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Microbiology and Molecular Biology Reviews, March 2002, p. 64-93, Vol. 66, No. 1
1092-2172/02/$04.00+0 DOI: 10.1128/MMBR.66.1.64-93.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
-Crystallin-Type Heat Shock Proteins: Socializing Minichaperones in the Context of a Multichaperone Network
Franz Narberhaus*
Institut für Mikrobiologie, Eidgenössische Technische Hochschule, CH-8092 Zürich, Switzerland
Summary: 
-Crystallins were originally recognized as proteins contributing to the transparency of the mammalian eye lens. Subsequently, they have been found in many, but not all, members of the Archaea, Bacteria, and Eucarya. Most members of the diverse -crystallin family have four common structural and functional features: (i) a small monomeric molecular mass between 12 and 43 kDa; (ii) the formation of large oligomeric complexes; (iii) the presence of a moderately conserved central region, the so-called -crystallin domain; and (iv) molecular chaperone activity. Since -crystallins are induced by a temperature upshift in many organisms, they are often referred to as small heat shock proteins (sHsps) or, more accurately, -Hsps. -Crystallins are integrated into a highly flexible and synergistic multichaperone network evolved to secure protein quality control in the cell. Their chaperone activity is limited to the binding of unfolding intermediates in order to protect them from irreversible aggregation. Productive release and refolding of captured proteins into the native state requires close cooperation with other cellular chaperones. In addition, -Hsps seem to play an important role in membrane stabilization. The review compiles information on the abundance, sequence conservation, regulation, structure, and function of -Hsps with an emphasis on the microbial members of this chaperone family.
Our knowledge about protein folding has increased substantially over the last 30 years. The traditional view that proteins fold spontaneously (9) was revised upon the finding that many proteins in a living cell will not fold correctly without the assistance of molecular chaperones. Chaperones have been defined as "a family of cellular proteins which mediate the correct folding of other polypeptides, and in some cases their assembly into oligomeric structures, but which are not components of the final functional structures" (75, 76). This definition implies that the predominant function of molecular chaperones is to transiently interact with other proteins, thereby preventing the formation of illegitimate interactions that might otherwise lead to deleterious protein aggregation. Chaperones generally seem to bind to exposed hydrophobic surfaces that will ultimately be buried in the folded state. Controlled release of a substrate protein from the chaperone, often driven by ATP hydrolysis, promotes folding into the native state. Repeated cycles of binding and release may be necessary for productive folding.
Enormous progress has been made in the elucidation of the major chaperones belonging to the Hsp60 (GroEL) and Hsp70 (DnaK) families. A wealth of information on the structural and functional properties of these chaperones has accumulated, and many excellent reviews on this topic are available (18, 40, 111, 196, 251, 257). However, our understanding of other chaperones is comparatively limited. Alternative chaperones are less abundant and less critical for protein folding than are DnaK and GroEL. However, since protein folding has been recognized as one of the central problems in biology, our knowledge about the contribution of minor chaperones to this process is catching up.
The purpose of this review is to draw attention to a family of low-molecular-mass chaperones, the -crystallin-type proteins. The bulk of our knowledge about this chaperone class comes from studies of the name-giving lenticular -crystallins. The two forms of -crystallin,A- and B-crystallin, prevent protein precipitation and cataract formation in the vertebrate eye lens (33). Interestingly, -crystallins not only are present in lenticular tissues but also have been found in many other tissues like heart, brain, and kidney (11). Because of the seminal finding of four -crystallin-type proteins in Drosophila (133), it is well established that -crystallins comprise a diverse protein family existing in most, but not all, animals, plants, bacteria, and archaea (43, 60, 192, 192a, 220, 349). The presence of -crystallin-type Hsps in all kingdoms clearly points to important biological functions beyond their specialized role in clear vision. The discovery that -crystallins can act as molecular chaperones (127, 138) boosted research on this class of stress proteins. The growing interest in this subject is reflected by a rapidly increasing amount of literature, which will be reviewed here.
Most information in this article will be presented from the perspective of a microbiologist. Whenever necessary and appropriate, complementary material on counterparts from higher organisms will be documented. More detailed information on eukaryotic -crystallins can be found in other articles (11, 62, 71, 193, 268a, 349). The present review first introduces general principles of protein quality control in Escherichia coli and then gives details on the regulation, structure, and function of -crystallin-type proteins. This information will finally be integrated into a model presenting how -crystallins cooperate in the framework of a complex multichaperone network.
Normal Conditions
One of the most critical events in the biogenesis of a protein is the conversion of its linear amino acid sequence into the properly folded three-dimensional structure. As soon as a nascent polypeptide chain emerges from the ribosome, it is prone to misfolding and subsequent aggregation. Although many proteins may fold spontaneously, the initial folding of a significant portion of cellular proteins requires the assistance of molecular chaperones (39, 229). It has been estimated that 20 to 30% of all proteins in the prokaryotic cytoplasm transit through the DnaK or GroEL chaperone machineries before adopting their final conformation (77, 316) (Fig. 1A). Chaperoning occurs both cotranslationally, while the polypeptide is being synthesized, and posttranslationally, after the complete amino acid chain has been released from the ribosome (39). Cotranslational folding requires DnaK and trigger factor, a ribosome-associated protein with peptidylprolyl-cis-trans isomerase and chaperone-like activities (66, 316). Both DnaK and GroEL contribute to posttranslational protein folding.
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FIG. 1. Simplified model of protein quality control under normal and heat shock conditions. (A) Under optimal growth conditions, the rate of transcription and translation is very high. As indicated by the thick arrow, most proteins fold spontaneously without the assistance of chaperones. Few proteins aggregate and are degraded by proteases. (B) After heat shock, the transcription and translation capacity is reduced. Temperature-induced unfolding returns previously folded proteins to the chaperone-dependent quality control system. Unfoldable proteins are removed by proteases. For simplicity, intermediate steps such as aggregation and disaggregation are not considered in this model.
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An alternative fate for misfolded proteins is proteolytic degradation. About 20% of all synthesized polypeptides never reach their final destination because fatal errors have occurred during transcription or translation or because correct folding has not been accomplished (357). Before such detrimental proteins accumulate, they are removed by proteases (Fig. 1A). Several universal ATP-dependent protease families have been described (96, 97). Interestingly, the analogy to the major chaperone classes extents beyond their common ATPase activity. Both chaperones and proteases are designed to recognize similar features that are found on unfolded proteins but not on native proteins, namely, surface-exposed hydrophobic patches. This related activity led to the evolution of a similar ring-like architecture of various chaperone and protease complexes (126, 357). Most intriguingly, the proteases involved in protein quality control have intrinsic chaperone activity, either within the same polypeptide (Lon, FtsH, and DegP) or located in associated components of a protease complex (ClpAP and ClpXP, where the ATP-dependent chaperone is underlined). The term "charonin" was invented for this type of multifunctional proteins or protein complexes (97, 272, 338). The tight combination of chaperone function with proteases suggests that the initial energy-dependent steps of the two processes are similar. Partial unfolding of substrate proteins is most probably the common prerequisite for both activities (304, 313, 351).
