Stress Responses: Heat

Stress Responses: Heat☆ SL Gomes, Universidade de Saõ Paulo, Saõ Paulo, Brazil RCG Simaõ, Universidade Estadual do Oeste do Parana´, Cascavel, PR, Brazil ã 2014 Elsevier Inc. All rights reserved.

Defining Statement Introduction Control of Heat Stress in Bacteria Positive Control by Alternative Sigma Factors The E. coli s32 regulon An mRNA thermosensor Control of s32 stability Role of DnaK in the turnoff of the HS response rpoH orthologues in other Gram-negative bacteria The sE regulon Orthologues of rpoE Negative Regulation of HS Genes CIRCE/HrcA HrcA acts as a repressor The repressor HspR The regulatory element ROSE The repressor RheA CtsR Bacterial HS Response Induced by Antibiotic-Stress HS response induced by antibiotic in Gram Positive Bacteria HS response induced by antibiotic in Gram Negative Bacteria HS Response in Archaea HSPs HS Control in Archaea The archaeal repressor Phr VapBC in Archaea Heat Stress in Eukaryotic Cells HSP Families The HSP100 family The HSP90 family The HSP70 family The HSP60 family The HSP27/28 family Proteases induced by HS Control of HSP Gene Expression in Eukarya Sensing the stress Final Remarks

Glossary Homologues Genes sharing common ancestry, often determined by structural comparison. HSE Heat shock element, it is a sequence in the promoter region of heat shock genes to which HSF binds. HSF Heat shock transcription factor. HSP Heat shock protein, chaperones, proteases, and regulatory proteins produced in response to temperature changes and other stresses.

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MDR Multidrug resistant microorganism. Operon Genes within the same transcriptional unit under the control of a common promoter element. Regulon A set of genes under the control of a common regulator. Repressor A protein or a regulatory element that inhibits transcription or translation, respectively. Sigma factor It is the subunit of bacterial RNA polymerase needed for initiation, it is the major influence on selection of promoters.

Change History: August 2014. SL Gomes and RCG Simaõ have updated the text and further reading.

Reference Module in Biomedical Research

http://dx.doi.org/10.1016/B978-0-12-801238-3.02459-4

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Stress Responses: Heat

Abbreviation CIRCE ECF ER HAIR HS HSE

controlling inverted repeat of chaperone expression extracytoplasmic function endoplasmic reticulum HspR-associated inverted repeat heat shock heat shock element

HSF HSPs RNAP ROSE SD TBP TFB

heat shock transcription factor heat shock proteins RNA polymerase repression of heat-shock gene expression Shine–Dalgarno TATA element-binding protein transcription factor B

Defining Statement All living cells, from microorganisms to human cells, respond to a temperature upshift by transiently increasing the rate of synthesis of a set of proteins denominated heat shock proteins (HSPs), which maintain protein homeostasis by alleviating protein misfolding defects and aggregation, thus protecting the cells from damage. The increase in HSP levels is mainly due to the induction of transcription of the corresponding genes. Although the HSPs are among the most conserved proteins in nature, the mechanisms responsible for the transcriptional control of heat shock (HS) genes vary greatly. Bacteria, for instance, can control HS gene expression either negatively or positively, using either repressors that act by inhibiting HS gene expression at normal temperatures being inactivated at high temperatures, or alternative sigma factors that once activated induce transcription during heat shock, or in some bacteria both mechanisms take place. HS response has also been linked to antimicrobial resistance in pathogenic bacteria. In eukaryotic microorganisms like Saccharomyces cerevisiae induction of the HS response occurs by the activation of a heat shock transcription factor (HSF1) which exists constitutively as a trimer bound to a specific DNA element, the heat shock element (HSE), present in multiple copies in HS gene promoters. In higher eukaryotes a family of HSFs is observed, with mammalian cells containing four members: HSF1, HSF2, HSF3 and HSF4. Each of them possesses unique and overlapping functions, exhibiting tissue-specific patterns of expression, multiple post-translational modifications and interacting protein partners. These HSFs are present as monomers under physiological conditions, differently from S. cerevisiae, with HSF1 being the most potent and dominant. Activation of HSF1 occurs by the formation of trimers and extensive post-translational modifications, followed by its binding to the HSEs in the HS gene promoters. The control of HS gene expression in Archaea is not well understood, but a repressor has been characterized which is inactivated by a temperature upshift. HSPs have shown to be involved in addition to the HS response in the control of the cell cycle. The evidence being that accumulation of misfolded proteins in both bacteria and yeast, for instance, has been observed to cause G1 arrest. HSPs have also been strongly associated with microbial infections and with human diseases such as cancer and neurodegenerative diseases making the study of these proteins of even greater importance.

Introduction The viability of all organisms crucially depends on the integrity of its constituent components, correctly folded proteins being one of the most important actors in all cellular processes. The folding state of proteins within cells is monitored by an energy-dependent quality control network of ATP-dependent proteases and molecular chaperones, which operates under all growth conditions but becomes extremely important during stress. In order to maintain homeostasis for protein folding, cells tightly regulate the expression of chaperones and proteases to compensate for environmental perturbations. Exposure of cells and organisms to high temperatures can cause misfolding and aggregation of proteins, which if not acted upon can lead to cell death. The heat shock (HS) response is the way by which cells counteract to prevent the potential damages due to temperature upshift and consists in the induction of almost all the universally conserved HS genes. These genes encode chaperones, proteases, and other stress-related proteins, which play important roles in helping cells cope with the toxic effects due not only to high temperature exposure but also to several other environmental stresses, such as starvation for carbon or amino acids, DNA-damaging agents, oxidative stress, and heavy metals. The first report of the HS response was by Ritossa (1962) who discovered the heat inducible chromosome puffs in the salivary glands of Drosophila melanogaster larvae. Subsequently, the puff-associated genes and proteins were identified initiating a rapidly expanding research field on HS. In bacteria, the HS response of Escherichia coli was the first to be characterized and the master regulator, the HS sigma factor s32, has been the first alternative sigma factor to be discovered in this organism. Later, many other bacteria have had their responses to high temperature investigated, and different control mechanisms have been revealed for the induction of HS genes, showing that even though the response is almost universally conserved, the molecular solutions for triggering gene induction can be quite distinct. The sequential molecular events in the HS response include drastic repression of normal transcription and translation pathways, and activation of transcription of HS genes. The increased expression of HS genes is transient, and as the heat shock proteins (HSPs) accumulate and repair or prevent the damages caused by temperature stress through their chaperone and protease activities, the


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cells can return to nearly normal growth conditions, downregulating HSP gene expression. Both prokaryotic and eukaryotic cells have established a number of complex strategies to tightly control the transcription of HS genes under any given condition and here we will show some of these mechanisms of control. In addition, the function of HSPs will also be discussed.

Control of Heat Stress in Bacteria Positive Control by Alternative Sigma Factors The HS response in the Gram-negative bacterium E. coli is the best-characterized stress response in bacteria, and its regulation involves the use of minor sigma factors. The HS sigma factor s32, also called sH, was the first alternative sigma factor to be uncovered in E. coli and it controls the major HS regulon in this bacterium, coping with cytoplasmic protein damage. The second HS regulon is controlled by sigma factor sE and protects cells against extracytoplasmic stress or extreme HS in E. coli. A third regulon of the stress response has been identified more recently and is under the control of sN or s54. This regulon includes the phage shock operon (pspABCDE) and the small HSP encoded by the ibpB gene. Its regulation is not well understood and will not be discussed further.

The E. coli s32 regulon

The s32 regulon includes the major HSPs, encoded in operons or single genes, such as DnaK, DnaJ, GrpE, GroES, GroEL, ClpB, ClpX, ClpP, Lon, FtsH, and several other proteins. The most important chaperone machines such as DnaK/DnaJ/GrpE, ClpB, and GroES/EL, which protect the cell against cytoplasmic stress, belong to this regulon, as well as the proteases ClpXP, Lon, and FtsH, which degrade the irreversibly denatured proteins unable to be recovered. Expression of these HSPs is rapidly enhanced upon temperature upshift due to the transient increase in the levels of s32, which occurs mainly by a higher rate of synthesis and temporary stabilization and activation of the HS sigma factor. The increased s32 levels leads to the formation of s32-RNA polymerase (RNAP) holoenzyme, which transcribes the HS genes. The higher rate of synthesis of s32 during HS is not due to increased transcription of the rpoH gene, which encodes the HS sigma factor, since its mRNA levels show only a small increase during temperature upshift. Even though the regulatory region of E. coli rpoH is rather complex, containing several distinct promoters, none of them is dependent on s32 itself.

