The small heat shock protein,HSPB6, in muscle function and disease

The small heat shock protein, HSPB6, is a 17-kDa protein that belongs to the small heat shock protein family. HSPB6 was identified in the mid-1990s when it was recognized as a by-product of the purification of HSPB1 and HSPB5. HSPB6 is highly and constitutively expressed in smooth, cardiac, and skeletal muscle and plays a role in muscle function. This review will focus on the physiologic and biochemical properties of HSPB6 in smooth, cardiac, and skeletal muscle; the putative mechanisms of action; and therapeutic implications.     

Versatility of the small heat shock protein HSPB6 (Hsp20)

The recently published review by Dreiza et al. (Cell Stress and Chaperones DOI ) dealing with the functional role of HSPB6 in muscle regulation is critically analyzed. Published data indicate that the chaperone-like activity of HSPB6 is comparable with that of HSPB5 and that phosphorylation of HSPB6 does not affect its oligomeric structure. Different hypotheses concerning the molecular mechanisms of HSPB6 action on smooth muscle contraction and on the reorganization of the cytoskeleton are compared, and it is concluded that although HSPB6 is not a genuine actin-binding protein, it can affect the actin cytoskeleton indirectly. Phosphorylated HSPB6 interacts with 14-3-3 and thereby displaces other binding partners of 14-3-3; among them, certain phosphatases, protein kinases, and various actin-binding proteins, which can participate in the reorganization of the actin cytoskeleton. In addition, HSPB6 seems to regulate the activity of certain protein kinases. All of these processes are dependent on HSPB6 phosphorylation which in turn might be regulated by the formation of heterooligomeric complexes of HSPB6 with other small heat shock proteins.     

Expression of APG-2 protein, a member of the heat shock protein 110 family, in developing rat brain

APG-2 protein is a member of the heat shock protein 110 family, and it is thought to play an important role in the maintenance of neuronal functions under physiological and stress conditions. However, neither the tissue-distribution of APG-2 protein nor developmental change of its expression has been studied at the protein level. Therefore, we generated an antiserum against APG-2 protein and studied expression of this protein in rat brain and other tissues by use of the Western blot method. The results showed a high expression of APG-2 protein in various regions of the central nervous system (cerebral cortex, hippocampus, striatum, midbrain, hypothalamus, cerebellum, medulla pons, and spinal cord) throughout the entire postnatal stage. Similarly, a high level of APG-2 protein was detected in the whole brain of rat embryos and in adult rat tissues such as liver, lung, spleen, and kidney. In contrast, its expression in heart was high at postnatal days 1 and 3, but thereafter drastically decreased to a low level. Furthermore, APG-2 protein was detected in neuronal primary cultures prepared from rat cerebral cortex, and its level did not change notably during neuronal differentiation. These results show that APG-2 protein is constitutively expressed in various tissues and also in neuronal cells throughout the entire embryonic and postnatal period. suggesting that it might play an important role in these tissues under non-stress conditions.     

Muscle develops a specific form of small heat shock protein complex composed of MKBP/HSPB2 and HSPB3 during myogenic differentiation