Protein synthesis in bacteria is much faster than in eukaryotes. As calculated from a synthesis rate of 15 to 20 amino acids per ribosome per s, E. coli produces some 30,000 polypeptides per min (39). This remarkable speed of translation requires a very efficient and accurate protein quality control system. Even if only 30% of the proteins need the attention of chaperones, almost 10,000 polypeptides per min are going through a chaperone cycle. Evidently, the decision of whether a polypeptide can be folded or must be degraded has to be made rapidly. Precise monitoring of the actual state of a protein is required to direct it to the appropriate pathway. It is not clear how the quality control system decides between (re)folding or breakdown when it faces a misfolded protein. Several tagging devices have evolved to sort out the hopeless cases that cannot be rescued by chaperones. Ubiquitinated polypeptides are removed by the proteasome in eucaryotes (47, 340). The N-end rule, variations of which apply to all known organisms, correlates the amino-terminal amino acid with the half-life of a protein (323, 339). Whereas certain residues confer a short-half life, others seem to stabilize a protein. The exposure of a destabilizing residue in damaged proteins directly routes them toward degradation, bypassing any refolding attempts. In E. coli, truncated polypeptides that result from abnormally terminated transcription are tagged by the SsrA system and thereby directed toward degradation (150, 329). A recent report demonstrates that abnormal proteins are susceptible to carbonylation, an unrepairable oxidative modification (70). This might provide the signal that a protein is irreparable and therefore destined for degradation. The associated chaperone components of the Clp proteases or the proteasome play a role in the decision whether a polypeptide can be salvaged (97, 357). Although important aspects of protein folding and degradation have been worked out over the last years, many details of the steps between the birth and death of a protein remain to be elucidated. The fate of every single protein in a cell constantly relies on a delicate balance between folding and degradation (121, 357). The rapid biogenesis of proteins in a growing cell puts a high pressure on the quality control machinery even under optimal conditions.
Stress Conditions
Once cellular proteins are folded, various stress conditions pose a serious threat to their integrity. Temperature variations, osmotic changes, antibiotics, solvents, or other chemicals not only interfere with transcription, translation, and protein folding but also often disrupt the faithfully acquired three-dimensional protein structure. The heat shock response elicited by a sudden increase in the ambient temperature is widely used as a model system for studying the impact of stress on biological systems. Proteins whose expression is induced on heat shock are generally called heat shock proteins (Hsps). It is not surprising that the classical chaperones and proteases are among them. All players involved in the regular protein quality control system described in the previous section are required to combat the damage inflicted by a thermal insult. On heat shock, a cell faces numerous problems. Transcription and translation slow because the RNA polymerase subunits RpoA, RpoB, and RpoD (
70) are thermolabile (23, 211) and because the translation factor EF-G is susceptible to aggregation at elevated temperatures (211) (Fig. 1B). Spontaneous folding of nascent polypeptides at high temperatures is error prone because interactive hydrophobic surfaces become exposed. Even subtle structural changes may suffice to inactivate cellular enzymes. It is evident that protein quality control under such circumstances becomes crucial for sustaining cellular metabolism and viability. In contrast to normal conditions, productive protein folding now occurs predominantly via the chaperone-mediated pathway due to a large number of thermolabile proteins (211). A large portion of already folded proteins gets partially or completely denatured and reenters the quality control system (Fig. 1B). When the rate of denaturation outpaces the refolding capacity, increasing amounts of proteolysis-sensitive substrates are being generated.
The additional demand for folding support during acute stress is met by two strategies: (i) the level of preexisting quality control proteins is elevated; and (ii) additional chaperones that are not expressed or are only weakly expressed under nonstress conditions are induced to counteract severe damage. Members of the first class, which are abundant under all metabolic conditions, are DnaK and GroEL. -Crystallin-type Hsps usually belong to the second class. Since these proteins are barely expressed at nonstress temperatures in most organisms, the induction factors after a temperature upshift can be very high (several hundred-fold [Table 1]) (258). Proteases are generally less abundant than chaperones regardless of the environmental conditions, indicating that protein refolding is preferred to proteolysis. Transcriptional induction after heat shock of most proteases was measured in the low to intermediate range (between 5- and 30-fold [Table 1]) (258). Although both chaperones and proteases are upregulated under stress conditions, many proteins seem to escape the quality control system and end up in aggregates. Polypeptides trapped in this state have long been considered dead-end products that cannot be rescued. Only recently, it was recognized that certain chaperones are able to solubilize aggregated proteins in vivo. The concerted action of Hsp104, Hsp70, and Hsp40 in yeast (92) or of ClpB and DnaK in E. coli (94, 211, 214, 347) recycles precipitated proteins into the folding pathway. Disaggregated material is released in a nonnative form that either can refold spontaneously or can be further processed by cellular chaperones.
In summary, nature has invented an arsenal of powerful strategies to maintain high-quality protein in the cell. Once a nascent polypeptide has folded correctly, it is not left alone. Problems arising within the life span of a protein are solved by chaperones and proteases, which assist in refolding or disposal of nonrefoldable molecules, respectively. The next section will look more closely at the different chaperone families that contribute to protein folding.
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MULTIPLE CHAPERONE FAMILIES
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Traditionally, Hsps have been grouped into five major families. They were designated Hsp100, Hsp90, Hsp70, Hsp60, and small Hsps according to their molecular masses (37, 40, 213). Several members of the Hsp100 family are chaperone subunits of protease complexes. Hence, there is good reason to include the single-chain charonins, which bear both chaperone and protease functions in a single polypeptide, in the chaperone list. Hsp33, a novel chaperone whose activity is redox regulated, recently joined the club (140). The updated list of chaperone families therefore comprises seven members (Table 1). It should be noted, however, that more than 30 functionally uncharacterized heat shock genes and proteins have been identified by global transcriptome and proteome analyses in E. coli (46, 258, 330). Whatever their function might be, one may anticipate a growing number of chaperone families in the future.
Hsp70 (DnaK) and Hsp60 (GroEL)
The two major chaperone families are Hsp70 and Hsp60. They are also the by far best-characterized chaperones. DnaK and GroEL, the bacterial representatives of these classes, are among the most abundant cellular proteins, indicating their important role in protein folding. In the gram-negative and gram-positive model organisms E. coli and Bacillus subtilis, respectively, groEL is essential whereas dnaK is not (80, 183, 240, 271). In both species, serious growth defects and inviability of dnaK mutants were observed only at high temperatures (210, 240). In Streptomyces coelicolor A3(2), Pseudomonas syringae pv. glycinea, and Bradyrhizobium japonicum, all attempts to construct dnaK deletion strains failed suggesting that in some organisms the DnaK function might also be essential under physiological conditions (34, 151, 206). Multiple hsp70 or hsp60 genes have been identified in many organisms (83, 189, 273). Often, Hsp70 or Hsp60 homologs are not temperature controlled but are either constitutively expressed or induced by environmental cues other than heat. In some cases, family members seem to be able to functionally replace each other, e.g., the GroEL proteins of B. japonicum (84). Other seemingly similar proteins appear to have more specialized functions and are unable to restore the defect caused by a deleted family member, e.g., DnaK and HscA (Hsc66) of E. coli (123).