An mRNA thermosensor The translational control of s32 is mediated by the secondary structure of the rpoH mRNA. Deletion analysis of a translational fusion rpoH–lacZ revealed two regions near the 50 -end portion of the mRNA (regions A and B) involved in thermoregulation. Region A (6–20 nt from the AUG initiation codon) and region B (112–208 nt) form a secondary structure by base pairing, which includes the AUG preventing ribosome binding and translation of the mRNA at normal temperatures (Figure 1). At HS temperatures, the base pairing is destabilized enhancing the translation of rpoH mRNA. Experimental evidences for such an RNA structure were generated by point mutations, deletion analysis, and structure probing correlating the RNA thermostability with the corresponding expression levels of s32. Furthermore, no evidence for the contribution of additional cellular factors was found suggesting the RNA structure itself as the molecular thermometer.

Control of s32 stability

During normal growth temperatures s32 is quite unstable, with a half-life of less than 1 min. However, a remarkable (at least eightfold) transient stabilization of the protein occurs immediately upon temperature upshift, which is responsible at least in part for the practically instantaneous induction of HSP synthesis. Degradation of s32 can be carried out by several proteases, however, FtsH, an ATP-dependent metalloprotease located in the cytoplasmic membrane, was the first to be implicated in this process. In addition, the absence of FtsH causes complete stabilization of s32 in vivo. Furthermore, the DnaK/DnaJ/GrpE chaperone machine seems to be involved in the turnover of s32, as absence of any of these proteins causes stabilization of the sigma factor at normal temperatures. However, in vitro degradation of s32 by FtsH is not affected by any of the members of DnaK chaperone system, indicating that the effect may be indirect.

Role of DnaK in the turnoff of the HS response Changes in the levels of DnaK affect not only the induction of the HS response in E. coli, but also the downregulation of the response. Cells with low levels of DnaK have a prolonged shutoff phase, whereas cells with high levels of this chaperone turn off the response more rapidly. Negative regulation of HSP gene induction by DnaK is mediated primarily by its direct association with s32, which leads to sigma factor inactivation and degradation by FtsH. This effect has been explained by the titration of DnaK by unfolded proteins during temperature upshift. When unfolded proteins are in excess relative to DnaK, DnaK dissociates from s32 to exert its chaperone activity. Free s32 then binds the RNAP core and transcription of the HS genes increases drastically, the dnaKJ genes among them. With the accumulation of DnaK/DnaJ and other HSPs, unfolded proteins are correctly folded, liberating DnaK to associate again with s32 inactivating it, and leading to sigma factor degradation and the turn off of the HS response (Figure 2). Recently, the involvement of the chaperones GroES/EL together with the DnaK chaperone system in the regulation of the HS response in E. coli has been described, indicating that a cellular network regulates the activity of the HS sigma factor s32.

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Figure 1 RNA thermometer controls the expression of rpoH gene. (a, b) Expression of Escherichia coli heat shock transcription factor s32, encoded by the rpoH gene, depends on five promoters differently modulated by three sigma factors (s70, sE, and s54) and the secondary mRNA structure is involved in its translational control. (b) At physiological temperatures (30 C), the Shine–Dalgarno (SD) sequence, the AUG start codon, and two specific regions (A and B) in the coding region of the rpoH gene generate a secondary structure that represses the translation process. (c) Melting of the secondary structure at high temperatures (42 C) permits ribosome access and translation initiation.

rpoH orthologues in other Gram-negative bacteria Orthologues of rpoH have been described in many other proteobacteria of a, b, and g subgroups, but not in the subgroups d and e or in Gram-positive bacteria. In most cases, only one rpoH gene has been found, but in some two or even three genes were characterized. In Agrobacterium tumefaciens and Caulobacter crescentus, bacteria from the a group, a single rpoH gene has been found. Interestingly, a marked increase in transcription is observed for the rpoH gene during HS in these bacteria, and the 50 portion of its mRNA is not predicted to form a secondary structure, unlike the situation observed in E. coli and other bacteria of the g subgroup. The increased transcription observed for the rpoH gene in Caulobacter and Agrobacterium is due to the presence of an RpoHdependent promoter. However, an essentially normal induction of HSPs was observed in Agrobacterium even with mutant strains unable to increase RpoH levels upon HS. These data indicate that controlling the activity rather than the amount of s32 is the most important mechanism for the induction of the HS response in this bacterium. Furthermore, even though s32 levels increase transiently during HS, DnaK-mediated control of s32 activity was demonstrated to play a primary role in the induction of the HSP synthesis. In addition, in Caulobacter, it was shown that downregulation of the response is independent of DnaK levels, whereas the chaperone ClpB is necessary for normal turnoff of HSP synthesis. This important role of ClpB in the shutoff of the HS response was suggested to be related to the capacity of this chaperone to renature the major Caulobacter sigma factor, names s73, in conjunction with the DnaK chaperone machine, which would then compete with s32 for the RNAP core, leading to the recovery of normal protein synthesis (Figure 2(c)). The root-nodulating bacteria Bradyrhizobium japonicum and Sinorhizobium meliloti contain three and two rpoH genes, respectively, which are regulated by distinct mechanisms. In B. japonicum, each rpoH gene is regulated differently. Transcription of rpoH1 is HS inducible by an unknown mechanism, and rpoH2 transcription decreases during HS, with both genes being transcribed from sigma 70-dependent promoters. The rpoH3 gene is in a probable operon with two genes coding for a classical two-component regulatory system, with transcription initiating from a promoter similar to s32-dependent promoters. In addition, the distinct RpoH proteins transcribe different sets of genes. RpoH2 seems to be essential for the synthesis of cellular proteins under physiological growth conditions, whereas RpoH1, and probably also RpoH3, are involved in their synthesis during the stress response. In S. meliloti, cells containing an rpoH1 gene mutation are impaired in growth at 37 C under free-living conditions and are defective in nitrogen fixation during symbiosis with alfalfa. In addition, expression of rpoH1 increases upon culture entry into the stationary phase of growth, but not under HS conditions. Cells containing an rpoH2 mutation have no apparent phenotype,


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Figure 2 Induction and downregulation of the heat shock (HS) response in bacteria. (a) The direct DnaK association with s32 leads to heat shock transcription factor inactivation and degradation by FtsH at normothermic temperatures. (b) Under HS stress, unassembled proteins are in excess, DnaK dissociates from s32 and exerts its chaperone activity. The transcription complex RNA polymerase (RNAP)–s32 is formed and transcription of HS genes increases. In Escherichia coli, the accumulation of chaperones, including DnaK, causes denatured proteins to acquire a correct folding, liberating DnaK to promote the negative regulation of s32 and downregulation of the HS response. (c) In Caulobacter crescentus, changes in the amount of DnaK/DnaJ only affect the induction phase of the HS response. Reactivation of heat-inactivated s73 (s70 homologue) in this bacterium is dependent on ClpB chaperone activity, and the competition between s32 and s73 for the RNAP core seems to be the most important factor during the recovery phase.

and its expression increases later in stationary phase. Since both rpoH1 and rpoH2 are induced in stationary phase, the proteins they encode could play roles in the general stress tolerance that develops in starved cells.

The sE regulon

The second HS sigma factor of E. coli was identified from studies of rpoH transcription at extreme temperatures (45–50 C). One of its promoters (P3) is activated at high temperatures and is dependent on sE, which places the s32 regulon under the control of sE under extreme heat stress. The sE regulon is involved in protection against extracytoplasmic stress, with its members including a periplasmic protease DegP and a periplasmic peptidyl prolyl isomerase FkpA, among others. Even though s32 is essential for E. coli growth only at high temperatures, sE is necessary at all temperatures. Besides heat and ethanol exposure, unfolded outer membrane or periplasmic proteins can induce the sE regulon. During normal growth conditions, activity of sE is inhibited due to its association with a membrane-bound anti-sigma factor, called RseA. Another periplasmic protein, called RseB, is also bound to RseA stabilizing the complex. Under extracytoplasmic stress, misfolded proteins bind to RseB, which releases RseA to be cleaved in its periplasmic domain by DegS. Additional proteolysis of transmembrane and cytoplasmic portions of RseA then frees the sE transcription factor, which directs the transcriptional response to extracytoplasmic stress. Transcription of rpoE is autoregulated, as

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Figure 3 The periplasmic response to heat is regulated by sE. Under normothermic growth conditions, sE is inhibited by the anti-sigma factor RseA. RseB stabilizes the RsaA–sE complex in the absence of heat stress. Perturbations in OMP folding due to extreme heat stress lead to disruption of the RseA–RseB interaction. The inner membrane-anchored DegS protease cleaves RseA in its periplasmic domain, the membrane-embedded protease RseP (YaeL) cleaves RseA near the inner membrane, and the released cytoplasmic RseA fragment is further degraded by protease ClpXP. The transcription factor sE is released in the cytoplasm and interacts with RNA polymerase core enzyme to promote transcription of genes involved in OMP assembly and degradation.

the operon rpoE-rseA-rseB has a sE-dependent promoter. Thus, once the stress conditions are no longer present, sE is again bound to RseA and inactivated, and the response is turned off (Figure 3).

