among the most abundant cell proteins – for example Hsp 90 accounts for about 1-2 % of total cell protein (Vierling, 1991; Leidhold and Voos, 2007, Patterson and Höhfeld, 2006) and are particularly abundant in cytosol, endoplasmic reticulum, mitochondria and chloroplasts. They are highly structured and undergo conformational changes linked to their function, recognizing and binding to exposed hydrophobic patches in the polypeptide chains, thus stabilizing proteins and promoting folding to the native state if possible or assisting recognition and removal by the proteasome systems (Liberek et al., 2008). To accomplish their essential functions, chaperones usually work in close cooperation with each other and with other proteins like co-chaperones and components of the proteasome systems (Leidhold and Voos 2007). Various chaperones act in concert in protecting proteins from stress, forming a network of chaperone machinery. There is a tight interplay between the cellular protein-folding and protein-degradation systems mediated by the chaperone network (Patterson and Höhfeld, 2006). They have essential functions in protein homeostasis in normal conditions and are highly responsive to various stresses (Wang et al., 2004).

Structural and Functional Diversity 

The striking molecular diversity of chaperones is related to the diversity of their functions in general and their different roles in non-stress and stress conditions. Each of the five major families of chaperones is characterised by amino acid sequence homology, approximate molecular weight, structure, mode of action and specific function - 12-40 kDa small Hsps which are widely present in plants; chaperonins (GroEL and Hsp60), the Hsp70/DnaK family, the Hsp90 family, and the Hsp100/Clp family. Except for the small Hsps, all other chaperones use ATP for processing client proteins. ATP binding and hydrolysis (the ATPase cycle) controls binding and release of the substrate protein through conformational changes in the chaperone (Wang et al., 2004; Patterson and Höhfeld, 2006).

Small Hsp Families

Higher plants possess at least 20 types of small Hsp (sHsp) families in the range of 12-40 kDa, all encoded by nuclear multigene families and divided into 6 classes – three (CI, CII and CIII) coding for sHsps located in the cytosol and nucleus and three (CIV, CV and CVI) in the endoplasmic reticulum, plastids and mitochondria (Mahmood et al., 2010). Cytosolic CI sHsps are the most abundant sHsps in plants. Sequence similarity in one class is up to 93% for different plant species while sHsps belonging to different classes in one species have only 50-75% sequence similarity (Vierling, 1991; Mahmood et al., 2010).

Chaperones of this group share a conserved 90 amino acid C-terminal domain – the α-crystallin domain or heat shock domain that contains several β-strands forming two β-sheets. This domain is involved in the oligomerization of sHsps to dimers and trimers, forming a dodecamer double ring (Liberek et al., 2008). In vivo, plant sHSPs form large spherical or cylindrical oligomeric structures of 200-300 kDa. Complex formation is ATP-independent but appears to be regulated by temperature and phosphorylation (Chang, 2009). The oligomeric complex is thought to be a storage form that continuously exchanges dimerized subunits. Dissociation may be necessary for effective chaperoning activity, the active form of sHsps being a dimer (Patterson and Höhfeld, 2006). Small Hsps have the ability to bind strongly to exposed hydrophobic regions in non-native proteins through hydrophobic interaction in an ATP-independent manner, thus stabilizing them and preventing irreversible aggregation. It appears that temperature-activated sHsps associate with heat-destabilized proteins in the aggregates, conferring different physicochemical properties on the aggregates (Liberek et al., 2008). Although sHsps are not able to refold non-native proteins, they could provide immediate protection under unfavourable conditions, maintaining a pool of substrates for subsequent refolding by the ATP-dependent chaperones of the Hsp70 and Hsp100 families.

Chaperonins (Hsp60 Family)

There are 2 subfamilies of chaperonins - Group I, E. c0l1 GroEL and the homologous Cpn60 in chloroplasts and mitochondria, and Group II, the CCT chaperonins (containing t-complex polypeptide 1) in Archea and the cytosol of eukaryotes. Folding of the most abundant chloroplast enzyme Rubisco is assisted by Rubisco binding protein (RBP) which belongs to the chaperonin family. Plastid Cpn60 in Arabidopsis is encoded by 7 genes, and 9 are predicted to encode the distinct CCT subunits in Arabidopsis (Wang et al., 2004).