Both major chaperones recognize hydrophobic surfaces of unfolded proteins. To finish the folding job, they require specific cochaperones and ATP (Table 1). The DnaK cycle is driven by the concerted action of DnaJ and GrpE (18, 40). Briefly, DnaJ accelerates the ATPase activity and substrate binding of DnaK whereas GrpE functions as nucleotide exchange factor, promoting the exchange of ADP for ATP. Although details of the GroEL cycle are still disputed, it seems widely accepted that binding of ATP and GroES trigger conformational changes in the GroEL ring and promote folding of the polypeptide captured in its central cavity. Subsequent ATP hydrolysis discharges GroES and the substrate from the complex (40, 111, 126, 257). Among the many details that have been worked out for the Hsp70 and Hsp60 machines are the structural properties not only of the chaperone components but also of their cochaperones (85, 110, 131, 243, 360, 368). GroEL assembles into a cylindrical complex consisting of two homoheptameric rings with a large cylindrical chamber that accomodates substrate proteins (360). Unlike GroEL, DnaK acts as a monomer. There is no high-resolution structure of a full-length Hsp70 protein yet, but the crystal structures of the isolated ATPase and substrate-binding domains have been solved (85, 368). The latter contains a polypeptide-binding channel with a hydrophobic pocket. The completely different architectures of DnaK and GroEL are reflected by different substrate specificities. DnaK binds to short hydrophobic segments of nonnative proteins, whereas the GroEL ring encloses the entire substrate protein. Due to the size limitation of the central cavity, typical GroEL substrates are in the small to medium size range from 20 to 60 kDa (130). DnaK, on the other hand, is able to handle large proteins because it acts on surface-exposed hydrophobic peptide chains (211).
Hsp100
Although DnaK and GroEL are the main actors on the folding stage, there are many other chaperones adding to the fidelity of protein folding. These chaperones have more specialized functions and extend the limits of the DnaK- and GroEL-based folding machineries. With few exceptions to the rule, neither of the alternative chaperones is crucial for survival under physiological growth conditions. Hsp100 proteins are ATP-hydrolyzing chaperones that assemble into ring-shaped structures (100, 152, 369). All members of this family use ATP to promote changes in the folding and assembly of other proteins (269). The subsequent fate of a substrate protein depends on the actual Hsp100 member with which it is interacting (Table 1). ClpB directly binds protein aggregates. ATP-induced structural changes in ClpB are assumed to shear aggregates, preparing them for refolding by the DnaK system (94). ClpA and ClpX represent the substrate recognition subunits of the two-component ClpAP and ClpXP proteases (97, 313, 351). Although much smaller than the other family members, ClpX (46 kDa) is considered as Hsp100 relative. Unlike other family members, ClpX has only one instead of two ATP-binding sites. Polypeptides destined for proteolysis by ClpP are first bound and remodeled by the ATPase components ClpA or ClpX before they are translocated into the heptameric proteolytic ring (135, 254, 291). In the absence of the peptidase subunits, the ATPase oligomer acts as a bona fide chaperone, releasing the substrate in a folding-competent form (352, 356). For completeness, another type of two-component protease, ClpYQ (HslUV), also consisting of an ATPase subunit and a proteolytic component, should be mentioned (209, 260). Although inference from ClpA and ClpX suggests that ClpY (HslU) might possess chaperone activity, this has not been demonstrated yet. The invention of several two-component proteases implies that the intimate association of chaperones together with proteases improves the efficiency of protein degradation by promoting the unfolding of otherwise inaccessible substrate proteins. In fact, it has been shown that ClpP has no proteolytic activity when it is separated from ClpA (132).
Single-Chain Charonins
The same principle applies to single chain charonins, in which chaperone and protease activities are combined on a single polypeptide. It is not trivial to separate the two activities experimentally, but again it seems that chaperone activity might be a prerequisite for efficient proteolysis. Several lines of evidence strongly suggest that the membrane-anchored metalloprotease FtsH has chaperone activity. E. coli FtsH binds to denatured proteins and is involved in membrane protein assembly (4, 5). Moreover, growth retardation of an ftsH mutant can be partially suppressed by the overexpression of other chaperones (286). Chaperone-like activity has been demonstrated most explicitly for the FtsH yeast homolog Yme1, which directly binds to unfolded polypeptides and suppresses their aggregation (180). A very peculiar protein is the periplasmic serine protease DegP. It undergoes a temperature-dependent activity switch and functions as a chaperone at low temperatures and as a protease at elevated temperatures (303). Whether the activity of other charonins is regulated in a similar temperature-dependent manner remains to be tested. A proteolytically inactive variant of the Lon protease retains its substrate-binding capacity, congruent with a chaperone-like activity (337). Whether active refolding of bound substrates can be achieved is not clear. Direct chaperone activity involved in the assembly of membrane protein complexes has been suggested for the mitochondrial Lon protease (256). It should be pointed out again that the primary function of charonins is not to release substrate proteins for subsequent refolding. Although FtsH, DegP, and Lon, like ClpA and ClpX, sequester other proteins and thereby prevent inappropriate interactions leading to aggregation, the primary purpose of these chaperones is not to rescue other proteins but to prepare them for final destruction.
Hsp90
Three chaperone families remain to be discussed, Hsp90, Hsp33 and -crystallin (Table 1). Of these proteins, only Hsp90 hydrolyzes ATP, and ATPase activity is essential for chaperone activity in vivo (241). ATP-independent chaperone-like activity can be demonstrated in vitro because Hsp90 bears two chaperone sites, one of which does not require ATP. Hsp90 is essential in yeast and Drosophila melanogaster but dispensable in E. coli and other bacteria (15, 36). In eukaryotes, Hsp90 is a dedicated chaperone involved in the folding of several signaling molecules including steroid hormone receptors (36, 137). The physiological function of HtpG, the bacterial Hsp90 protein, has not been discerned yet. In vitro, HtpG prevents aggregation of unfolded citrate synthase (CS) by transiently interacting with early unfolding intermediates (139). ATP hydrolysis is not necessary for this activity. A recent report indicates that HtpG has chaperone activity in vivo and is necessary for the optimal folding of certain cytoplasmic proteins in stressed E. coli cells (318).