Orthologues of rpoE Homologues of sE have been classified as extracytoplasmic function (ECF) sigma factors and are present in different numbers in distinct bacteria. Similarly to sE, they are usually found in an operon with the anti-sigma factor gene, which inactivates the ECF sigma factor by binding to it. Under stress conditions, the anti-sigma factor is inactivated by different mechanisms, liberating the sigma factor to bind the RNAP initiating transcription of their regulons. The ECF sigma factors are involved not only in HS but also in a variety of types of environmental stresses, including saline and osmotic stress, oxidative stress, antimicrobial biosynthesis, heavy metal exposure, and bacterial virulence. Interestingly, in alpha-proteobacteria certain ECF sigma factors, whose activation is regulated by a two-component system (sensor histidine kinase/ response regulator), have been shown to control the general stress response. In C.crescentus, for instance, the sensor histidine kinase PhyK is autophosphorylated in response to stress then transferring this phosphate group to the response regulator PhyR, which binds the anti-sigma factor NepR liberating the ECF sigma factor sT to transcribe the general stress response regulon.


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Negative Regulation of HS Genes In contrast to the positive regulation by alternative sigma factors observed only in proteobacteria, negative regulatory systems are more widespread in prokaryotes, typically involving repressor proteins that bind to cis-acting regulatory sites located in the 50 region of the genes encoding chaperones and proteases. Evidence for additional regulatory mechanisms transpired from the finding that several stress genes in Bacillus subtilis, a Gram-positive bacterium, including the dnaK and groE chaperone operons, remained heat inducible in a sigB mutant – the gene encoding an alternative sigma factor involved in stress responses. In addition, inspection of the corresponding promoter sequences revealed typical housekeeping promoters (SigA-dependent promoters) for the HS operons, and analysis of a temperature-sensitive sigA mutant revealed that groESL transcription is SigA-dependent. Transcription from vegetative promoters was subsequently demonstrated in a number of bacteria, and regulatory DNA elements were found in the promoter regions, being proposed as putative repressor-binding sites responsible for transcriptional repression under normal growth conditions. Presently, it appears as if negative control of HS genes is more prominent in eubacteria than positive regulation by alternative sigma factors. HS regulation by HrcA and CIRCE (controlling inverted repeat of chaperone expression), the repressor and its binding site, was the first to be characterized and this regulatory system is widespread in Gram-positive bacteria, proteobacteria, and cyanobacteria.

CIRCE/HrcA A 9-base inverted repeat with a consensus sequence of TTAGCACTC-N9-GAGTGCTAA was first noticed upstream of groESL and/or dnaK operons of mycobacteria, Clostridium acetobutylicum and B. subtilis. Initially thought to be exclusive to Gram-positive bacteria, this regulatory element in now believed to be one of the most conserved in nature. As it had been reported exclusively in association with groE and dnaK operons, it was named CIRCE. However, its presence was more recently detected upstream of some other HS genes indicating that this element can have a greater regulatory potential than initially expected. CIRCE is frequently located in the DNA region corresponding to the 50 -untranslated region of the mRNA, but it can also be positioned upstream or overlapping the transcription start site. The variability in the position of CIRCE in conjunction with the observation that this element is able to heat-control transcription from a position upstream of the promoter indicates a role mainly of the DNA rather than of the mRNA level. A negative role for CIRCE in the expression of HS genes was proposed based on the observation that mutations in this element resulted in elevated transcription of the genes located downstream from it.

HrcA acts as a repressor The elucidation of CIRCE as a potential repressor-binding site began with a search for the cognate repressor. Evidences showing that a deletion mutant in the first gene of the B. subtilis dnaK operon (orf39) resulted in elevated levels of groE mRNA and that regulatory mutants affecting B. subtilis groE and dnaK expression were mapped in orf39 strongly suggested this gene as encoding the CIRCE cognate repressor, which was named HrcA for heat regulation at CIRCE. Subsequently, the binding of HrcA to CIRCE was investigated by gel shift assays, and it was shown that the presence of GroEL is important for HrcA to acquire its active repressor conformation, both in vitro and in vivo. These results led to the proposal of a GroEL titration model for the CIRCE/HrcA regulatory system. In this model, HS leads to depletion of the GroEL pool due to the necessity of refolding denatured proteins. This depletion would cause inactivation of HrcA and derepression of transcription of the chaperone genes regulated by the CIRCE/HrcA system. Once the levels of the chaperones increase, sufficient amounts of GroEL would be present in the cell and HrcA would be activated and capable of binding to CIRCE again, turning off transcription of groE and dnaK operons (Figure 4). Interestingly, in certain proteobacteria including A. tumefaciens, B. japonicum, C. crescentus, and Xylella fastidiosa, both rpoH and CIRCE/HrcA are present regulating groESL operons, but in no other HSP gene. The groE genes are negatively controlled by HrcA at normal temperatures and positively regulated by rpoH during HS.

The repressor HspR HspR is an autoregulatory repressor protein discovered initially in Streptomyces, a genus of the high G + C actinomycete bacterial group. The HspR repressor/operator system was later found in other actinomycetes including Mycobacterium tuberculosis and in Helicobacter pylori and Aquifex aeolicus. However, HspR is much less widespread than HrcA, in particular it is not found in any low G + C Gram positive as B. subtilis. HspR acts as a repressor by binding at the HspR-associated inverted repeat (HAIR) site (consensus sequence: 50 -TTGAY-N7-ACTCAA-30 ) within the promoter regions of the genes and operons it controls. In Streptomyces coelicolor, studies in vitro and in vivo have provided evidences the DnaK functions as a corepressor of the HspR regulon, which is shown to include lon and the dnaK and clpB operons. Thus, DnaK negatively regulates its own expression. Although 17 genes were shown to be upregulated more than twofold in a S. coelicolor hspR disruption mutant, transcriptome and genome sequence analyses indicated that the above three transcription units are probably the only direct targets of HspR in this bacterium. Interestingly, in the genus Streptomyces besides the HspR/HAIR regulatory system, the HrcA/CIRCE system has also been described regulating the groEL1/ES operon and the groEL2 gene, and the RheA repressor protein (which will be described below) negatively regulating the transcription of hsp18 in Streptomyces albus. This is a clear example of multiple regulatory systems acting in conjunction to control HSP gene expression in bacteria.

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Figure 4 The activity of the repressor HrcA depends on the GroEL system. In the absence of heat stress, GroESL converts the HrcA protein into its active form. HrcA binds controlling inverted repeat of chaperone expression (CIRCE) regions on heat shock (HS) genes inhibiting the RNA polymerase holoenzyme access. During temperature upshift, unfolded proteins titrate the GroESL chaperone system, leading to an increase in the amount of inactive HrcA. HS gene transcription is induced leading to activation of repressor HrcA that binds to CIRCE turning off transcription of the HS operons.