The main function of this group is to assist the proper folding of newly synthesized and membrane-translocated proteins into their native conformation. Cpn60 molecules form a multisubunit structure of nearly one million Daltons, composed of double back-to-back rings, each of 7 subunits of about 60 kDa, surrounding a central cavity large enough to refuge a protein of up to 60-70 kDa (Bukau and Horwich, 1998). The central cavity functions in two states – binding-active and closed. The former is open at the end, exposing a flexible hydrophobic lining that binds the non-native protein, provoking additional partial unfolding, in that way removing the bound protein from a kinetic trap. The closed state is formed by complexing with a co-chaperone (Hsp10 or Hsp20), resulting in dramatic en bloc upstream movements of the hydrophobic parts, enlarging the central cavity and exposing the now hydrophilic inner surface, thus promoting the transition of the trapped protein into its native state (Bukau and Horwich, 1998). Chaperonins thus appear to function through alternating exposure of hydrophobic and hydrophilic inner surfaces. The complex with co-chaperone is stable with bound ATP and unstable with bound ADP. ATP hydrolysis triggers substrate release, enabling the next cycle of chaperone action (Bukau and Horwich, 1998). Under heat shock conditions the folding capacity of the chaperonins is suppressed while their binding affinity towards the unfolded client proteins is increased (Llorca et al., 1998). That way chaperonins could protect their client proteins from unfavourable conditions by sequestering them, releasing them only when the stress is relieved. CCT chaperonins form 8-9 subunit rings, each subunit being encoded by a distinct but related gene. They assist the folding of actin and tubulin in the cytoplasm (Wang et al., 2004).

Hsp70 Family

This family is highly evolutionarily conserved (about 50% identity between E.c0l1 and eukaryotic Hsp70s) and includes two subfamilies: Hsp70/Hsp40 and Hsp110/SSE (Grigorova 2010). The Arabidopsis genome contains 18 genes coding for Hsp70 chaperones – 14 of the DnaK subfamily and 4 of the Hsp110/SSE subfamily (Wang et al., 2004). Some members of this family are phosphorylated and/or methylated (Chang, 2009).

Hsp70 family is engaged in assisting folding of newly synthesized proteins, enabling translocation of proteins across membranes of organelles, disassembling oligomeric protein structures, refolding misfolded and aggregated proteins, facilitating proteolytic degradation of unstable proteins by targeting them to proteasomes, controlling the activity of regulatory proteins (Bukau and Horwich, 1998). Hsp70 chaperones exhibit common structural features - a highly conserved N-terminal ATPase domain (nucleotide binding domain, NBD) of 44 kDa, a central peptide-binding cleft, and a C-terminus that forms a lid over the peptide-binding cleft. Crystallographic analysis has revealed the formation of a substrate-binding hydrophobic pocket. Hsp70 binds with high affinity to short hydrophobic segments in extended conformation. The consensus motif recognized in the client proteins consists of 4-5 hydrophobic residues, flanked at each side by basic residues. Such a motif occurs frequently in buried β-strands and is exposed in non-native proteins. ATP binding to the N-terminal domain of Hsp70 drives conformational changes in the C-terminal domain – the substrate binding pocket is open allowing rapid exchange of substrates. In the ADP-bound state the substrate binding site is closed, with high affinity and slow exchange rate for substrates. Hsp70 prevents protein aggregation and promotes proper folding by shielding hydrophobic segments in the protein substrate. ATP hydrolysis is the rate limiting step in the ATPase cycle of Hsp70 and this step is subjected to control by co-chaperones (Bukau and Horwich, 1998). Co-chaperones of the Hsp40 family stimulate the ATP hydrolysis step and promote substrate binding. The Hsp70-interacting protein Hip stabilizes the ADP-bound conformation, whereas the co-chaperone BAG-1 stimulates nucleotide exchange.

Hsp90 Family

The Hsp90 family is distinct from other families by its active involvement in signal transduction networks and cell cycle control. Besides managing protein folding of signal transduction proteins, Hsp90 interacts with 26S proteasome and plays a principal role in its assembly and maintenance (Wang et al., 2004). Plant Hsp90s exhibit 63-71% amino acid identity with yeast and animal Hsp90s (Krishna and Gloor, 2001). In Arabidopsis, 7 genes code for members of the Hsp90 family – 4 for cytoplasmic Hsp90 proteins, and 3 for Hsp90s located in plastids, mitochondria and the endoplasmic reticulum (Krishna and Gloor, 2001; Wang et al., 2004).

This group of chaperones binds to substrates at a late stage of folding to assist activation of signalling proteins, by accepting partially folded proteins from Hsp70 for further processing (Pearl et al., 2008). The functional form of Hsp90 is a phosphorylated dimer containing 2 to 3 covalently bound phosphate molecules per monomer. Biochemical and electron microscopic studies indicate that Hsp90 contains two clearly distinguishable domains, attached to each other by a relatively flexible, highly charged loop. The C-terminal domain itself may also have a bilobal structure (Krishna and Gloor, 2001). The N-terminal ATP-binding domain is a highly twisted, eight-stranded β-sheet covered on one side by α-helices, with a deep pocket site for ATP/ADP binding at the centre of the helical side. This domain is involved in binding sasaran proteins. The central, highly charged region of Hsp90 contains alternating lysine and glutamic acid residues (“KEKE-motifs”) which may be involved in protein-protein interactions and serve as a binding site for the proteasome. The C-terminal domain has a binding site for calmodulin and is engaged in constitutive Hsp90 dimerization. ATP binding and hydrolysis are coupled to transient N-domain dimerization in an ATP-driven molecular clamp mechanism. The N-terminal and central domains, which both bind protein-kinase client protein (bivalent interaction), move relative to each other during the ATPase cycle, thus forcing a change of the mutual orientation of the domains in the substrate protein for its activation (Saibil, 2008). Besides various structurally unrelated proteins, Hsp90 client proteins are mostly protein kinases and transcription factors (Pearl et al., 2008). That way Hsp 90 integrates multiple regulatory signals in signalling networks.