Previously, we identified a new mammalian sHSP, MKBP, as a myotonic dystrophy protein kinase-binding protein, and suggested its important role in muscle maintenance (Suzuki, A., Sugiyama, Y., Hayashi, Y., Nyu-i, N., Yoshida, M., Nonaka, I., Ishiura, S., Arahata, K., and Ohno, S. (1998) J. Cell Biol. 140, 1113-1124). In this paper, we develop the former work by performing extensive characterization of five of the six sHSPs so far identified, that is, HSP27, alphaB-crystallin, p20, MKBP/HSPB2, and HSPB3, omitting lens-specific alphaA-crystallin. Tissue distribution analysis revealed that although each sHSP shows differential constitutive expression in restricted tissues, tissues that express all five sHSPs are only muscle-related tissues. Especially, the expressions of HSPB3, identified for the first time as a 17-kDa protein in this paper, and MKBP/HSPB2 are distinctly specific to muscles. Moreover, these sHSPs form an oligomeric complex with an apparent molecular mass of 150 kDa that is completely independent of the oligomers formed by HSP27, alphaB-crystallin, and p20. The expressions of MKBP/HSPB2 and HSPB3 are induced during muscle differentiation under the control of MyoD, suggesting that the sHSP oligomer comprising MKBP/HSPB2 and HSPB3 represents an additional system closely related to muscle function. The functional divergence among sHSPs in different oligomers is also demonstrated in several ways: 1) an interaction with myotonic dystrophy protein kinase, which has been suggested to be important for the maintenance of myofibril integrity, was observed only for MKBP/HSPB2; 2) a myotube-specific association with actin bundles was observed for HSP27 and alphaB-crystallin, but not for MKBP/HSPB2; and 3) sHSPs whose mRNAs are induced by heat shock are alphaB-crystallin and HSP27. Taken together, the results suggest that muscle cells develop two kinds of stress response systems composed of diverged sHSP members, and that these systems work independently in muscle maintenance and differentiation.     

Molecular structure and dynamics of the dimeric human small heat shock protein HSPB6

ATP-independent small heat-shock proteins (sHSPs) are an essential component of the cellular chaperoning machinery. Under both normal and stress conditions, sHSPs bind partially unfolded proteins and prevent their irreversible aggregation. Canonical vertebrate sHSPs, such as the α-crystallins, form large polydisperse oligomers from which smaller, functionally active subspecies dissociate. Here we focus on human HSPB6 which, despite having considerable homology to the α-crystallins in both the N-terminal region and the signature α-crystallin domain (ACD), only forms dimers in solution that represent the basic chaperoning subspecies. We addressed the three-dimensional structure and functional properties of HSPB6 in a hybrid study employing X-ray crystallography, solution small-angle X-ray scattering (SAXS), mutagenesis, size-exclusion chromatography and chaperoning assays. The crystal structure of a proteolytically stable fragment reveals typical ACD dimers which further form tetrameric assemblies as a result of extensive inter-dimer patching of the β4/β8 grooves. The patching is surprisingly mediated by tripeptide motifs, found in the N-terminal domain directly adjacent to the ACD, that are resembling but distinct from the canonical IxI sequence commonly binding this groove. By combining the crystal structure with SAXS data for the full-length protein, we derive a molecular model of the latter. In solution, HSPB6 shows a strong attractive self-interaction, a property that correlates with its chaperoning activity. Both properties are dictated by the unstructured yet compact N-terminal domain, specifically a region highly conserved across vertebrate sHSPs.     

Why proteins without an α-crystallin domain should not be included in the human small heat shock protein family HSPB

The presence of an α-crystallin domain documents the evolutionary relatedness of the ubiquitous family of small heat shock proteins. Sequence and three-dimensional structure provide no evidence for the presence of such a domain in HSPC034, recently proposed as the 11th member of the human HSPB family. Also, phylogenetic analyses detect no relationship between HSPC034 and the human HSPB1–10 sequences. Arguments are provided as to why inclusion in the HSPB family of proteins like HSPC034, which resemble small heat shock proteins in being heat-inducible and having chaperone-like properties and a low monomeric mass, but are evolutionarily unrelated, is misleading and confusing.     

Ubiquitous and cell-specific members of the avian small heat shock protein family.

Small heat shock proteins (sHsps) have been suggested to act as molecular chaperones for many kinds of substrates and have protective roles in cells exposed to external stresses. Unlike other major Hsps such as Hsp70 and Hsp90, expression of many vertebrate sHsps is restricted to the muscle tissues and/or eye lens. Among the sHsps, the heat-inducible human Hsp27 (hHsp27) homologue is believed to be expressed ubiquitously in various cell types. Here, we distinguished the chicken homologue of hHsp27 (cHsp24) from the chicken major heat-inducible protein of molecular size 25 kDa (cHsp25). cHsp25 is not expressed in the absence of stress, but is highly expressed after hyperthermia in all tissues of developing embryos. In contrast, expression of cHsp24 is restricted to some specific tissues even in the presence of stress. Thus, cHsp25 is the first member of the sHsps in vertebrates the expression of which is ubiquitous in tissues exposed to external stresses similar to Hsp70.     