The regulatory element ROSE The cis-acting regulatory element repression of heat shock gene expression (ROSE) contains a conserved sequence of about 100 nucleotides in the 50 -untranslated region of multiple small HS genes including rpoH in several bacteria. The inhibitory effect of ROSE is based on experimental evidences that indicated this regulatory element as a putative site to bind to a repressor protein. Extracts from non-HS cells of B. japonicum retarded full-length ROSE in gel shift assays. However, a regulatory protein with a potential ability of binding DNA containing ROSE element was never isolated. In fact, computer calculations indicated that ROSE predicted to fold into a complex structure occluding both the Shine–Dalgarno (SD) sequence and the translation start site AUG, suggesting the possibility of ROSE acting as a RNA thermosensor similar to the rpoH thermometer. The thermoresponsive structures folded in the 50 -UTR region of mRNAs are known to control translation of HS and virulence genes in bacteria. The potential role of this RNA structure was determined by mutational studies with ROSE cloned upstream the hspA–rpoH operon from B. japonicum. Mismatches introduced into unpaired regions of the stem-loop IV of the thermometer structure were completely neutral, but when mismatches were introduced in the paired regions of the same loop, an increased expression of a translational lacZ fusion was observed. Besides this, compensatory mutations restored repression of translation. The correct function of ROSE thermometer depends on the presence of a conserved G residue opposite to the SD, its elimination made the thermosensor irresponsive to high temperatures. The G residue closes the loop of the thermometer by a weak GG base pair that breaks at elevated temperatures making the ribosome-binding site accessible to initiation of the translation process.

The repressor RheA An open reading frame (rhea) located 150 bp uspstream in the opposite orientation of the small HS gene hsp18 participates in negative regulation in the Gram-positive bacterium S. albus. The small HSP, Hsp18, is involved with acquisition of thermotolerance in the bacteria of Streptomyces genera. The S. albus rheA-null mutant displays high hsp18 mRNA levels at physiological temperature. On the other hand, overexpression of the rheA gene restores repression of hsp18 in rheA mutant cells. Curiously, the hsp18 mRNA is translated only after temperature


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upshift, suggesting a posttranscriptional control to hsp18 gene expression in this bacterium. One possible model for this posttranscriptional control involves changes in mRNA conformation as described for the ROSE element and the rpoH gene. Denaturation of the secondary structure of the mRNA would therefore lead to an increase in ribosome binding and translation initiation. Two similar inverted–repeated sequences are present in the promoter region of the hsp18 gene (GTCATC-N5-GATGAC) and in the 50 untranslated region of the rheA gene (GTCGTC-N5-GATGAC), indicating that both the genes are controlled in a coordinated way and probably by the same regulator. In gel shift assays, when DNA/RheA sample was incubated under normothermic temperature, a shift was observed in the mobility of the DNA fragment. However, no complex formation was observed at HS temperatures. In fact, RheA is an autoregulatory protein and its activity is inhibited at elevated temperatures, but the temperatureinduced derepression by RheA is reversible. At low temperature, RheA binds its target in the promoter region, preventing hsp18 expression. At high temperature, RheA is inactivated derepressing the hsp18 gene. Then, RheA acts as a cellular thermometer in hsp18 expression, sensing increases in temperature and derepressing hsp18 gene transcription.

CtsR The CtsR regulon consists of three transcriptional units: the tetracistronic clpC operon containing ctsR, mcsA, mcsB, and clpC genes and two monocistronic clpP and clpE operons. The clpC and clpE genes code for ATPase subunits and clpP codes for a proteolytic subunit of the two ClpCP and potential ClpEP ATP-dependent proteases. McsA and McsB are kinases that act as modulators of CtsR. In Gram-positive bacteria, CtsR is a repressor that controls the class III HS genes, class I genes are regulated primarily by the repressor HrcA, class II genes are regulated by the alternative sigma factor sB, and the two-component system CssRs controls the class V genes. The repressor CtsR is a dimeric protein that contains a classical helix-turn-helix DNA-binding motif, which binds to a highly conserved heptanucleotide direct repeat (A/GGTCAAAnAnA/GGTCAAA), located upstream of clpP, clpE, and the clpC operons. Besides this, CtsR contains two other functional domains: a dimerization domain (the first 24 domains) and the central glycinerich region (aa 64–67) involved in heat sensing. All three transcriptional units regulated by CtsR are preceded by two promoters each, clpE by two sA-dependent promoters and clpC and clpP operons by sB- and sA-type promoters. The dimeric protein CtsR binds the 35 and 10 regions of the sA promoter preventing the association of the sA RNAP holoenzyme to it. At physiological temperatures (37 C), the expression of CtsR maintains a certain steady-state level, which leads to a low expression of the genes from CtsR regulon. Upon exposure to heat stress, not only a rapid degradation of CtsR by ClpCP proteases occurs but also an induction in the expression of HS genes. ClpE was reported as a new independent type of the HSP100 ATPase belonging to the CtsR regulon in Gram-positive bacteria. ClpEP was found to destabilize CtsR after HS in vivo. Coherent with this, it was recently reported that a clpE-null mutant displays a significant delay of protein disaggregation and retardation of CtsRdependent gene induction. All the genes of clpC operon are involved in the regulation of the activity of CtsR. McsB is at the center of a network interacting with the McsA, ClpC, and CtsR. McsB is a tyrosine kinase, which utilizes a guanidine kinase domain, known only from eukaryotic kinases. McsB needs to be activated by McsA, which becomes concurrently phosphorylated by McsB. McsA activates the kinase activity of McsB that when phosphorylated causes the phosphorylation of CtsR and a stronger CtsR–DNA-binding inhibiting activity. On the other hand, ClpC inhibits the kinase activity of McsB. Upon HS in vivo, CtsR is degraded and this HS-induced degradation depends on the presence of McsA.

Bacterial HS Response Induced by Antibiotic-Stress Pathogenic bacteria have the ability to persist in the human host and medical environment where they encounter an excess of stressors like heat shock and antimicrobials. Heat shock response activation by antimicrobials has been reported in different Gram negative multi drug resistant bacteria (MDR) such as E. coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Burkholderia cenocepacia and in Gram positive bacteria such as Staphylococcus aureus and Streptomyces coelicolor. A large amount of recent data have shown that molecular chaperones clearly play important roles in bacterial stress tolerance, but their role in antibiotic tolerance is still starting to be clarified.

HS response induced by antibiotic in Gram Positive Bacteria In S. aureus, a knockout in the dnaK gene increased the susceptibility of the meticillin-resistant strain COL to the antibiotics oxacillin and methicillin. Chaperones are known to be involved in responses to antibiotics because they are induced when the cell wall is subjected to antibiotic stress in S. aureus. On the other hand, ciprofloxacin treatment in S. coelicolor caused reduced levels of the chaperones GroESL and DnaK at both, transcript and protein levels. In a transcriptomic study of S. coelicolor (strain A3-2) in the presence of ciprofloxacin the htpG gene was up regulated. This increase is supposed to occur in compensation of the decrease in GroESL and DnaK proteins levels. The role of HtpG chaperone has not been clearly defined in bacteria yet. However, it is suggested that HtpG could become the primary folding mechanism under stress caused by antimicrobials, when other chaperones are down regulated. Despite the data mentioned above, recent proteomic studies for S. coelicolor indicated the down regulation of the chaperone HtpG and ClpB upon exposure to ciprofloxacin. In this bacterium, ClpB and DnaK belong to the same regulon, reflecting their functional cooperation.

