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Protection of hair cells by HSP70 released by supporting cells is reported by May et al. in this issue of the JCI. Their findings suggest a new way to reduce ototoxicity from therapeutic medications and raise larger questions about the role and integration of heat shock proteins in non–cell-autonomous responses to stress. Increasing evidence suggests an important role for extracellular heat shock proteins in both the nervous system and the immune system. The work also suggests that defective chaperones could cause ear disease and supports the potential use of chaperone therapeutics.

Ulcerative colitis (UC) is a form of inflammatory bowel disease (IBD) characterized by damage of large bowel mucosa and frequent extra-intestinal autoimmune comorbidities. The role played in IBD pathogenesis by molecular chaperones known to interact with components of the immune system involved in inflammation is unclear. We previously demonstrated that mucosal Hsp60 decreases in UC patients treated with conventional therapies (mesalazine, probiotics), suggesting that this chaperonin could be a reliable biomarker useful for monitoring response to treatment, and that it might play a role in pathogenesis. In the present work we investigated three other heat shock protein/molecular chaperones: Hsp10, Hsp70, and Hsp90. We found that the levels of these proteins are increased in UC patients at the time of diagnosis and decrease after therapy, supporting the notion that these proteins deserve attention in the study of the mechanisms that promote the development and maintenance of IBD, and as biomarkers of this disease (e.g., to monitor response to treatment at the histological level).

Background: Hsp70 chaperones are required for key cellular processes and response to environmental changes and survival but they have not been fully characterized yet. The human hsp70-gene family has an unknown number of members (eleven counted over ten years ago); some have been described but the information is incomplete and inconsistent. A coherent body of knowledge encompassing all family components that would facilitate their study individually and as a group is lacking. Nowadays, the study of chaperone genes benefits from the availability of genome sequences and a new protocol, chaperonomics, which we applied to elucidate the human hsp70 family., Results: We identified 47 hsp70 sequences, 17 genes and 30 pseudogenes. The genes distributed into seven evolutionarily distinct groups with distinguishable subgroups according to phylogenetic and other data, such as exon-intron and protein features. The N-terminal ATP-binding domain (ABD) was conserved at least partially in the majority of the proteins but the C-terminal substrate-binding domain (SBD) was not. Nine proteins were typical Hsp70s (65-80 kDa) with ABD and SBD, two were lighter lacking partly or totally the SBD, and six were heavier (>80 kDa) with divergent C-terminal domains. We also analyzed exon-intron features, transcriptional variants and protein structure and isoforms, and modality and patterns of expression in various tissues and developmental stages. Evolutionary analyses, including human hsp70 genes and pseudogenes, and other eukaryotic hsp70 genes, showed that six human genes encoding cytosolic Hsp70s and 27 pseudogenes originated from retro-transposition of HSPA8, a gene highly expressed in most tissues and developmental stages., Conclusion: The human hsp70-gene family is characterized by a remarkable evolutionary diversity that mainly resulted from multiple duplications and retrotranspositions of a highly expressed gene, HSPA8. Human Hsp70 proteins are clustered into seven evolutionary Groups, with divergent C-terminal domains likely defining their distinctive functions. These functions may also be further defined by the observed differences in the N-terminal domain.

Hsp70 (DnaK) is a highly conserved molecular chaperone present in bacteria, eukaryotes, and some archaea. In a previous work we demonstrated that DnaK from the archaeon Methanosarcina mazei (DnaK(Mm)) and the DnaK from the bacterium Escherichia coli (DnaK(Ec)) were functionally similar when assayed in vitro but DnaK(Mm) failed to substitute for DnaK(Ec) in vivo. Searching for the molecular basis of the observed DnaK species specificity we compared substrate binding by DnaK(Mm) and DnaK(Ec). DnaK(Mm) showed a lower affinity for the model peptide (a-CALLQSRLLS) compared to DnaK(Ec). Furthermore, it was unable to negatively regulate the E. coli sigma32 transcription factor level under heat shock conditions and poorly bound purified sigma32, which is a native substrate of DnaK(Ec). These observations taken together indicate differences in substrate specificity of archaeal and bacterial DnaKs. Structural modeling of DnaK(Mm) showed some structural differences in the substrate-binding domains of DnaK(Mm) and DnaK(Ec), which may be responsible, at least partially, for the differences in peptide binding. Size-exclusion chromatography and native gel electrophoresis revealed that DnaK(Mm) was found preferably in high molecular mass oligomeric forms, contrary to DnaK(Ec). Oligomers of DnaK(Mm) could be dissociated in the presence of ATP and a substrate (peptide) but not ADP, which may suggest that monomer is the active form of DnaK(Mm).