Testis-specific human small heat shock protein HSPB9 is a cancer/testis antigen, and potentially interacts with the dynein subunit TCTEL1

Searching EST databases for new members of the human small heat shock protein family, we recently identified HSPB9, which is expressed exclusively in testis as determined by Northern blotting (Kappé et al., Biochim. Biophys. Acta 1520, 1-6, 2001). Here we confirm this testis-specific expression pattern by RT-PCR in a larger series of normal tissues. Interestingly, while screening HSPB9 ESTs, we also noted expression in tumours, which could be verified by RT-PCR. Protein expression of HSPB9 was also detected in normal human testis and various tumour samples using immunohistochemical staining. We thus conclude that HSPB9 belongs to the steadily growing number of cancer/testis antigens. To get a better understanding of the function of HSPB9, we performed a yeast two-hybrid screen to search for HSPB9-interacting proteins. TCTEL1, a light chain component of cytoplasmic and flagellar dynein, interacted in both the yeast two-hybrid system and in immunoprecipitation experiments with HSPB9. Additionally, immunohistochemical staining showed co-expression of HSPB9 and TCTEL1 in similar stages of spermatogenesis and in tumour cells. The possible functional significance of this interaction is discussed.     

A small heat shock protein cooperates with heat shock protein 70 systems to reactivate a heat-denatured protein

Small heat shock proteins (sHsps) are a diverse group of heat-induced proteins that are conserved in prokaryotes and eukaryotes and are especially abundant in plants. Recent in vitro data indicate that sHsps act as molecular chaperones to prevent thermal aggregation of proteins by binding non-native intermediates, which can then be refolded in an ATP-dependent fashion by other chaperones. We used heat-denatured firefly luciferase (Luc) bound to pea (Pisum sativum) Hsp18.1 as a model to define the minimum chaperone system required for refolding of a sHsp-bound substrate. Heat-denatured Luc bound to Hsp18.1 was effectively refolded either with Hsc/Hsp70 from diverse eukaryotes plus the DnaJ homologs Hdj1 and Ydj1 (maximum = 97% Luc reactivation with k(ob) = 1.0 x 10(-2)/min), or with prokaryotic Escherichia coli DnaK plus DnaJ and GrpE (100% Luc reactivation, k(ob) = 11.3 x 10(-2)/min). Furthermore, we show that Hsp18.1 is more effective in preventing Luc thermal aggregation than the Hsc70 or DnaK systems, and that Hsp18.1 enhances the yields of refolded Luc even when other chaperones are present during heat inactivation. These findings integrate the aggregation-preventive activity of sHsps with the protein-folding activity of the Hsp70 system and define an in vitro system for further investigation of the mechanism of sHsp action.     

Self-association of a small heat shock protein

Human Hsp27 oligomerizes in vivo in a phosphorylation-dependent manner that regulates the functional activity of the protein. We have studied the self-association of wild-type Hsp27 by both sedimentation velocity and sedimentation equilibrium analysis and established that the protein forms an equilibrium mixture of monomers/dimers, tetramers, 12-mers and 16-mers (20 mM Tris-HCl (pH 8.4), 100 mM NaCl, 20 degrees C). Corresponding analysis of the S15D/S78D/S82D triple variant, which is believed to mimic the behavior of phosphorylated Hsp27, establishes that this form of the protein forms primarily monomers and dimers but also forms a small fraction of very large oligomers. Variants in which critical N-terminal sequences have been deleted exhibit oligomerization behavior that is intermediate between that of the triple variant and the wild-type protein. On the other hand a C-terminal sequence deletion variant forms larger oligomers than does the wild-type protein, but also exhibits a greater fraction of smaller oligomers. Notably, the presence of an N-terminal His6-tag induces formation of much larger oligomers than observed for any other form of the protein. The results of this work establish that the wild-type protein forms smaller oligomers than previously believed, define the roles played by various structural domains in Hsp27 oligomerization, and provide improved molecular probes with better-defined properties for the design of future experiments.     