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HS response induced by antibiotic in Gram Negative Bacteria Antimicrobial peptides (AMPs) are antibiotic agents commonly found in animals, plants and microorganisms, and they have been suggested as the future of antimicrobial chemotherapies. Resistant and susceptible strains of E. coli ATCC 8739 to the AMP named magainin I, were compared by proteomic analysis. The authors report the observation that alterations of DnaK and GroEL expression in the metabolism of resistant strains occurred only in a non-stress environment. The arousing decrease in the levels of the intact forms of the chaperones DnaK and GroEL and the increase of their forms with reduced molecular masses were also observed only in the magainin-resistant strains, showing that individual metabolic alterations could be occurring among the resistant strain group suggesting that in E. coli could take place posttranslational modifications induced by AMPs in the HSP group. Quantitative proteomic was also used to compare the expression programs of two clonal isolates of B. cenocepacia retrieved from patient presenting chronic respiratory infection. The isolate IST439 was the first bacterium recovered while the clonal variant IST4113 was obtained after three years of persistent infection and intravenous therapy with ceftazidime/gentamicin antimicrobials. However, both isolates exhibited a MDR profile towards different classes of drugs. A higher content of the molecular chaperones ClpB, DnaK and GroEL was observed in the isolate IST4113 compared with IST439 and it is also consistent with higher robustness of isolate IST4113 against environmental stresses and in particular antibiotic-induced stress. In the case of A. baumannii (ATCC 19606), cells pretreated for 30 min at heat shock temperatures (45 C) proved to be more thermotolerant and resistant to aminoglycoside streptomycin than bacteria pretreated at physiological temperatures (37 C). In addition, DnaK levels increased more than 4-fold after 1 hour of exposure to a subinhibitory concentration of streptomycin, and GroEL levels doubled. Incubation of a MDR A. baumanii RS4 strain in the presence of different antibiotics resulted in a significant increase in the production of DnaK mRNA and protein. A similar result was observed by comparative proteomics for isolates of A. baumannii under carbapenen stress, which increased the synthesis of three different chaperonins. Multidrug-resistant A. baumannii strains have been considerably studied at nucleic acid sequence and proteomic level. The proteome of the multidrug resistant strain BAA-1605 was compared with the proteome of the drug-sensitive strain ATCC 17978. Not all stress-related proteins are more abundant in the MDR strain, but ClpA/B chaperone and DnaK were 7-fold and 2-fold higher in abundance in the MDR strain than in the drug-sensitive strain, respectively. In P. aeruginosa, for instance, both the aminoglycoside tobramycin and heat shock induce the expression of Lon protease AsrA mediated by the heat shock sigma factor s32. However, overexpression of the asrA transcript makes P. aeruginosa cells just moderately resistant to the aminoglycoside. The reason for the connection between the heat shock response and aminoglycosides is the fact that this antimicrobial impacts cytosolic protein folding leading to accumulation of insoluble proteins in the cell. Overexpression of GroESL from E. coli (strain MG1655) reduced protein misfolding and rescued the membrane potential, cell growth and survival. In contrast, inhibition of GroESL expression increased aminoglycoside sensitivity allowing the collapse of the membrane potential abridging cell life. Overexpression of the DnaK/DnaJ/GrpE chaperone system increases survival, but less efficiently than the GroES/EL system because it does not promote growth of aminoglycoside-exposed bacteria. Thus, it is supposed that HSPs can eliminate unfolded proteins targeting the atypical mistranslated polypeptides that are produced by aminoglycoside-disrupted ribosomes and that insert into and disturb bacterial membranes. Aminoglycoside-mediated membrane damage has been described and is supposed to be a key-step in the lethal activity of these agents. Then, elimination of atypical polypeptides would therefore reduce their toxicity to bacterial cells. In fact, HSPs help bacteria cope with early exposure to aminoglycosides (Figure 5).

Figure 5 Chaperone effects on bacterial survival during early aminoglycoside exposure. (a) Aminoglycoside uptake and binding to ribosome causing translational misreading. Misread polypeptides are encountered by chaperones acting in succession. Limiting chaperone capacity causes protein misfolding, membrane permeabilization, depletion of essential cytosolic functions and cell killing. (b) In contrast, chaperone availability (mainly GroESL) ensures protein folding and increased bacterial survival (KJ: component of DnaK-DnaJ-GrpE chaperone system).


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HS Response in Archaea HSPs The three domains of life on Earth include the two prokaryotic groups, Archaea and Bacteria. The Archaea domain presents three phyla, Crenarchaeota, Euryarchaeota, and Korarchaeota. All of them are distinguished from Bacteria based on differences in tRNA and rRNA sequences, in the cytoplasmic membrane and cell wall composition, in the transcription and translation apparatus, and their restriction to unusual habitats. In addition, members of archaea present a general capacity to dominate or out-compete bacteria in niches in which chronic energy stress is a characteristic, including environmental stresses (temperature, acidity, and salinity) and low energy availability. The ability to cope with environmental stress is essential to all microorganisms, including those thriving in extreme temperatures like hyperthermophiles archaea, they also have thermal limits and display a classic heat shock-response when thermally stressed. However, genome sequence information indicates that some archaea lack HSPs that were previously known to be ubiquitous in Eucarya and Bacteria. Hyperthermophiles archaea lack several HSPs, to name a few, HSP90, HSP70/DnaK, DnaJ, GrpE, HSP33, and HSP10 homologues. The minimal protein-folding machinery present in these organisms may represent a prototype of antistress systems in early life. However, it remains unclear whether archaea members use other proteins or proteases associated to resolubilize and eliminate the aggregates formed following stress by high temperature. Despite the fact that not all archaea have DnaK or the other component of the KJE (DnaK-DnaJ-GrpE) chaperone system, chaperonins, also known as Hsp60 or GroEL, are present, in the genome of all archaea and their up-regulation is part of multiprotein complexes known as thermosomes during stress by heat shock. Thermosomes are constituted by large oligomeric ring containing 7–9 subunits that assist in the protein folding and other functions. These proteins are more similar to the type II chaperonins found in the eukaryotic cytosol than to the type I chaperonins found in chloroplasts, mitochondria and bacteria. However, some archaea species also possess type I chaperonin homologues Genome analysis in the archaea species has revealed that these organisms do not always contain hsp70 or the other two genes of the chaperone machine triad, hsp40 and grpE. Nevertheless, the full genome sequence of Methanobacterium thermoautotrophicum and Methanosarcina mazei S-6 demonstrates the presence of the genes from the Hsp70 chaperone machinery. The amino acid sequence of the archaeal HSP70 resembles more the bacterial than the eukaryal counterpart. In addition, the activity of M. mazei HSP70 is enhanced by interaction with bacterial cochaperone DnaJ, but not by the eukaryotic homologue, indicating that archaeal hsp70 gene may have been received from bacteria by lateral gene transfer. In addition, in vitro experiments demonstrated functional similarities between the M. mazei HSP70 and E. coli DnaK, but they do not present complete functional resemblance. M. mazei HSP70 functions as a chaperone in luciferase renaturation in vitro, and it requires bacterial-type chaperones like DnaJ and GrpE to perform its function. However, E. coli dnaK mutants were not complemented by the hsp70 M. mazei gene. In the thermoacidophilic archaeon Sulfolobus sulfataricus, Hsp20 gene is heat-inducible and the protein possesses chaperone activity and ability to recognize and bind unfolded proteins to prevent their aggregation in vitro. In addition, S. fulfataricus Hsp20 expression in an E. coli system protects the cells from thermic stress. The Crenarchaeota species has a separate class of HSP60 chaperonins related to the eukaryotic protein and only distantly related to the highly conserved bacterial GroEL. As mentioned above chaperonins are organized into two groups: class I and class II. Class I is found in Eubacteria, mitochondria, and chloroplasts. Class II is found in Archaea and the eukaryotic cytosol. Class II chaperonins are oligomeric proteins that have two rings containing eight or nine subunits each. Some chaperonins are heat inducible, but others are regulated at low temperatures. A Lon protease gene homologous to the bacterial lon gene was also identified in the Crenarchaeota Archaeoglobus fulgidus by sequence analysis, but the ATP-binding domain was absent in the predicted protein. In general, some HSPs are absent in members of the Crenarchaeota, although they are found in some members of the Euryarchaeota. Furthermore, transcription factors that control thermotolerance are missing in crenarchaeotal genomes

HS Control in Archaea Archaeal hsp genes respond to HS by an increase in the production of their monocistronic transcripts, as one would expect for stress genes. Maximum transcript levels are reached after longer exposures to high temperature than those that would induce a peak response in bacteria, in agreement with what is observed in eukaryotes. The mechanism of transcription initiation for archaeal genes differs from those known to operate in bacteria and must involve factors that are not of a bacterial type, thus different from s factors. In fact, transcription initiation in archaea closely resembles eukaryal class II transcription initiation and is mediated by a single RNAP and two general transcription factors, TATA elementbinding protein (TBP) and transcription factor B (TFB). Archaeal cells do not have sigma factors or homologues of eukaryotic heat shock transcription factors (HSFs) or sequences similar to heat shock elements (HSEs), which will be described below, and this suggests that Archaea have evolved a unique mechanism to control the HS response, but the components involved in this process are not obvious until the present time.