Hsp70s are chaperone proteins that are conserved in evolution and present in all prokaryotic and eukaryotic organisms. In the archaea, which form a distinct kingdom, the Hsp70 chaperones have been found in some species only, including Methanosarcina mazei. Both the bacterial and archaeal Hsp70(DnaK) chaperones cooperate with a GrpE co-chaperone which stimulates the ATPase activity of the DnaK protein. It is currently believed that the archaeal Hsp70 system was obtained by the lateral transfer of chaperone genes from bacteria. Our previous finding that the DnaK and GrpE proteins of M. mazei can functionally cooperate with the Escherichia coli GrpE and DnaK supported this hypothesis. However, the cooperation was surprising, considering the very low identity of the GrpE proteins (26%) and the relatively low identity of the DnaK proteins (56%). The aim of this work was to investigate the molecular basis of the observed interspecies chaperone interaction. Infrared resolution-enhanced spectra of the M. mazei and E. coli DnaK proteins were almost identical, indicating high similarity of their secondary structures, however, some small differences in band position and in the intensity of amide I' band components were observed and discussed. Profiles of thermal denaturation of both proteins were similar, although they indicated a higher thermostability of the M. mazei DnaK compared to the E. coli DnaK. Electrophoresis under non-denaturing conditions demonstrated that purified DnaK and GrpE of E. coli and M. mazei formed mixed complexes. Protein modeling revealed high similarity of the 3-dimensional structures of the archaeal and bacterial DnaK and GrpE proteins.

The stress chaperone protein Hsp70 (DnaK) (abbreviated DnaK) and its co-chaperones Hsp40(DnaJ) (or DnaJ) and GrpE are universal in bacteria and eukaryotes but occur only in some archaea clustered in the order 5'-grpE-dnaK-dnaJ-3' in a locus termed Locus I. Three structural varieties of Locus I, termed Types I, II, and III, were identified, respectively, in Methanosarcinales, in Thermoplasmatales and Methanothermobacter thermoautotrophicus, and in Halobacteriales. These Locus I types corresponded to three groups identified by phylogenetic trees of archaeal DnaK proteins including the same archaeal subdivisions. These archaeal DnaK groups were not significantly interrelated, clustering instead with DnaKs from three bacterial lineages, Methanosarcinales with Firmicutes, Thermoplasmatales and M. thermoautotrophicus with Thermotoga, and Halobacteriales with Actinobacteria, suggesting that the three archaeal types of Locus I were acquired by independent events of lateral gene transfer. These associations, however, lacked strong bootstrap support and were sensitive to dataset choice and tree-reconstruction method. Structural features of dnaK loci in bacteria revealed that Methanosarcinales and Firmicutes shared a similar structure, also common to most other bacterial groups. Structural differences were observed instead in Thermotoga compared to Thermoplasmatales and M. thermoautotrophicus, and in Actinobacteria compared to Halobacteriales. It was also found that the association between the DnaK sequences from Halobacteriales and Actinobacteria likely reflects common biases in their amino acid compositions. Although the loci structural features and the DnaK trees suggested the possibility of lateral gene transfer between Firmicutes and Methanosarcinales, the similarity between the archaeal and the ancestral bacterial loci favors the more parsimonious hypothesis that all archaeal sequences originated from a unique prokaryotic ancestor.