Hsp33
E. coli Hsp33 is at present the only member of a novel redox-regulated chaperone class (140). Database searches suggested, however, that Hsp33 homologs are present in a wide variety of microorganisms. Hsp33 prevents the aggregation of thermally or oxidatively damaged proteins very efficiently in an ATP-independent manner (140). A feature that distinguishes Hsp33 from all other known chaperones is the reversible regulation of its chaperone activity by the redox potential of the cellular environment. Reduced Hsp33 is monomeric and inactive, whereas the oxidized chaperone is dimeric and functional (156a, 344a). The activity switch is brought about by the reversible disulfide bond formation between two pairs of cysteines that coordinate zinc in the reduced state (14). Zinc release and concomitant dimerization of Hsp33 induces structural rearrangements that lead to the formation of potential binding sites for unfolded proteins. Since it is thought that heat shock causes oxidative protein damage (21, 58, 197), it may not be surprising that a redox-sensitive chaperone is under heat shock control.
-Heat Shock Proteins
The remaining tools in the protein-folding inventory that need to be discussed are the -crystallin-type Hsps. The widely used nomenclature "small Hsps" for these chaperones is somewhat unfortunate because other Hsps, e.g., Hsp33, GroES, GrpE, and Hsp15 (a ribosome-associated Hsp [161]), are also small heat-inducible proteins but bear no resemblence to -crystallin. To avoid any confusion, in the remainder of this review the more appropriate term "-Hsp," as suggested by de Jong et al. (61), will be used for the small Hsps that contain the characteristic -crystallin domain.
As the (just discarded) designation "small Hsps" indicates, -Hsps are low-molecular-mass proteins. Their size ranges from 12 to 43 kDa, with the majority being between 14 and 27 kDa. Although these proteins are small, their active entity usually is a large oligomer consisting of multiple subunits. The overall sequence homology between -Hsps is much lower than in other chaperone classes. A common attribute of all -Hsps is the presence of a conserved sequence of about 80 amino acids, which is generally referred to as the -crystallin domain (43). This domain is preceded by an N-terminal region of variable length and considerable sequence diversity. In most cases, a short C-terminal tail extends downstream of the -crystallin domain (Fig. 2).
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FIG.2. Amino acid alignment of bacterial and archaeal -crystallins. Human A- and B-crystallins were added for comparison at the bottom. Abbreviations: Mjannas, M. jannaschii; Eco, E. coli; Vcholer, V. cholera; Ccr, C. crescentus; Mlo, M. loti; Bja, B. japonicum; Paerugi, P. aeruginosa; Avinela, A. vinelandii; Lpneumo, L. pneumoniae; Buchner, Buchnera sp. strain APS; Caceto, C. acetobutylicum; Ooeni, O. oeni; Mtu, M. tuberculosis; Xfastid, X. fastidiosa; Sth, S. thermophilus; Phoriko, P. horikoshii; Pabysii, P. abysii; Mleprae, M. leprae; Mintr; M. intracellulare; Maviu, M. avium; Salbus, S. albus; Synecho, Synechocystis sp. strain PCC 6803; Svulcan, S. vulcanus; Aaeolic, A. aeolicus; Tmarit, T. maritima; Afulg, A. fulgidus; Saurant, S. aurantiaca; Bha, B. halodurans; Dra, D. radiodurans; Mthermo, M. thermoautotrophicum; Ape, A. pernix; Tac, T. acidophilum; Hal, Halobacterium sp. NRC-1; Rprowaz, R. prowazekii; Bsu, B. subtilis; Homo-aA, Homo sapiens A-crystallin; Homo-aB, Homo sapiens B-crystallin. The initial alignment was constructed with CLUSTAL W (322) and then imported into the multiple sequence alignment editor and shading utility GeneDoc (www.psc.edu/biomed/genedoc) and further refined manually. White letters shaded in black or gray indicate amino acids that are identical in at least 80 or 60% of all proteins, respectively. The consensus sequence below the alignment lists these residues in capital and lowercase letters, respectively. Shaded residues printed in black are identical in at least 40% of all sequences. Structural features of M. jannaschii Hsp16.5 are depicted on top of the alignment according to crystal structure data (155). For comparison, see Fig. 5B. At the bottom, the secondary-structure assignment of human A-crystallin is provided (162). -Crystallin regions that were labeled by bis-ANS or related fluorescent probes are indicated in yellow (280). The highlighted M. jannaschii region is equivalent to the labeled peptide of pea Hsp18.1 (175). Segments that cross-linked to target proteins are shaded in blue (278, 281). Peptides identified in the labeling and cross-linking technique are represented in green.
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During growth under optimal conditions, most bacteria produce either no or negligible amounts of -Hsps. Under stress conditions, the induction factors both at the mRNA level and at the protein level can be dramatic (13, 115, 142, 178, 205, 220, 263, 276). Microarray-based expression profiling revealed that transcription of ibpA and ibpB, encoding the E. coli -Hsp members, was heat induced by a factor of 300 (258). This was by far the highest increase among all heat shock genes (Table 1). IbpA and IbpB received their designation of "inclusion body-associated proteins" because they were initially characterized as E. coli proteins tightly associated with overexpressed recombinant proteins that had formed insoluble inclusion bodies (6). In general, -Hsps seem to be dispensable. A temperature-sensitive growth defect of E. coli ibpAB mutants is barely detectable but becomes more pronounced in the additional absence of the dnaK gene (158, 319). Similarly, disruption of hsp30 in the fungus Neurospora crassa produces no clear phenotype at elevated temperatures but, the hsp30 mutant was extremely sensitive if heat stress was combined with carbohydrate limitation (248). Deletion of hsp16.6, the single -Hsp gene in the cyanobacterium Synechocystis sp. strain PCC 6803, resulted in significantly decreased growth rates and in reduced photosynthetic activity on heat treatment (178). Probably the most drastic phenotype to date has been reported in mice, in which disruption of the eye lens A-crystallin led to cataract formation due to rapid inclusion body formation (33).
While most chaperones consume energy, -Hsps are generally believed to be ATP-independent chaperones (138). As a consequence, they lack the refolding capacity of the major chaperones because unfolding and release of folding intermediates cannot be triggered. Nevertheless, -Hsps have been added to the catalogue of molecular chaperones because they bind to denatured proteins and thereby suppress inappropriate interactions leading to the precipitation of aggregates. The action of -Hsps maintains substrate proteins in a folding-competent state (72, 175). The reservoir of substrate proteins associated with -Hsps remains amenable to subsequent refolding by the major chaperone machineries (341). It is important to consider that -Hsps alone will not efficiently "mediate the correct folding of other polypeptides," according to the chaperone definition given above (75, 76). Only in the context of a multichaperone network are they able to enhance protein folding.