The archaeal repressor Phr Interestingly, in the hyperthermophiles Pyrococcus furiosus there is the first transcriptional repressor (Phr) characterized until now in Archaea that shows little similarity to eukaryotic or bacterial regulators. P. furiosus grows optimally near 100 C and undergoes a

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heat shock response at 105 C mediated in part by the repressor Phr. The N- and C-terminal domains in Phr exhibit bacterial and eukaryal features, respectively. In addition, Phr amino acid sequence displays 23% of identity to the human BAG domain. The conserved eukaryotic BAG proteins bind to the ATPase domain of HSP70 and regulate the chaperone activity. In spite the fact that no homologue to HSP70 is encoded in Pyrococcus, HSP60-like chaperonins and AAA+ chaperones are detected and could be potential binding targets of the BAG-like domain in Phr. Phr exists as a homodimer and recognizes a nucleotide sequence overlapping the transcription start site of some HS genes (sequence consensus: 5´TTTAnnnACnnnnnGTnAnnAAAA3´), thereby inhibiting RNAP recruitment. Phr does not affect binding of TBP and TFB to the promoter, but abrogates RNAP recruitment to the TBP/TFB complex at optimal temperature. Three of the nine genes described to be regulated by Phr in P. furiosus are annotated as encoding conserved hypothetical proteins. Two of them have homologues in bacteria and archaea and only one has homologues just in bacteria. This finding suggests a lateral gene transfer from bacteria to P. furiosus genome. Another Phr target, is the gene encoding the enzyme myo-inositol-phosphate synthase that is involved in the production of the compatible solute di-myo-inositol-1,1´-phosphate (DIP). This protein has been shown to accumulate to high concentrations in the cytoplasm of P. furiosus at suboptimal growth temperatures and to stabilize proteins at extreme temperatures. The other proteins regulated by the repressor include a ferritin/ ribonucleotide-like family and a heat shock inducible phosphoesterase that is supposed is involved in the synthesis of organic phosphates. In addition, Phr inhibits specifically cell-free transcription of its own gene, HSP20, and of an AAA+ ATPase. The aaa+atpase and phr mRNA levels are induced after HS and during stationary growth phase in P. furiosus, indicating that the transcription of these genes is also affected by general stress and starvation. By contrast, the levels of the protein Phr are only slightly elevated during heat stress. In vitro experiments have shown that at high temperature (103 C) Phr loses its functional conformation. The dissociation of the protein from its operator sequence may account for the high increase of phr mRNA levels detected after temperature upshift. Then, in Pyrococcus there is a simple model for HS regulation: Phr binds promoter regions of HS genes at normothermic temperature inhibiting transcription by blocking RNAP recruitment. Subsequent release of Phr along with elevated temperatures leads to activation of HS genes.

VapBC in Archaea VapBC (virulence-associated protein BC) proteins belong to toxin-antitoxin (TA) system and are thought to promote survival during environmental stress. TA system consists of an intracellular protein toxin co-expressed with a cognate protein antitoxin. The two component system genes are usually contiguous and encoded on the genome or plasmids. Cognate toxins are inactivated by antitoxin that is susceptible to cleavage by proteases due to its natively unfolded C-terminal domain. Free-toxins are able to modulate protein synthesis acting as ribonucleases. Protein expression in the cell is controlled by the concentration of stable toxin and unstable antitoxin. Three TA families are described in archaea: RebBE, Phd/Doc and VapBC. The VapBC family is the most abundant TA system among prokaryotic genomes and is present in high numbers in the archaea including hyperthermophiles and thermoacidophiles. VapB is the unstable antitoxin and VapC is the stable toxin. VapC toxin has a PIN domain (PitN-terminal) which is suggestive of RNase activity. In the archaea S. solfataricus, for instance, VapC toxin appears to regulate abundance of its cognate antitoxin. In contrast, VapC transcript abundance is reduced when VapB is deleted. Apart from the above, the significance of the TA loci in the Archaea remains unknown at the moment.

Heat Stress in Eukaryotic Cells In eukaryotes, many of the HSPs and molecular chaperones are represented as large gene families with functional homologues in each cellular compartment, including cytosol, nucleus, and specialized organelles, such as endoplasmic reticulum (ER), mitochondria, and chloroplasts. There are families of proteins for HSP100, HSP90, HSP70, HSP60 (which are restricted to mitochondria and chloroplasts), and HSP27/28. Among the questions still outstanding concerning this great number of proteins is to which extent the different homologues vary in their ability to carry out their various chores, such as preventing protein aggregation, maintaining the unfolded state necessary for protein transport among different cellular compartments, disaggregating protein aggregates, and regulation of the HS response.

HSP Families

The HSP100 family The HSP100 protein family includes the HSP104 from yeast and ClpB from bacteria, which are required for induced thermotolerance to extreme temperatures. HSP104/ClpB belongs to the AAA + protein superfamily, which includes the ‘ATPase associated with a variety of cellular activities’ (AAA) proteins and Clp/HSP100 proteins. These two protein classes exhibit considerable sequence homology in their ‘AAA’ domains, which are important for ATP hydrolysis and oligomerization. Usually AAA + proteins form ring-shaped homohexamers, which drive the assembly and disassembly of macromolecular complexes by ATP-dependent remodeling of their substrates.


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The main target of the proteins from the HSP100 family is protein aggregates, and they act together with HSP70 proteins as a bi-chaperone system (HSP104/HSP70 or ClpB/DnaK) in the refolding of aggregated proteins. The cooperation DnaK/HSP70 chaperone system in the disaggregation process was proposed to be either by acting sequentially after ClpB/HSP104 or simultaneously. Protein aggregates of reduced size, generated by ClpB/HSP104, could serve as substrates for DnaK/HSP70, which has a disaggregation activity toward small protein aggregates.

The HSP90 family Concerning the HSP90 family, the abundance and essentiality for cell growth of HSP90 proteins, found in the cytoplasm and ER, suggests that they perform important biological functions in eukaryotes. However, the bacterial homologue HtpG is not found in many bacteria analyzed, and when present it is not essential for growth. Among the major HSPs, HSP90 is unique in its functions, since it is not required for the maturation or maintenance of most proteins in vivo. The majority of its identified cellular targets are signal transducers and cell cycle and developmental regulators whose conformational instability is relevant to their roles as molecular switches. Through low-affinity interactions characterized by repeated cycles of binding and release, HSP90 keeps these unstable signaling proteins poised for activation until they are stabilized by conformational changes associated with signal transduction. Studies of yeast illustrate the specificity of HSP90: at normal temperatures, reduction on HSP90 levels that have no apparent effects on cell growth or metabolism, but can completely abolish signaling through HSP90-dependent pathways. Conditions that cause general protein damage can divert HSP90 from its normal targets to other partially denatured proteins, and due to its dual involvement, HSP90 may link developmental programs to environmental contingency.

The HSP70 family The HSP70 protein family is the best characterized and contains the largest number of members localized in many different compartments of the cell, including cytosol, mitochondria, and ER. The HSP70 is, by far, the most conserved protein in evolution. It is found in all organisms from archaea and plants to humans, and the prokaryotic HSP70 protein DnaK shares approximately 50% amino acid identity with eukaryotic HSP70 proteins. Functionally, the organellar HSP70s are better understood, having roles in protein translocation and folding. They are constituents of what is called the HSP70 (DnaK) chaperone machine, together with the co-chaperones HSP40 (DnaJ) and GrpE. The DnaK and DnaJ members appear to be bona fide chaperones: DnaK prefers to bind unfolded polypeptides and DnaJ binds to the more compact intermediate, termed molten globule. In some instances, the DnaK/ DnaJ proteins appear to bind synergistically to an unfolded substrate and then hand it off to the second most important chaperone system formed by GroEL/GroES (HSP60/HSP10). The DnaJ and GrpE proteins dramatically regulate the otherwise weak ATPase activity of the DnaK partner, with DnaJ specifically accelerating the rate of hydrolysis of the DnaK-bound ATP and GrpE accelerating the ADP/ATP exchange by causing the release of DnaK-bound nucleotide.

The HSP60 family The HSP60/GroEL chaperone machine consists of the GroEL (HSP60) and GroES (HSP10) family members, also called chaperonins. This chaperonin system is restricted to eubacteria, mitochondria, and chloroplasts. The GroEL protein appears to bind unfolded polypeptide substrates when present in the compact molten globule state, which corresponds to a proposed folding intermediate that still exhibits hydrophobic groups. The GroES protein has a pivotal role in substrate maturation by coordinating the ATPase activity of the GroEL subunits. The GroEL subunits are arranged as two stacked heptameric rings with a central cavity where folding takes place. The GroES subunits form a single heptameric ring that binds to one of the GroEL rings regulating its function. ADP is tightly bound to the subunits in the ring adjacent to GroES and with lower affinity in the opposite ring. Unfolded protein binds to GroEL ring opposite to GroES and triggers ADP dissociation from GroEL subunits. This in turn results in the release of GroES. ADP–ATP exchange weakens the affinity for the bound protein. GroES rebinds GroEL in the ATP state and may cover the ring that contains the bound substrate. Cooperative ATP hydrolysis releases the substrate protein for folding in the ring cavity. GroES binding becomes stabilized in the regained ADP state, and partially folded protein may reassociate for another round of interaction. Even though no direct homologues of HSP60 exist in the cytosol, a different class of chaperonins was described in thermophilic archaebacteria (TF55) and found to have homology with the TCP-1 protein present in the cytosol of most eukaryotic cells forming a heterooligomeric ring structure complex named TriC. Although initially proposed as specialized for the folding and assembly of cytoskeletal proteins, it is now becoming clear that TriC has a more general chaperonin function.