Archaea are prokaryotes but some of their chaperoning systems resemble those of eukaryotes. Also, not all archaea possess the stress protein Hsp70(DnaK), in contrast with bacteria and eukaryotes, which possess it without any known exception. Further, the primary structure of the archaeal DnaK resembles more the bacterial than the eukaryotic homologues. The work reported here addresses two questions: Is the archaeal Hsp70 protein a chaperone, like its homologues in the other two phylogenetic domains? And, if so, is the chaperoning mechanism of bacterial or eukaryotic type? The data have shown that the DnaK protein of the archaeon Methanosarcina mazei functions efficiently as a chaperone in luciferase renaturation in vitro, and that it requires DnaJ, and the other bacterial-type chaperone, GrpE, to perform its function. The M. mazei DnaK chaperone activity was enhanced by interaction with the bacterial co-chaperone DnaJ, but not by the eukaryotic homologue HDJ-2. Both the bacterial GrpE and DnaJ stimulated the ATPase activity of the M. mazei DnaK. The M. mazei DnaK-dependent chaperoning pathway in vitro is similar to that of the bacterium Escherichia coli used for comparison. However, in vivo analyses indicate that there are also significant differences. The M. mazei dnaJ and grpE genes rescued E.coli mutants lacking these genes, but E.coli dnaK mutants were not complemented by the M. mazei dnaK gene. Thus, while the data from in vitro tests demonstrate functional similarities between the M. mazei and E.coli DnaK proteins, in vivo results indicate that, intracellularly, the chaperones from the two species differ.

A brief survey is presented of salient findings on transcription in the Archaea, focussing on stress genes of the hsp70(dnaK locus, which code for the molecular chaperones Hsp70(DnaK), Hsp40(DnaJ), and GrpE. Archaeal basal factors and some recently characterized regulators pertinent to non-stress genes are presented first to show their similarities and differences with equivalents in organisms of the other two phylogenetic domains, Bacteria and Eucarya, and to reveal clues on how these or similar factors might transcribe and regulate the archaeal stress genes. The second part of the article deals with the hsp70(dnaK)-locus genes, particularly those from Methanosarcina mazeii, because they are virtually the only ones within the methanogenic Archaea whose patterns of constitutive and stress-induced expressions have been studied. Therefore, these genes provide a standardized model system to elucidate transcription initiation and regulation at the molecular level in this phylogenetic group. Promoters, and other cis-acting sites that are, or might be, involved in stress-gene expression are described. Conformational changes of basal transcription factors after interaction with stress-gene promoters are discussed that suggest ways for generating a large diversity of initiation complexes using a few factors and DNA sites in different combinations. Likewise, the effects of stress on DNA topology and on TBP-TFB-promoter complex formation and tightness are described, which might also contribute to the generation of transcription-initiation complex diversity. This diversity would be key to differential gene expression, namely, which genes are transcribed, when (basal, steady expression vs. sporadic stress-induced expression), and to what level. Future research should investigate this diversity, and the mechanism of complex formation and action at the atomic, molecular, and supramolecular levels, to elucidate the dynamics of transcription initiation in real time.

The hsp70(dnaK) locus of the moderate thermophilic archaeon Methanosarcina thermophila TM-1 was cloned, sequenced, and tested in vitro to measure gene induction by heat and ammonia, i.e., stressors pertinent to the biotechnological ecosystem of this methanogen that plays a key role in anaerobic bioconversions. The locus' genes and organization, 5'-grpE-hsp70(dnaK)-hsp40 (dnaJ)-trkA-3', are the same as those of the closely related mesophile Methanosarcina mazei S-6, but different from those of the only other archaeon for which comparable sequence data exist, the thermophile Methanobacterium thermoautotrophicum deltaH, from another genus, in which trkA is not part of the locus. The proteins encoded in the TM-1 genes are very similar to the S-6 homologs, but considerably less similar to the deltaH proteins. The TM-1 Hsp70(DnaK) protein has the 23-amino acid deletion--by comparison with homologs from gram-negative bacteria first described in the S-6 molecule and later found to be present in all homologs from archaea and gram positives. The genes responded to a temperature elevation in a manner that demonstrated that they are heat-shock genes, functionally active in vivo. Ammonia also induced a heat-shock type of response by hsp70(dnaK), and a similar response by trkA. The data suggest that the moderate thermophile TM-1 has an active Hsp70(DnaK)-chaperone machine in contrast to hyperthermophilic archaea, and that trkA is a stress gene, inasmuch as it responds like classic heat-shock genes to stressors that induce a typical heat-shock response.