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-HEAT SHOCK PROTEINS IN ARCHAEA, BACTERIA, AND EUCARYA
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-Crystallins are widely distributed among all kingdoms, and their existence has been reported in numerous organisms from bacteria to humans. It is noteworthy, however, that not all organisms contain -Hsps. Owing to the steadily growing number of whole-genome sequences, it is now possible to determine the exact number of -Hsp genes in a diverse array of organisms. Table 2 illustrates that this number can vary substantially. With the exception of Halobacterium sp. strain NRC-1, whose genome encodes five putative -Hsps, all other archaea investigated so far have genomes that encode either one or two. The mere presence of these proteins in thermophilic archaea seems puzzling since many of them are devoid of DnaK, DnaJ, and GrpE homologs (192), which are generally believed to be more important for protein folding than are -Hsps. The universal occurrence of -crystallins in the archaeal kingdom might be indicative of an early phylogenetic origin of this protein family. Perhaps they were established before the more efficient DnaK machinery was invented. In fact, it has been proposed that archaea that contain dnaK, dnaJ, and grpE genes acquired them from bacterial donors by lateral gene transfer (99).
Not only thermophilic archaea but also the bacterial thermophiles Thermotoga maritima and Synechococcus vulcanus each contain a single -Hsp (Table 2). The S. vulcanus protein HspA accumulates after heat treatment (263). Apart from global genome and proteome analyses, detailed information about the heat shock response in thermophilic organisms is scarce. Representative thermophiles from all three phylogenetic domains are capable of mounting a heat shock response (327). It appears that organisms adapted to permanent growth at ferocious temperatures between 60 and 100°C still require responsiveness to temperatures beyond their optimal growth range.
The complete absence of -crystallin genes in a number of bacteria became evident during mining of genome-sequencing data (Table 2). What do the microorganisms lacking -Hsps have in common? First, they have rather small genomes, ranging from 0.58 to 2.27 Mb. The 0.58-Mb genome of Mycoplasma genitalium demonstrated that the minimal gene set of self-replicating organisms comprises some 470 coding regions (88). As one might predict, a streamlined genome like this devotes only few resources to superfluous functions such as regulation, DNA repair, and protein quality control. The minimal Hsp set consists of GroEL and GroES, DnaK, DnaJ and GrpE, ClpB, Lon, and FtsH. Both Mycoplasma species sequenced lack genes coding for ClpA, ClpP, ClpX, HtpG, DegP, and -Hsps (88, 124). Treponema pallidum and Helicobacter pylori, with genomes approximately twice and three times the size of the M. genitalium genome, respectively, contain the classical set of Hsps listed in Table 1 with the exception of -Hsps (89, 324). Since -Hsps are unable to productively refold denatured proteins, they are probably the chaperones of choice for omission by a cell with a limited genome. In line with the assumption that small genomes go along with the absence of -Hsps is the presence of multiple -Hsps in organisms with relatively large genomes such as B. subtilis, rhizobia, and eukaryotes (Table 2). The larger the genome, the more genes seem to be devoted to the adaptation to atypical conditions. It should be pointed out, however, that a small genome does not strictly coincide with the absence of -Hsps. Buchnera sp. strain APS (0.64 Mb) and Rickettsia prowazekii (1.1 Mb) each carry an -crystallin-type protein (Table 2). Deducing the absence of these proteins from the limited genome size of these two species would have been misleading.
Why are -Hsp genes present in some organisms but absent in others? Probably more important than the genome size is the life-style of a bacterium and the ambient temperature it usually faces. Buchnera species are obligate endosymbionts of aphids. Their limited gene reservoir renders them completely dependent on a mutualistic relationship with the insect (285). Rickettsia species, the living relatives of mitochondria, also are intracellular parasites. Although their life-style resembles that of Chlamydia species, rickettsias are normally found in arthropods such as lice and ticks. Only occasionally do they infect humans and cause serious diseases (7). The preference for insects, whose body temperature fluctuates with the ambient temperature, might explain why Rickettsia and Buchnera species carry an -Hsp gene in their limited gene set. Most strikingly, all presently known microorganisms without any -Hsps are parasites and/or pathogens of human and animals. Some of them are degenerated to such an extent that they have become obligate intracellular human parasites, e.g., the Chlamydia species (252). In their more or less isothermal niche, the mammalian host, an elaborate system responding to temperature fluctuations might become obsolete. Incidentally, the absence of -Hsps goes along with the absence of cold shock proteins (CSPs) in most cases. The only exception, Haemophilus influenzae, encodes just a single CSP, whereas -Hsp-containing organisms, such as E. coli, B. subtilis, and Lactobacillus lactis, generally produce multiple CSPs during cold stress (98, 361). CSPs function as RNA chaperones, facilitating the translation of mRNA that tends to form secondary structures after a temperature downshift. Assuming that the simultaneous lack of -Hsps and CSPs is no coincidence, it seems as if living inside mammalian hosts poses few thermal challenges and therefore obliterates the need for sophisticated temperature-responsive systems.
B. subtilis, Halobacterium sp. strain NRC-1, and rhizobial species are exceptional among bacteria and archaea in that they encode more than two -Hsps (Table 2). Of the three putative -crystallin-type proteins of B. subtilis, only YocM and CotM were recognized as family members during the initial open reading frame annotation (167). Like many potential -crystallins in other annotated genome sequences, B. subtilis YdfT was categorized as a protein with unknown function. In fact, the similarity of CotM, YocM, and YdfT to other -crystallins is low (Fig. 2). CotM is a dedicated -crystallin involved in the assembly of the outer spore coat (120). Evidence for a chaperone-like activity of CotM was not found. CotM, YdfT, and YocM all might have rather specialized functions that do not require all conserved -crystallin residues.
Multiple -Hsps were detected during proteome analysis of various plant root-nodulating bacteria (205, 220, 226). A more detailed investigation of B. japonicum extracts uncovered at least 10 heat-induced small proteins belonging to the -crystallin family (219). On the basis of their primary amino acid sequence, the -Hsps were tentatively divided into two classes, class A and class B (220). Class A proteins (HspA, HspB, HspD, HspE, and HspH) are closely related to the E. coli IbpA and IbpB proteins (Fig. 2). Class B -Hsps (HspC and HspF) are distinguished from class A proteins by their much longer N-terminal region and a shorter C-terminal extension. They have little similarity to E. coli -Hsps. The presence of two -Hsp classes was also found in Bradyrhizobium sp. (Parasponia) and in Rhizobium sp. strain NGR234 (235). Additional evidence for the presence of multiple -Hsps and for the existence of distinct classes in rhizobia was provided by the determination of the entire genome sequence of Mesorhizobium loti and Sinorhizobium meliloti (89, 143). Eight -Hsp genes encoding four class A and four class B proteins were identified in the first organism, and five genes encoding three class A and two class B proteins were identified in the latter. In this respect, the situation in rhizobial cells is similar to that in plant cytosol, in which three classes of -Hsps (class I, II, and III) have been described (268a, 349).