The HSP27/28 family An interesting aspect of the HSP27/28 class of proteins, also called small HSP (sHSP), is its structural and functional similarity to that of a-crystallins. They encompass a large number of related proteins that share some structural features common to the lens protein a-crystallin and are present in virtually all organisms. Analyses of several members of this family of proteins have led to the conclusion that in unstressed cells each sHSP has its own tissue-specific expression and particular intracellular localization. In contrast, during stress, most of the sHSPs analyzed are expressed in every cell and at least some of them were demonstrated to have protective functions. This indicates that they belong to the stress-induced machinery that protects against and/or repairs cellular damages. In fact, it has been shown that sHSPs of E. coli cooperate with ClpB and the DnaK system in reversing protein aggregation.

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Proteases induced by HS Many HSPs are proteases or makeup components of a protease system. This indicates that when the chaperone activities of the HSPs are not sufficient to renature the heat-denatured proteins, the proteolytic systems of the cells act by degrading them. In eukaryotes, the bulk of the ATP-dependent proteolysis is carried out by the ubiquitin system. In this system, polypeptides to be degraded are covalently attached to ubiquitin, which is an extremely conserved HSP. In addition, many of the ubiquitin-conjugating enzymes are also under HS or stress regulation. The degradation is mediated by the 26S proteasome, a large machine composed of two different complexes. The 20S core complex forms a hollow cylinder that is composed of four heptameric rings, which harbor the proteolytic active sites in its interior chamber. The substrates need to be unfolded and translocated before they can reach the proteolytic chamber. This task is executed by the 19S complex, which is located at either end of the 20S proteolytic complex. The base of each 19S complex contains AAA + proteins that promote the ATP-dependent unfolding and threading of substrates into the proteolytic chamber of the 20S complex. In prokaryotes, proteolysis of misfolded proteins is mediated by proteasome-like machines, which also consist of an ATPase module (i.e., ClpA and ClpX), and a proteolytic component, which is either covalently attached (i.e., Lon) or diffusible (i.e., ClpP). The peptidase ClpP of E. coli does not exhibit sequence homology to the subunits of the eukaryotic 20S complex. However, it forms a structure of similar architecture, consisting of two heptameric rings that generate a barrel-shaped proteolytic core with active sites hidden in an interior chamber. Access to these sites is controlled by narrow pores, which do not allow the passage of folded polypeptides. Substrate unfolding and translocation is mediated by various HSP100 proteins (i.e., ClpA), which associate with either end of the ClpP core. Recently, it has been observed that eukaryotic cells exhibit homologues of the Lon protease mitochondrially located.

Control of HSP Gene Expression in Eukarya The HS response in eukaryotic cells is positively controlled at the transcriptional level by the ubiquitous HSFs that bind to arrays of 5-bp DNA consensus element nGAAn (HSEs) in the HSP gene promoter regions. A single gene (HSF1) encodes the HSF in the yeast Saccharomyces cerevisiae and in other eukaryotic microorganisms, whereas three to four different HSFs can coexist in plant, avian, and mammalian cells. However, HSF1 is the master regulator in vertebrates. Hsf1-knockout mouse and cell models have revealed that HSF1 is a prerequisite for the transactivation of HSP genes, maintenance of cellular integrity during stress and development of thermotolerance. All HSFs share common structural motifs, including a conserved DNA-binding domain, a trimerization domain, and a C-terminal transactivation domain, but are quite variable between species. Nevertheless, examples of functional interchangeability of HSFs from organisms as diverse as yeast, fruit, fly, and man have been demonstrated. The HSF is synthesized constitutively in all eukaryotic organisms, its activity, however, is regulated post-translationally. Furthermore, there is diversity in the mechanisms of regulation of HSF activity. For example, under nonstressed conditions, metazoans sequester the majority of HSF as chaperone-bound monomers within the cytoplasm. Upon HS, HSF monomers undergo an intramolecular rearrangement that allows HSF subunits to trimerize. HSF trimers then enter the nucleus and bind DNA at the HSEs, activating transcription of the HSP genes. Despite the apparent simplicity, transcriptional activation is not a necessary consequence of DNA binding. Treatment of human cells with salicylate induces HSF to trimerize and bind DNA but not to activate transcription. This suggests that there are at least two activation steps for metazoan HSF, one of which occurs subsequent to DNA binding. In fact, the DNA-binding and transactivation capacity of HSF1 are coordinately regulated through multiple posttranslational modifications (including phosphorylation, sumoylation and acetylation), protein-protein interactions and subcellular localization. HSF1 also has an intrinsic stress sensing capacity, as both D. melanogaster and mammalian HSF1 can be converted from a monomer to a trimer in vitro in response to thermal or oxidative stress. There is strong evidence that HSF1 interacts with multiple HSPs at different phases of its activation. Trimeric HSF1 can be kept inactive when its regulatory domain is bound by a multi-chaperone complex containing HSP90. Elevated levels of both HSP90 and HSP70 negatively regulate HSF1 and prevent trimer formation upon heat shock. The negative feedback from the end products of HSF1-dependent transcription (the HSPs) provides an important control step in adjusting the duration and intensity of HSF1 activation according to the levels of chaperones and presumably the levels of nascent and misfolded peptides. Furthermore, evidence is accumulating that HSFs are very versatile transcription factors that, in addition to protecting cells against proteotoxic stress, are vital for many physiological functions, especially during development. Genome-wide gene expression studies have revealed that numerous genes not classified as HSP genes or molecular chaperones are under HSF1-dependent control. Although mice lacking HSF1 can survive to adulthood, they exhibit multiple defects, such as increased prenatal lethality, growth retardation and female infertility. HSF1 from S. cerevisiae and Kluyveromyces cerevisiae (but not HSF1 from Schizosaccharomyces pombe) behaves differently from metazoan HSFs. Budding yeast HSF1 trimerizes and binds to promoters before stress. Nonetheless, upon HS, the level of HSF1dependent transcription is enhanced. This suggests that yeast HSF1 can exist in two conformations – an unstressed ‘low-activity’ conformation and a stressed ‘high-activity’ conformation. S. cerevisiae HSF1 regulates transcription under normal physiological conditions as well as under different stress conditions, including heat, oxidative stress, glucose starvation, and pH, and it is essential for cell viability. The genes targeted by HSF1 encode proteins that function in a broad range of biological processes, including protein folding and degradation, detoxification, energy generation, carbohydrate metabolism, and cell wall organization. HSF1 is phosphorylated concomitant with its heat shock-induced activation, and this covalent modification is intra-molecularly controlled by two domains of the protein: CE2 and CTM. The CE2 region functions negatively to restrict the activity of HSF1 at low


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temperature and/or to convert the protein from an active to an inactive form. The CTM domain is required for extensive phosphorylation, but this requirement is bypassed in an hsf1 mutant that lacks CE2, suggesting that the CTM domain alleviates the repressive effect of CE2 with respect to hyperphosphorylation. In addition, CTM regulates the activating ability of HSF1 and thereby affects recruitment of the transcription machinery. Cooperative protein–DNA and protein–protein interactions are important for fine-tuning transcriptional levels. The cooperative binding of HSF1 molecules to the HSEs is connected with the binding affinity to DNA and the strength of transcriptional activation.