Members of the Hsp70 and 90 families are able to cooperate with the ubiquitin-proteasome system (described in section 5.1) with the mediation of the CHIP protein, which is an E3 ligase that interacts with molecular chaperones through its N-terminal tetratricopeptide domain (Murata et al., 2003). In this way the CHIP protein assists interaction between the chaperone system and the ubiquitin-proteasome system.

Hsp100/Clp Family

Chaperones of this family are members of the large AAA ATPase superfamily of enzymes that catalyze mechanical processes such as locomotion, unwinding, disassembly and unfolding of other macromolecules (Maurizi and Xia, 2004). Hsp100/Clp chaperones are divided into two classes. Class 1 contains proteins with two AAA modules including Hsp104, ClpB, mitochondrial Hsp78, plant Hsp101, ClpA and ClpC. Class 2 comprises proteins having only one AAA module, such as ClpX. Class 1 is divided into 2 subfamilies - ClpB/Hsp104, which possesses disaggregating activity coupled with the refolding activity of Hsp70 chaperones, and the ClpA subfamily whose unfolding activity is coupled with ATP-dependent proteolysis by ClpP subunits. The Arabidopsis genome contains 8 genes coding for Hsp100 proteins, 5 of which have predicted plastidial localization signals (Agarwal et al., 2001). Five Hsp100 genes are present in rice, 4 of them predicted to have chloroplast transit peptides (Batra et al., 2007).

All members of this family have a conserved structural core – the AAA module that consists of 2 subdomains – a large α/β domain consisting of five-stranded parallel β-sheets, flanked by α-helices and connected by a mobile linker to a smaller helical C-terminal domain. ATP binds between the two domains in a crevice containing Walker A and B motifs (the catalytic residues for ATP hydrolysis) and sensors 1 and 2 which respond to the nucleotide state (Maurizi and Xia, 2004). ClpA and ClpB have two AAA modules in tandem (NBD1 and NBD2) linked to a large helical N-domain, as well as a ClpP-binding loop in the C-terminal module. ClpB also possesses an additional intermediate or middle domain (I-domain or M-domain) situated near the junction of NBD1 and NBD2, and composed of two coiled-coils running in opposite directions from the point of attachment, like a two-bladed propeller. The I-domain undergoes displacements in response to nucleotide hydrolysis by NBD1 and plays a substantial role in the re-solubilization of protein aggregates (Barends et al., 2010). Hsp100/Clp chaperones form hexameric rings with a central channel of 25 Å in Clp proteins (Doyle and Wickner, 2008). ClpA and ClpB, which contain two AAA domains, form a bilayered structure with two homomeric rings formed by NBD1 and NBD2, both acting in the same direction for vectorial translocation of the unfolded protein substrate through the central channel. The channel is lined with highly dynamic pore loops containing conserved tyrosine residues, implicated in binding unfolded substrate and translocating it through the channel (Saibil, 2008; Barends et al., 2010). Hsp100 chaperones act as unfoldases – hydrolysis of ATP is coupled to the mechanical action of unravelling the folded structure, beginning from a loosely folded region of the polypeptide substrate. Members of the Hsp100/Clp family function as chaperone parts of the ATP-dependent Clp proteases (see section 5.2) in plant organelles (Leidhold and Voos, 2007).