-Hsps are not the only rhizobial chaperones that are present in multiple copies. Several groESL operons or groEL genes were found not only in Rhizobium leguminosarum, S. meliloti, B. japonicum, and M. loti but also in many other microorganisms (83, 105, 144, 238, 345). What could be the underlying rationale for the evolution of such multigene families? At least four different reasons can be envisaged. (i) Simultaneous induction of redundant copies of a stress gene rapidly elevates the cellular pool of that protective protein class. Such a coordinated parallel induction of -Hsps was observed in B. japonicum (220). (ii) Providing individual family members with different promoter sequences permits induction in response to a variety of stimuli, a strategy used in the case of rhizobial groESL operons (12, 83). (iii) A multigene family relieves the individual genes from functional constraints. Therefore, each gene is free to evolve, allowing it to acquire new functions. The B. subtilis CotM protein, for example, might be the product of such mutational diversification resulting in its role in sporulation. (iv) Given that a protein functions as an oligomer (as GroEL and -Hsps do), slightly different versions of that protein might assemble into hetero-oligomers. Mixed oligomers probably possess substrate specificities or functions slightly different from homo-oligomeric complexes (312). Whatever the exact reason for a gene family might be, it is easily conceivable that iterated stress genes allow a more flexible response to changing conditions than a single gene.
Eukaryotes are well known for containing multiple -crystallins. Saccharomyces cerevisiae inhabits the lower end of the scale with two -Hsps (Hsp26 and Hsp42) (93), whereas plants lie at the upper end. The Arabidopsis thaliana genome exodes 19 -Hsps. Six families of plant -Hsps can be distinguished based on their sequence similarities and their cellular localization. Three families (I, II, and III) reside in the cytosol; one is localized to the chloroplasts, one is localized to the mitochondria, and one is localized to the endoplasmic reticulum (268a, 348, 349). Nine human -Hsps, including A- and B-crystallin, have been described (144). Once thought to be eye lens-specific proteins, mammalian -crystallins have now been found in many cell types (11, 193). In contrast to plant -Hsps, the mammalian family members seem to be restricted to the cytosol and to the nucleus and do not localize to other cellular compartments.
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SEQUENCE DIVERGENCE OF ARCHAEAL AND BACTERIAL -CRYSTALLINS
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Members of the -Hsp superfamily consist of three regions, a characteristic -crystallin domain flanked by a poorly conserved N-terminal region and a short C-terminal extension (43, 61). Extensive alignments and phylogenetic analyses of plant and animal -Hsps can be found elsewhere (43, 61, 268a, 348-350). An overall alignment of 65 -Hsps from archaea and bacteria reveals only very few highly conserved positions (Fig. 2). The prototype -crystallins, humanA- and B-crystallin, are listed for comparison. The sequence comparison clearly demonstrates that -Hsps are related but quite distinct. Pairwise alignments between Methanococcus jannaschii Hsp16.5, E. coli IbpA, or B. subtilis CotM and human A-crystallin show that the sequence identity is as low as 23, 21, and 19%, respectively. Closely related proteins like E. coli IbpA and IbpB or human A- and B-crystallins have not more than 52 or 58% identical amino acids. Rhizobial -Hsps show the highest degree of sequence similarity, provided that they belong to the same class. For example, 74% the amino acids in B. japonicum HspA and HspB (class A) and 61% of those in HspC and HspF (class B) are identical.
As can be seen in Fig. 2, not a single amino acid is entirely conserved throughout all the proteins examined. Only five residues (corresponding to G62, L111, A122, G127, and L129 of M. jannaschii Hsp16.5) are present in more than 80% of all sequences. The last three residues occur in an A-x-x-x-n-G-v-L consensus motif toward the end of the -crystallin domain; this motif is the most significant indicator of the domain (43, 61, 349). Almost universally conserved are the G127 and L129 residues, which occur in all but one protein. In CotM from B. subtilis, the residue equivalent to G127 is a glutamine. In addition, CotM carries replacements of other conserved residues (e.g., the amino acids equivalent to P61 and G62 in Hsp16.5 are lysine and histidine in CotM). Probably owing to this diversification, CotM has no discernible chaperone activity and serves a unique function in spore coat formation (120). Hsp1 from Halobacterium sp. strain NRC-1 contains a cysteine instead of the conserved leucine at position 129. As for most of the sequences listed in Table 2, nothing is known about the functional role of this putative protein. It is possible that Hsp1, like CotM, has structural and functional properties different from standard -Hsps.
All five highly conserved amino acids reside in the -crystallin domain, which carries a few additional moderately conserved residues. The flanking regions are even more divergent. Large deviations in sequence and length are evident in Fig. 2. An alignment can be enforced only at the expense of numerous gaps, in particular in the N-terminal region. Very few residues are retained in more than 40% of all sequences. The only exceptions are two characteristic isoleucines (I144 and I146 in Hsp16.5) in the C-terminal extension. They are separated by a single variant amino acid and play an important structural and functional role, at least in M. jannaschii Hsp16.5, wheat Hsp16.9, and both classes of B. japonicum -Hsps (see below) 155, 338a; S. Studer, M. Obrist, N. Lentze, and F. Narberhaus, submitted for publication).
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REGULATION OF -HEAT SHOCK PROTEINS
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As indicated in the quality control section (see above), it is important that the cellular level of chaperones and proteases be adjusted to the prevailing environmental conditions. The cellular level of chaperones and proteases must be strictly controlled because both excessive and limited amounts compromise the fitness of a cell. The regulatory mechanisms controlling their expression are very diverse. Regulation in most cells occurs at the level of transcription. In eukaryotes, elevated expression of heat shock genes is accomplished by transcriptional activation. With some modifications to the theme, the general principle is conserved in yeast, vertebrates, and plants. In brief, an inert non-DNA-binding heat shock transcription factor (HSF) is converted upon heat shock to a transcription-competent activator that binds to a cis-acting heat shock element (HSE) in the promoter region of heat shock genes (212, 236). The activation process of the HSF goes along with its trimerization and phosphorylation. Stress-induced molecular chaperones, most importantly Hsp70, autoregulate the heat shock response by direct binding to the activation domain of HSF (212, 284).
Heat shock regulation in archaea is largely unsolved. The archaeal proteins involved in basal transcription are related to those of the eukaryotic transcription machinery (19, 179, 300). However, archaea have neither an identifiable HSF nor an HSE in heat shock gene promoter regions (192). Bacterial heat shock promoters or regulatory elements are also absent in archaeal genomes. Transcription of the heat-inducible cct1 (chaperonin-containing Tcp-1) gene in the archaebacterium Haloferax volcanii requires only sequences within the core promoter region (320). The finding of multiple variants of the promoter-binding TATA-binding protein led to the hypothesis that alternative forms of TATA-binding protein are responsible for transcription under heat shock conditions (321).