Sensing the stress In metazoans, HSPs and co-chaperones are assembled into multi-chaperone complexes to regulate HSF1 activity. HSP90containing multi-chaperone complexes appear to be the most relevant repressors of HSF1 activity. Because HSP90-containing multi-chaperone complexes interact not only with HSF1 but also with nonnative proteins, the concentration of nonnative proteins can influence the assembly on HSF1 of HSP90-containing multi-chaperone complexes that repress activation, and may play a role in inactivation of HSF1. Since yeast HSF1 is under negative regulatory control in the absence of stress, a model similar to the one in metazoans, where in the basal state HSF1 would be tied up with HSPs in a regulatory loop was investigated. This model however has been challenged by the observation that overexpression of Ssa2, one of the HSP70 homologues highly induced by HS in yeast, and thus a prominent candidate molecule in this feedback regulation, did not inhibit the HS response. Nevertheless, a role for HSP70 proteins in the down-regulation of the response cannot be discarded. In contrast, evidences were provided indicating that in S. cerevisiae and in D. melanogaster HS and oxidative stress can act directly on HSF1 by causing conformational changes capable of inducing its activation. In addition, it has been shown that the transcriptional activity of HSF1 during the HS response depends on trehalose levels in S. cerevisiae. Strains with low levels of the disaccharide trehalose have a diminished transcriptional response to HS, while strains with high levels of trehalose have an enhanced transcriptional response to HS. The enhanced transcriptional response does not require the other heat-responsive transcription factors present only in yeast, Msn2 and Msn4, but is dependent upon heat and HSF1. In addition, the phosphorylation levels of HSF1 correlate with both transcriptional activity and the presence of trehalose. These in vivo results support a new role for trehalose (which was previously known to be correlated with tolerance to adverse conditions in yeast, and suggested to act as a chemical co-chaperone), where trehalose directly modifies the dynamic range of HSF1 activity and therefore influences HSP mRNA levels in response to stress (Figure 6). Even though the HS response has been better characterized in S. cerevisiae, many studies with heat stress have been carried out with other fungi and a number of different eukaryotic microbes. Presently, genomic and postgenomic tools, which have become available for high-throughput studies in several microorganisms, will certainly improve significantly our knowledge of HSP function and control in these organisms.

Final Remarks For a variety of microbial pathogens, the expression of HS genes has been observed to be upregulated following host cell infection. Concurrent with this fact is the observation that HSPs are immunodominant antigens for a number of infectious organisms including bacteria, fungi, parasites, and protozoa. However, the exact role of HSPs during infection has not been clearly demonstrated yet. For instance, studies with parasites and protozoa revealed that surface-expressed HSP90 is immunogenic in Chagas’ disease, ascariasis, leishmaniasis, toxoplasmosis, Trichinella spiralis, and infection due to Schistosoma mansoni. In addition, Trypanosoma cruzi HSP90 can functionally complement yeasts, and its inhibition by the HSP90 inhibitor geldanamycin demonstrated that it was essential for cell division, as epimastigotes were arrested in the G1 phase of cell cycle. Recombinant Leishmania HSP90 and HSP70 were recognized by sera from patients with visceral leishmaniasis but not by Chagas’ disease patients. HSP90 inhibitors led to

Figure 6 Activation of HSF1 during heat stress in budding yeast. In yeast, HSF1 trimerizes and binds to HSE in the HS gene promoters before stress, but it remains in a low-activity form, probably inhibited by HSP70 or other chaperones at normal temperatures. During heat shock and in the presence of high levels of the chemical cochaperone trehalose, HSF1 undergoes hyperphosphorylation and conformational changes and becomes active producing a dramatic increase in the expression of HSP genes.

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growth arrest and the differentiation from the promastigote to amastigote. HSP70 and HSP83 have been suggested as distracting the immune system to a nonspecific activation of immune cells leading to immunosuppression. In pathogenic fungi, similar results were found and this knowledge has been used in novel therapies against these microorganisms. For instance, the immunodominance of HSP90 in patients who survived disseminated candidiasis and invasive aspergillosis made this molecule a natural target for immunotherapy. In addition, immunization with a membrane fraction from Candida albicans containing HSP90 in combination with fluconazole protected mice from infection. Finally, the involvement of HSPs in a number of human diseases including cancer and neurodegenerative disorders, as well as aging, gives further importance for the studies of these universally conserved proteins.

Further Reading Akerfelt M, Morimoto RI, and Sistonen L (2010) Heat shock factors: integrators of cell stress, development and lifespan. Nature Reviews Molecular Cell Biology 11: 545–555. Bonner JJ, Carlson T, Fackenthal DL, Paddock D, Storey K, and Lea K (2000) Complex regulation of the yeast heat shock transcription factor. Molecular Biology of the Cell 11: 1739–1751. Bucca G, Brassington AME, Hotchkiss G, Vassilios M, and Smith CP (2003) Negative feedback regulation of dnaK, clpB and lon expression by the DnaK chaperone machine in Streptomyces coelicolor, identified by transcriptome and in vivo DnaK-depletion analysis. Molecular Microbiology 50: 153–166. Bulman AL and Nelson HCM (2005) Role of trehalose and heat in the structure of the C-terminal activation domain of the heat shock transcription factor. Proteins 58: 826–835. Cardoso K, Gandra RF, Wisniewski ES, Osaku CA, Kimiko MK, Felipach-Neto V, Haus LFA, and Simaõ RCG (2010) DnaK and GroEL are induced in response to antibiotic and heat shock in Acinetobacter baumannii. Journal of Medical Microbiology 59: 1061–1068. da Silva ACC, Simaõ RCG, Susin MF, Baldini RL, Avedissian M, and Gomes SL (2003) Down-regulation of heat shock response is independent of DnaK and s32 levels in Caulobacter crescentus. Molecular Microbiology 49: 541–553. Duguay AR and Silhavy TJ (2004) Quality control in bacterial periplasm. Biochimica et Biophysica ACTA 1694: 121–134. Fiester SE and Actis LA (2013) Stress responses in the opportunistic pathogen Acinetobacter baumannii. Future Microbiology 8: 353–365. Golterman L, Good L, and Bentin T (2013) Chaperonins Fight Aminoglycoside-induced Protein Misfolding and Promote Short-term Tolerance in Escherichia coli. The Journal of Biological Chemistry 288: 10483–10489. Keese AM, Schut GJ, Ouhammouch M, Adams MWW, and Thomm M (2010) Genome-wide identification of targets for the archeal heat shock regulator Phr by cell-free transcription of genomic DNA. Journal of Bacteriology 192: 1292–1298. Lindquist S and Craig EA (1998) The heat-shock proteins. Annual Review of Genetics 22: 631–677. Liu W, Vieke G, Wenke A, Thomm M, and Ladenstein R (2007) Crystal structure of the archaeal heat shock regulator from Pyrococcus furiosus: A molecular chimera representing eukaryal and bacterial features. Journal of Molecular Biology 369: 474–488. Lourenço RF, Kohler C, and Gomes SL (2011) A two-component system, an anti-sigma factor, and two paralogous ECF sigma factors are involved in the control of general stress response in Caulobacter crescentus. Molecular Microbiology 80: 1598–1612. Narberhaus F (1999) Negative regulation of bacterial heat shock genes. Molecular Microbiology 31: 1–8. Narberhaus F, Krummenacher P, Fischer HM, and Hennecke H (1997) Three disparately regulated genes for sigma 32-like transcription factors in Bradyrhizobium japonicum. Molecular Microbiology 24: 93–104. Poole K (2012) Stress responses as determinants of antimicrobial resistance in Gram negative bacteria. Trends in Microbiology 20: 227–234. Ritossa F (1962) A new puffing pattern induced by temperature chock and DNP in Drosophila. Experientia 18: 571–573. Schumann W (2003) The Bacillus subtilis heat shock stimulon. Cell Stress & Chaperones 8: 207–217. Simaõ RCG, Susin MF, Alvarez-Martinez CE, and Gomes SL (2005) Cells lacking ClpB display a prolonged shutoff phase of the heat shock response in Caulobacter crescentus. Molecular Microbiology 57: 592–603. Staron A and Mascher T (2010) General stress response in alpha-proteobacterium: PhyR and beyond. Molecular Microbiology 78: 271–277. Susin MF, Baldini RL, Gueiros-Filho F, and Gomes SL (2006) GroES/GroEL and DnaK/DnaJ have distinct roles in stress responses and during cell cycle progression in Caulobacter crescentus. The Journal of Bacteriology 188: 8044–8053. Voellmy R (2004) On mechanisms that control heat shock transcription factor activity in metazoan cells. Cell Stress & Chaperones 9: 122–133. Weibezahn J, Tessarz P, Schlieker C, et al. (2004) Thermotolerance requires refolding of aggregated proteins by substrate translocation through the central pore of ClpB. Cell 119: 653–665. Young JC, Agashe VR, Siegers K, and Hartl FU (2004) Pathways of chaperone-mediated protein folding in the cytosol. Nature Reviews Molecular Cell Biology 5: 781–791. Yura T, Kanemori M, and Morita MT (2000) The heat shock response: Regulation and function. In: Storz G and Hengge-Aronis R (eds.) Bacterial stress responses, pp. 1–18. Washington, DC: ASM Press.