A major finding within the field of archaea and molecular chaperones has been the demonstration that, while some species have the stress (heat-shock) gene hsp70(dnaK), others do not. This gene encodes Hsp70(DnaK), an essential molecular chaperone in bacteria and eukaryotes. Due to the physiological importance and the high degree of conservation of this protein, its absence in archaeal organisms has raised intriguing questions pertaining to the evolution of the chaperone machine as a whole and that of its components in particular, namely, Hsp70(DnaK), Hsp40(DnaJ), and GrpE. Another archaeal paradox is that the proteins coded by these genes are very similar to bacterial homologs, as if the genes had been received via lateral transfer from bacteria, whereas the upstream flanking regions have no bacterial markers, but instead have typical archaeal promoters, which are like those of eukaryotes. Furthermore, the chaperonin system in all archaea studied to the present, including those that possess a bacterial-like chaperone machine, is similar to that of the eukaryotic-cell cytosol. Thus, two chaperoning systems that are designed to interact with a compatible partner, e.g., the bacterial chaperone machine physiologically interacts with the bacterial but not with the eucaryal chaperonins, coexist in archaeal cells in spite of their apparent functional incompatibility. It is difficult to understand how these hybrid characteristics of the archaeal chaperoning system became established and work, if one bears in mind the classical ideas learned from studying bacteria and eukaryotes. No doubt, archaea are intriguing organisms that offer an opportunity to find novel molecules and mechanisms that will, most likely, enhance our understanding of the stress response and the protein folding and refolding processes in the three phylogenetic domains.

The dnaK locus of Methanosarcina mazei S-6, a mesophilic organism of the phylogenetic domain Archaea, contains the heat-shock genes 5'-grpE-dnaK-dnaJ-3'. Parameters known to affect the response of these genes in organisms of the other two domains, Bacteria and Eucarya, were tested to determine their effects on the archaeal homologs. The mRNA from the three genes increased after heat shock more in lamina than in single cells (these S-6 morphologic stages can be grown in the same substrate). Single cells in early stationary phase showed the highest levels of dnaK mRNA after heat shock, as compared with cells in exponential, or in late stationary, phase. The dnaK mRNA always had the size of a monocistronic transcript. dnaK was also found in the thermophileMethanosarcina thermophila TM-1, and its response to heat shock showed distinctive characteristics. However, dnaK was not revealed in other archaea: three hyperthermophiles (Methanothermus fervidus,Methanococcus jannaschii, and Sulfolobus sp.), and one mesophilic methanogen (Methanospirillum hungateii).

In some eubacteria with a dnaK locus in which grpE is close upstream of dnaK, grpE is preceded by an open reading frame (orf) believed to be a heat-shock gene. We also found an orf, orf16, upstream of grpE in the archaeon Methanosarcina mazei S-6, but this gene differs from the eubacterial counterpart: it is shorter, does not respond to a temperature upshift as heat-shock genes do, and the deduced protein Orf16, does not resemble the proteins coded by the eubacterial equivalents. orf16 is expressed monocistronically, with a transcription initiation site 24 bases upstream of the translation start codon, 22 bases downstream of a putative promoter identical to the consensus promoter for genes in methanogens. This initiation site is used by heat-shocked and non-heat-shocked cells in the two morphologic stages of M. mazei S-6 tested, i.e., packets and single cells. Three transcription termination sites were identified, one of which is detectable only in non-heat-shocked cells. Data from comparative analyses of the Orf16 deduced amino acid sequence and those of other known proteins, as well as the apparent biochemical characteristics of Orf16, suggest that the latter is a membrane molecule.

The organization and expression of the first archeael dnaK-dnaJ gene cluster cloned and sequenced have been elucidated. The work focused on the methanogen Methanosarcina mazei strain S-6, but a survey of two other strains (JC3 and LYC) and species (Methanosarcina sp. JCV and Methanosarcina acetivorans) showed that the findings are pertinent to other mesophilic methanosarcinas as well. The organization and some expression features of the archaeal genes resemble eubacterial equivalents for which comparable sequence information is available. However, the archaeal genes also display characteristics that are distinct from those of eubacterial and eucaryotic homologs. dnaK and dnaJ are transcribed into monocistronic messages. The initiation site is the same for transcription under optimal cell-growth conditions, and under stress due to a temperature upshift. The two genes are expressed constitutively at lower levels than those observed after heat shock. The constitutive and post-heat-shock expression levels are higher for dnaK than for dnaJ. Both genes withstand heat shocks of at least one and a half hours without a decline in transcript levels. While the transcription termination signals are to some extent reminiscent of those of eubacteria, the initiation signals are not. These have archaeal characteristics, which resemble those of eukaryotes. The intergenic dnaK-dnaJ region contains inverted repeats. These have the potential to build firm stem-loops in the transcript and in single-stranded DNA.