The latter strategy is strikingly reminiscent of heat shock regulation in E. coli. It uses alternative transcription factors, so-called sigma factors, to modulate heat shock gene expression. Bacterial sigma factors are subunits of the RNA polymerase that confer promoter specificity to the core enzyme (103, 134, 358). Specific transcription of the majority of heat shock genes in E. coli depends on the sigma factor 32 (RpoH) (102). The cellular concentration of 32 increases transiently after a temperature upshift, primarily as the result of elevated translation of rpoH mRNA and of increased stability of the sigma factor. At normal temperatures, 32 is rapidly degraded by FtsH. The requirement of DnaKJ for 32 proteolysis couples the availability of chaperones to heat shock gene transcription and provides a homeostatic heat shock control mechanism (38, 102, 366). The ibpAB operon, which encodes both -Hsps of E. coli, belongs to the 32 regulon (6, 46) (Fig. 3A). Immunodetectable levels of IbpA and IbpB proteins in rpoH mutants suggest, however, that a 32-independent pathway participates in the expression of these chaperones (6, 170). Recently, it was found that ibpB alone can be transcribed from a 54 (RpoN)-dependent promoter (164a).
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FIG. 3. Regulation of bacterial -Hsp genes. A representative organisms for each regulatory strategy is listed. The regulated genes and corresponding regulators are indicated. Promoters are presented as -10 and -35 regions. Known repressor-binding sites occur as inverted repeats (IR) or directed repeats (DR). Details are presented in the text.
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Regulation by alternative sigma factors has also been described for two other bacterial -Hsp genes (Fig. 3A). Legionella pneumophila gspA is transcribed from a 70-type and a 32-type promoter (2, 3). Induction of gspA expression by stress stimuli and during intracellular infection occurs predominantly via the latter promoter. Hsp16.3 of Mycobacterium tuberculosis is, at least in part, dependent on SigF, one of many stress-responsive sigma factors in this organism (194). Hsp16.3 (Acr) is not heat shock responsive but accumulates in the transition to stationary phase, during hypoxia and infection of macrophages (364, 365). Many conditions that enhance the SigF level have no effect on hsp16.3 expression, indicating that SigF is not the sole regulator of this gene. The involvement of the two-component regulatory pair Rv3133c/Rv3132c has recently been inferred from a microarray approach (283).
During investigation of other heat shock regimes, it turned out that positive control by alternative sigma factors is not typical of many other bacteria. Rather, transcriptional repression of heat shock genes is a widespread strategy found in many phylogenetically distinct bacteria. Overlapping or nearby repressor and RNA polymerase-binding sites prevent transcriptional initiation under circumstances where the repressor is competent for DNA binding. A number of heat shock gene repressors have been identified recently (114, 222). Some of them also control -Hsp genes (Fig. 3B). An intriguingly simple mechanism was discovered in Streptomyces albus. At low temperatures, RheA (for "repressor of hsp eighteen"; formerly OrfY) inhibits the transcription of hsp18 (276, 277). As the result of a thermally induced conformational change, the repressor is unable to interact with its target sequence (an inverted repeat) at elevated temperatures (274). The transition between the active and inactive forms is fully reversible. RheA thus acts as a cellular thermosensor, directly correlating heat shock gene expression to the ambient temperature.
hsp18 of Clostridium acetobutylicum, hsp18 of Leuconostoc oenos (Oenococcus oeni), and hsp16.4 of Streptococcus thermophilus are preceded by putative CtsR target sites (64). CtsR (for class three stress gene repressor) was initially identified in B. subtilis as negative regulator of the clpC operon (64, 163). It blocks transcription of target genes at normal temperatures through binding to a conserved heptanucleotide repeat (Fig. 3B). CtsR is specifically degraded by the ClpCP protease during stress. Since expression of the Clp components (and some additional regulatory factors) is under CtsR control, proteolysis of the repressor constitutes a built-in autoregulatory loop (65, 164). Putative ctsR homologs and potential CtsR-binding sites upstream of heat shock genes were documented in a number of gram-positive microbes (64). Temperature-regulated transcription from housekeeping promoters was demonstrated for the C. acetobutylicum and O. oeni hsp18 genes (142, 267). However, experimental proof that CtsR mediates heat shock control of -Hsp genes in these and other gram-positive bacteria remains to be generated.
Neither of the three -crystallins of B. subtilis is temperature regulated. CotM is induced at late stages during sporulation and maintains the structural integrity of the outer spore coat (120). Both an alternative sigma factor (the late sporulation sigma factor K) and a repressor protein (GerE) contribute to its proper temporal and spatial expression (Fig. 3C). Similar dual control of ydfT led to its alternative designation, cotP (255). Although expression of CotM and CotP is restricted to sporulation, they seem to be dispensable for this process.
A number of bacterial -Hsp genes and operons are regulated by mechanisms that cannot be assigned to either transcriptional activation or repression. Long untranslated regions (5'UTRs) were found upstream of various rhizobial -Hsp genes and upstream of hspA of Synechococcus vulcanus (223, 224, 235, 263) (Fig. 3D). Temperature-regulated transcription of all these genes was initiated at vegetative promoters. A novel regulatory mechanism controlling mRNA stability in a temperature-dependent fashion was inferred from the fact that the hspA transcript of S. vulcanus is more stable at 63 than 50°C. Unknown factors might be involved in this process (263). The 5'UTR of at least 15 small heat shock operons from B. japonicum, Bradyrhizobium sp. (Parasponia), Rhizobium sp. strain NGR234, and Mesorhizobium loti was designated ROSE, acknowledging its role in repression of heat shock gene expression (223, 235). Although a repressor-type mechanism was favored initially, there now is accumulating evidence that ROSE acts at the posttranscriptional level. RNA structure predictions suggest that the highly conserved 3' region of all known ROSE sequences folds into a stem-loop structure that masks the ribosome-binding site (RBS) (234). A detailed mutagenesis study on a representative ROSE element demonstrated that the exchange of individual nucleotides potentially involved in base pairing relieves -Hsp gene repression at low temperatures. The current model holds that mRNA folding at low temperatures has two consequences. It blocks access of ribosomes to the RBS and thereby impairs translation. The presumed secondary structure probably also promotes RNase cleavage and rapid decay of ROSE-containing mRNAs, explaining the minute amounts of -Hsp transcripts at low temperatures. According to this model, high temperatures would melt the hairpin structure, allowing ribosomes to access the RBS. Ribosome entry then would initiate translation and simultaneously protect ROSE-containing mRNA from degradation.