A heat shock protein localized to chloroplasts is a member of a eukaryotic superfamily of heat shock proteins.

We have isolated cDNA clones from soybean and pea that specify nuclear-encoded heat shock proteins (HSPs) which localize to chloroplasts. The mRNAs for these HSPs are undetectable at control temperatures, but increase approximately 150-fold during a 2-h heat shock. Hybridization-selection followed by in vitro translation demonstrates that these HSPs are synthesized as precursor proteins which are processed by the removal of 5-6.5 kd during import into isolated chloroplasts. The nucleotide sequence of the cDNAs shows the derived amino acid sequences of the mature pea and soybean proteins are 79% identical. While the predicted transit peptide encoded by the pea cDNA has some characteristics typical of transit sequences, including high Ser content, multiple basic residues and no acidic residues, it lacks two domains proposed to be important for import and maturation of other chloroplast proteins. The carboxy-terminal region of the chloroplast HSP has significant homology to cytoplasmic HSPs from soybean and other eukaryotes. We hypothesize that the chloroplast HSP shares a common structural and functional domain with low mol. wt HSPs which localize to other parts of the cell, and may have evolved from a nuclear gene.     

Interaction of human HSP22 (HSPB8) with other small heat shock proteins

Mammalian small heat shock proteins (sHSP) are abundant in muscles and are implicated in both muscle function and myopathies. Recently a new sHSP, HSP22 (HSPB8, H11), was identified in the human heart by its interaction with HSP27 (HSPB1). Using phylogenetic analysis we show that HSP22 is a true member of the sHSP superfamily. sHSPs interact with each other and form homo- and hetero-oligomeric complexes. The function of these complexes is poorly understood. Using gel filtration HPLC, the yeast two-hybrid method, immunoprecipitation, cross-linking, and fluorescence resonance energy transfer microscopy, we report that (i). HSP22 forms high molecular mass complexes in the heart, (ii). HSP22 interacts with itself, cvHSP (HSPB7), MKBP (HSPB2) and HSP27, and (iii). HSP22 has two binding domains (N- and C-terminal) that are specific for different binding partners. HSP22 homo-dimers are formed through N-N and N-C interactions, and HSP22-cvHSP hetero-dimers through C-C interaction. HSP22-MKBP and HSP22-HSP27 hetero-dimers involve the N and C termini of HSP22 and HSP27, respectively, but appear to require full-length protein as a binding partner.     

MafT, a new member of the small Maf protein family in zebrafish

Small Maf proteins play critical roles on morphogenesis and homeostasis through associating with CNC proteins. To date, three small Maf proteins, MafF, MafG, and MafK, have been reported in vertebrates, which share redundant functions. In this study, we tried to identify and characterize small Maf proteins in zebrafish to elucidate their conservation and diversity in the fish kingdom. We identified homolog genes of MafG and MafK but not MafF in zebrafish, indicating the former two are conserved among vertebrates. In addition, a novel type of small Maf protein MafT was identified. MafT protein bound MARE sequence as a homodimer or heterodimers with zebrafish Nrf2 or p45 Nfe2. Co-overexpression of MafT and Nrf2 synergistically activated MARE-mediated gene expression in zebrafish embryos. These results indicated that MafT is a new member of small Maf proteins and involved in the Nrf2-dependent gene regulation in cellular defense system.     

A protein antigen of Mycobacterium leprae is related to a family of small heat shock proteins.

The gene encoding an immunologically important 18-kilodalton protein antigen of Mycobacterium leprae has been sequenced, and the amino acid sequence of the antigen has been deduced. The 18-kilodalton antigen is strikingly similar in size and sequence to a family of eucaryotic heat shock proteins.