Relation of Expression to Function

Some chaperones are constantly expressed and are referred as heat shock cognates (Hsc) – a term that indicates Hsps expressed constitutively to serve protein homeostasis in the absence of stress. They are involved in folding of de novo synthesized polypeptides and the import/translocation of precursor proteins. Proper folding is mediated by Hsc70 for about 10-20% of the nascent proteins; for more complex proteins, especially in organelles (10-15% of the cases), the assistance of the chaperonins is necessary (Liberek et al., 2008). While, in prokaryotes, Hsp70 and Hsp60 chaperones have both housekeeping and stress-responsive roles, in eukaryotes a distinction exists between chaperones linked to protein synthesis and those induced by stress (Liberek et al., 2008). Chaperone concentrations are highly responsive to diverse stresses. The induction of chaperones is regulated by heat shock transcription factors (HSFs) which interact with the heat shock element sequence in the promoter region of the HSP genes. HSFs are expressed constitutively but are stored in inactive form in the cytoplasm. Stress provokes oligomerization of HSFs, with translocation to the nucleus (Grigorova, 2010). Some Hsps are expressed predominantly under stress, their major function being to assist refolding/ degradation of stress modified proteins. Hsp100/Clp proteins perform essential functions in plants and are constitutively expressed, however their expression is also developmentally regulated and induced by environmental stresses such as heat, cold, drought, salinity, dark-induced etiolation, as well as in the post-stress phase during recovery from stress (Wang et al., 2004). Hsp100 chaperones act in collaboration with Hsp70 and sHSPs to rescue proteins from stress-induced aggregation (Liberek et al., 2008).

Involvement of Chaperones in Response to Drought

Generally Hsps are expressed under normal conditions and accumulate in response to high temperature, nevertheless they could be induced or increasingly expressed to protect against various other stresses including drought (Shinozaki and Yamaguchi-Shinozaki, 1996; Campalans et al., 2001). Feder and Hofmann (1999) postulated that all known stresses, if sufficiently intense, induce Hsp expression. It has been demonstrated that Hsps can be induced by water deficit, preventing protein aggregation and denaturation during the stress; they may also play a role in desiccation tolerance (Almoguera and Jordano, 1992; Alamillo et al., 1995). The drought induced Hsps with chaperone function include different classes of Hsps - Hsp100 or Clp, Hsp90, Hsp70, Hsp60 and sHsps below 30 kDa (Wang et al., 2004).

The effect of drought on gene expression of some Hsps has been analyzed in tobacco plants and Arabidopsis thaliana by Rizhsky et al. (2002, 2004). The highest Hsp expression was established under combined drought and heat stress in wheat plants (Grigorova et al., 2011a, 2011b). During drought stress, alone or combined with high temperature stress, the drought-tolerant cultivar exhibited higher Hsp70 and sHsp contents than the sensitive cultivar. Recently, Akashi et al. (2011) studied dynamic changes in the leaf proteome of a wild watermelon. Using PCR and immunoblot analyses they found that 15 of the 23 up-regulated proteins under water deficit (65% of annotated up-regulated proteins) were Hsps. Moreover, 10 out of the 15 up-regulated Hsps belonged to the sHsp family. Other stress-induced proteins included those related to anti-oxidative defence and carbohydrate metabolism. According to the authors these observations suggest that the defence response of wild watermelon may involve orchestrated regulation of a diverse array of functional proteins related to cellular defence and metabolism, of which Hsps may play a pivotal role in protecting the plant under water deficit (Akashi et al., 2011). The protective chaperone activities of Hsp70 help to confer tolerance to heat, glucose deprivation, and drought. Overexpression of Hsp70 in many organisms correlates with enhanced thermotolerance, altered growth, and development. In antisense transgenic Nicotiana tabacum plants subjected to heat stress and drought, the results suggested the indirect functions of Nt-Hsp70 in defence mechanisms (Cho and Choi, 2009). Cho and Hong (2006) considered that over-expression of tobacco NtHsp70-1 contributes to drought-stress tolerance. Alvim et al. (2001) also detected enhanced accumulation of Hsp70 in transgenic tobacco plants after a reduction of its relative water content to 65%, which confers tolerance to water stress.

Pareek et al. (1997) found two proteins from the Hsp90 family that were expressed on exposure of rice seedlings to water stress and elevated salinity. Three At-Hsp90 isoforms - cytosolic AtHsp90.2, chloroplast-located At-Hsp90.5 and endoplasmic reticulum (ER)-located At-Hsp90.7 - were characterized by constitutively overexpressing their genes in Arabidopsis thaliana (Song et al., 2009). It was concluded that the overexpression of At-Hsp90 isoforms enhances plant sensitivity to salt and drought stresses. The overexpression of At-Hsp90 isoforms may shift the equilibrium of Hsp90s with their client-bound states, disrupt ABA-dependent or Ca²⁺ pathways, and thus impair plant tolerance to abiotic stresses, suggesting that proper homeostasis of Hsp90 is critical for a cellular stress response and/or tolerance in plants.

Sato and Yokoya (2008) suggested that overproduction of sHsp17.7 could increase drought tolerance in transgenic rice seedlings. The authors observed that, although no significant difference was found in water potential of seedlings between transgenic lines and wild-type plants at the end of drought treatments, only transgenic seedlings with higher expression levels of sHsp17.7 protein could resume growth after rewatering. Overproduction of At-Hsp17.6A could increase salt and drought tolerance in Arabidopsis thaliana (Weining et al., 2001). Wehmeyer and Vierling (2000) proposed that the expression of sHsps suggests a general protective role in desiccation tolerance.

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