A noteworthy feature of several -Hsp genes is their extrachromosomal location. Small plasmids carry the low-molecular-mass stress proteins of Streptococcus thermophilus (239, 298, 299). On plasmids pER16 (4.5 kb), pER35 (10 kb), pER36 (3.7 kb), and pER341 (2.8 kb) originating from different S. thermophilus strains, the only other open reading frame encodes a rolling-circle replication protein (298, 299). The 6.5-kb plasmid pCI65st from S. thermophilus NDI-6 codes for two almost identical -Hsps (Hsp1 and Hsp2; an alignment is given in Fig. 2), a replication protein, a putative enolase, and HsdS, a putative type 1 restriction modification enzyme (239). These streptococcal replicons are readily lost at low temperatures but stably maintained at optimal growth temperatures of 42°C (239, 298). The plasmid stability suggests that the -Hsps are beneficial to lactic acid bacteria during fermentation of dairy products.
Sequencing of rhizobial genomes revealed that some of their multiple -Hsp genes are also located on extrachromosomal replicons. M. loti contains the megaplasmids pMLa (352 kb) and pMLb (208 kb). The latter bears the locus mll9627, coding for one of eight -crystallins (143, 235). In Sinorhizobium meliloti, three of five -Hsps are encoded on symbiotic megaplasmids. The locus SMa1118 lies on pSymA (1.4 Mb), and SMb21294 and SMb21295 are on pSymB (1.7 Mb) (89). The presence of several -Hsps in bacteroids suggests that these proteins play a role in establishing a successful plant-microbe interaction (226). A plasmid-borne -crystallin gene was also found in Buchnera aphidicola, the endosymbiont of aphids (334). Other genes harbored by this 8.5-kb plasmid are involved in leucine biosynthesis. A remarkably similar clustering of an ibpB-like gene with leucine biosynthetic genes was observed on the chromosome of Azotobacter vinelandii (195). Whether the close association of -Hsp genes with amino acid metabolism genes is a coincidence or functionally relevant is unclear.
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OLIGOMERIZATION OF -HEAT SHOCK PROTEINS
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Homo-Oligomeric Complexes
A hallmark of -Hsps is their tendency to "socialize," or in other words, to assemble into large oligomeric complexes. There is ample evidence that oligomerization is a structural prerequisite for chaperone activity of the vast majority of -Hsps. Neither naturally occurring multimerization-incompetent -crystallins nor mutated variants that have lost the ability to form large complexes are efficient chaperones 160, 181, 182, 331; Studer et al., submitted). In many cases in which bacterial -Hsps have been studied biochemically, they were reported to build complexes consisting of approximately 24 subunits (156, 204, 262, 310). Formal proof for such an organization stems from the crystal structure of the M. jannaschii Hsp16.5. A total of 24 monomers form a spherical complex with 14 open windows (155) (see Fig. 5). Electron microscopy (EM) images also revealed similar spherical complexes for human B-crystallin, human Hsp27, native bovine -crystallin, and yeast Hsp26 (107, 112).
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FIG. 5. Overall structure and subunit interactions of Methanococcus jannaschii Hsp16.5. (A) In the space-filling model of the hollow sphere, each tetramer is represented in one color with different shadings. At the bottom, the interior of the sphere is viewed along the threefold axis (left) and the fourfold axis (right). The front one-third of each sphere is cut off. (B) Topology of the secondary structure of a Hsp16.5 dimer. The first and last residue numbers for each secondary-structure element are indicated in the left monomer, which is indicated by a dotted frame. The structural elements are labeled in the right monomer. (Reprinted from reference 155 with permission of the publisher.)
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Examples of both smaller and larger assemblies than 24-mers have been documented. M. tuberculosis Hsp16.3 forms a triangular structure comprising a nonamer composed of a trimer of trimers (44). Pea Hsp18.1 and Hsp17.7, two representatives of cytosolic plant -Hsps, produce discretely sized complexes containing 12 identical subunits (174). Murine Hsp25 exists predominantly as a hexadecamer (72, 74). At the opposite extreme of the oligomeric scale are the proteins that form large and heterogenous assemblies. -Crystallin from vertebrate eye lenses is normally isolated as an 800-kDa complex comprising 32 subunits. However, various other assemblies from 280 kDa to 10 MDa have been reported (101). The majority of E. coli IbpB is eluted from gel filtration columns as globular structures exceeding 2 MDa (282, 341). Electron micrographs reveal pronounced size heterogeneity from small roughly spherical complexes with a diameter of 15 nm (approximately 600 kDa) to 100-200-nm structures that interact over time to form loose aggregates of micrometer size (282).
Regardless of the subunit composition, all those diverse assemblies described above efficiently protect other proteins from aggregation, illustrating that a composition of 24 subunits is not a structural prerequisite for chaperone activity. The astounding diversity of -Hsp assemblies also is the reason why the minimal oligomeric requirement for chaperone activity is not clear-cut. The monomeric Hsp12.6 from Caenorhabditis elegans is unable to prevent the aggregation of test substrates (181). Hsp12.2 and Hsp12.3, two other exceptionally small members of the -crystallin family of C. elegans, assemble into tetramers that are devoid of any chaperone activity (160). In contrast, Hsp20 from rats tends to form dimers that act as poor chaperones (331). A truncated version of human B-crystallin (B57-157) that contains only the isolated -crystallin domain builds dimers with significant chaperone activity (81). Unlike other -Hsps, yeast Hsp26 requires temperature-assisted dissociation of a 24-mer into dimeric species in order to become an efficient chaperone (112). Tetramers of murine Hsp25 that occur as intermediates in the assembly of 16-mers have been shown to bind troponin T, a structurally labile marker protein for myocardial cell damage (73). This short list of observations already implies that the correlation between the oligomeric status and chaperone activity is different for individual -Hsps.
Hetero-Oligomeric Complexes
The tendency of -Hsps to generate higher-level structures poses the question whether oligomers always contain identical subunits or whether mixed oligomers can be formed (provided that different -Hsps occur in the same cell or cellular compartment). Native -crystallin isolated from the eye lens is composed of the closely related subunits A- and B-crystallins in a 3:1 stoichiometry (129). Under in vitro conditions, however, any ratio can be formed (333). Several reports demonstrate that A- and B-crystallins are able to interact with other members of the -crystallin family. B-crystallin copurifies with human Hsp28 or with rat Hsp27 (146, 367). On mixing of purified components in one study, heteropolymers from any combination of bovine A-crystallin, B-crystallin, and mouse Hsp25 could be detected (202). An interaction between Hsp27 and B-crystallin was demonstrated by the yeast two-hybrid system (27, 190). Finally, fluorescence-labeled A-crystallin readily exchanged with B-crystallin and with Hsp27 (31). All these findings strongly suggest that hetero-oligomerization is a common phenomenon in mammalian cells.
The formation of mixed -Hsp complexes in plants and bacteria appears to be more restricted than in mammals. Most of the multiple -Hsps that are being produced in plants are prevented from interacting with each other because they are housed in different cellular compartments (349). However, three distinct classes occur in the cytoplasm. The recombinant pea proteins Hsp18.1 and Hsp17.7 (representative class I and class II members, respectively) were analyzed for complex formation in vitro (119). Both proteins strictly assembled into homo-oligomeric complexes and did not
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