Transcription of the heat shock gene grpE was studied in two different morphologic stages of the archaeon Methanosarcina mazei S-6 that differ in resistance to physical and chemical traumas: single cells and packets. While single cells are directly exposed to environmental changes, such as temperature elevations, cells in packets are surrounded by intercellular and peripheral material that keeps them together in a globular structure which can reach several millimeters in diameter. grpE transcript levels determined by Northern (RNA) blotting peaked after a 15-min heat shock in single cells. In contrast, the highest transcript levels in packets were observed after the longest heat shock tested, 60 min. The same response profiles were demonstrated by primer extension experiments and S1 nuclease analysis. A comparison of the grpE response to heat shock with those of dnaK and dnaJ showed that the grpE transcript level was the most increased, closely followed by that of the dnaK transcript, with that of the dnaJ gene being the least augmented. Transcription of grpE started at the same site under normal and heat shock temperatures, and the transcript was consistently approximately 700 bases long. Codon usage patterns revealed that the three archaeal genes use most codons and have the same codon preference for 61% of the amino acids.

A grpE heat-shock gene was found by sequencing in the genome of the methanogenic archaeon Methanosarcina mazei S-6. It is the first example of grpE from the phylogenetic domain Archaea. Since the other seven sequenced homologs are from the domain Bacteria, it may be concluded that grpE appeared early in evolution, before the two domains separated. The archaeal grpE is located in the dnaK locus, 431 base-pairs upstream of dnaK, which is followed downstream by the dnaJ gene. The organization of these three genes is known for Bacillus subtilis, Clostridium acetobutylicum, Borrelia burgdorferi and Mycobacterium tuberculosis. The archaeal locus organization, grpE-dnaK-dnaJ, is similar to that of the former three bacteria, but different from that of M. tuberculosis. This, and sequence homologies, suggest that the M. tuberculosis GrpE belongs, together with the Streptomyces coelicolor homolog, to a subgroup of the GrpE proteins. The M. mazei grpE gene encodes a protein of 209 amino acid residues. The deduced amino acid sequence shows 28.2 to 34.6% identities, and 50.3 to 58.9 similarities (identities plus conservative substitutions) with the other six complete GrpE sequences available. These percentages fall within the range observed for the other GrpEs. Two regions in the second and fourth quarters of the GrpE molecule show higher homology, particularly in three stretches of nine, six and nine amino acid residues, respectively. The archaeal gene uses all codons but three, whereas the bacterial homologs lack higher numbers of codons. The M. mazei grpE responded to heat-shock by increasing transcription, in a manner similar to that of the nearby heat-shock gene dnaK.

The first trkA gene homolog in the phylogenetic domain Archaea is reported. The gene is located near the dnaK-dnaJ gene cluster in the genome of Methanosarcina mazei S-6, and encodes a protein homologous to the only other TrkA known, i.e., that of the bacterium Escherichia coli, involved in K+ transport. This finding supports an essential, evolutionarily early, and conserved role for this gene in cell survival and adaptation.

A fragment of genomic DNA cloned from the methanogenic archaebacterium, Methanosarcina mazei strain S6, was found to contain an 1857-bp open reading frame (ORF). A sequence matching the consensus ribosome-binding sequence determined for other methanogens was found upstream from the ORF. The amino acid (aa) sequence encoded by the ORF was compared with reference sequences and was found to be related to six DnaK sequences determined for five species of eubacteria (none exist for archaebacteria). The M. mazei S6 aa sequence was over 61% identical and over 77% similar (identities plus conservative substitutions) to the closest four reference sequences, which were all DnaKs. The gene described here is therefore proposed to be the first member of the dnaK family sequenced from the archaebacterial kingdom (Archaea). This finding confirms that DnaK proteins are highly conserved, occurring not only in eubacteria (Bacteria) and eukaryotes (Eucaria), but also in archaebacteria (Archaea).