The expanding small heat-shock protein family, and structure predictions of the conserved "alpha-crystallin domain"

The ever-increasing number of proteins identified as belonging to the family of small heat-shock proteins (shsps) and alpha-crystallins enables us to reassess the phylogeny of this ubiquitous protein family. While the prokaryotic and fungal representatives are not properly resolved, most of the plant and animal shsps and related proteins are clearly grouped in distinct clades, reflecting a history of repeated gene duplications. The members of the shsp family are characterized by the presence of a conserved homologous "alpha-crystallin domain," which sometimes is present in duplicate. Predictions are made of secondary structure and solvent accessibility of this domain, which together with hydropathy profiles and intron positions support the presence of two similar hydrophobic beta-sheet-rich motifs, connected by a hydrophilic alpha-helical region. Together with an overview of the newly characterized members of the shsp family, these data help to define this family as being involved as stable structural proteins and as molecular chaperones during normal development and induced under pathological and stressful conditions.     

NMR spectroscopy of alpha-crystallin. Insights into the structure, interactions and chaperone action of small heat-shock proteins

The subunit molecular mass of alpha-crystallin, like many small heat-shock proteins (sHsps), is around 20 kDa although the protein exists as a large aggregate of average mass around 800 kDa. Despite this large size, a well-resolved 1H NMR spectrum is observed for alpha-crystallin which arises from short, polar, highly-flexible and solvent-exposed C-terminal extensions in each of the subunits, alpha A- and alpha B-crystallin. These extensions are not involved in interactions with other proteins (e.g. beta- and gamma-crystallins) under non-chaperone conditions. As determined by NMR studies on mutants of alpha A-crystallin with alterations in its C-terminal extension, the extensions have an important role in acting as solubilising agents for the relatively-hydrophobic alpha-crystallin molecule and the high-molecular-weight (HMW) complex that forms during the chaperone action. The related sHsp, Hsp25, also exhibits a flexible C-terminal extension. Under chaperone conditions, and in the HMW complex isolated from old lenses, the C-terminal extension of the alpha A-crystallin subunit maintains its flexibility whereas the alpha B-crystallin subunit loses, at least partially, its flexibility, implying that it is involved in interaction with the 'substrate' protein. The conformation of 'substrate' proteins when they interact with alpha-crystallin has been probed by 1H NMR spectroscopy and it is concluded that alpha-crystallin interacts with 'substrate' proteins that are in a disordered molten globule state, but only when this state is on its way to large-scale aggregation and precipitation. By monitoring the 1H and 31P NMR spectra of alpha-crystallin in the presence of increasing concentrations of urea, it is proposed that alpha-crystallin adopts a two-domain structure with the larger C-terminal domain unfolding first in the presence of denaturant. All these data have been combined into a model for the quaternary structure of alpha-crystallin. The model has two layers each of approximately 40 subunits arranged in an annulus or toroid. A large central cavity is present whose entrance is ringed by the flexible C-terminal extensions. A large hydrophobic region in the aggregate is exposed to solution and is available for interaction with 'substrate' proteins during the chaperone action.     

Development of a high-volume in situ mRNA hybridization assay for the quantification of gene expression utilizing scintillating microplates

The 94-kDa glucose-regulated protein (GRP94) is a member of the 90-kDa heat-shock protein (HSP90) family. In this study, we expressed the barley (Hordeum vulgare L.) GRP94 and the alpha isoform of human HSP90 (HSP90 alpha) in Escherichia coli and compared their dimer-forming abilities. Native polyacrylamide gel electrophoresis revealed that GRP94 (amino acids 69-809) and the full-length form of HSP90 alpha existed in the dimeric state. The C-terminal 326 amino acids of GRP94 or the C-terminal 200 amino acids of HSP90 alpha were sufficient for the dimerization. Limited proteolysis of the C-terminal half of GRP94 with thrombin revealed a 16-kDa fragment, which was derived from the C-terminus of GRP94 through the cleavage of either the Arg710-His711 or the Arg735-Leu736 bond. These cleavage sites were nearly, if not completely, equivalent to the proteolyzed region of HSP90 alpha. Their structural similarity prompted us to investigate, by use of a coexpression system, the possibility that the two proteins form a heterodimeric complex. A two-step affinity chromatography that specifically trapped only the complex revealed that the C-terminal 200 amino acids of HSP90 alpha and the C-terminal 326 amino acids of GRP94 associated with HSP90 alpha and GRP94, respectively. However, the C-terminal 326 amino acids of GRP94 failed to form a complex with HSP90 alpha. In conclusion, these results indicate the similarity of the general dimeric conformation of the two HSP90 family member proteins, but show that the similarity is not sufficient to allow heterodimer formation.     

Zipper protein, a B-G protein with the ability to regulate actin/myosin 1 interactions in the intestinal brush border

We recently identified a 28-kDa protein in the intestinal brush border that resembled tropomyosin in terms of size, homology, and alpha helical content. This protein contained 27 heptad repeats, nearly all of which began with leucine, leading to its name zipper protein. Subsequent analysis, however, indicated that both a 49-kDa and a 28-kDa immunoreactive protein existed in intestinal brush-border extracts. Using 5'-rapid amplification of cDNA ends analysis, we extended the N-terminal sequence of zipper protein to the apparent translation start site. This additional sequence contained a putative transmembrane domain and two potential tryptic cleavage sites C-terminal to the transmembrane domain which would release a 28-kDa cytoplasmic protein if utilized. The additional sequence was highly homologous to members of the B-G protein family, a family with no known function. Immunoelectron microscopy showed that zipper protein was confined to the membrane of the microvillus where it was in close association with brush-border myosin 1 (BBM1). Recombinant zipper protein (28-kDa cytoplasmic portion) blocked the binding of actin to BBM1 and inhibited actin-stimulated BBM1 ATPase activity. In contrast, zipper protein had no effect on endogenous or K/EDTA-stimulated BBM1 ATPase activity. Furthermore, zipper protein displaced tropomyosin from binding to actin, suggesting that these homologous proteins bind to the same sites on the actin molecule. We conclude that zipper protein is a transmembrane protein of the B-G family localized to the intestinal epithelial cell microvillus. The extended cytoplasmic tail either in the intact molecule or after tryptic cleavage may participate in regulating the binding and, thus, activation of BBM1 by actin in a manner similar to tropomyosin.     

Binding of G protein beta gamma-subunits to pleckstrin homology domains

Ligand-induced activation of many receptors leads to dissociation of the alpha- and beta gamma-subunit complexes of heterotrimeric G proteins, both of which regulate a variety of effector molecules involved in cellular signaling processes. In one case, a cytosolic enzyme, the beta-adrenergic receptor kinase (beta ARK) binds to the dissociated, prenylated, membrane-anchored beta gamma-subunits of heterotrimeric G proteins (G beta gamma) and is thereby targeted to its membrane-bound receptor substrate. Quite recently, numerous proteins involved in cellular signal transduction have been shown to contain sequences homologous with a "domain" originally identified in the protein "pleckstrin" (pleckstrin homology domain; PH domain) and subsequently found in the G beta gamma interaction region of the beta ARK sequence. Here we demonstrate that glutathione S-transferase-fusion proteins, containing sequences encompassing the PH domain of nine proteins from this group, bind G beta gamma to varying extents. Binding of G beta gamma to these fusion proteins was documented either by a direct binding assay or by ability to block G beta gamma-mediated membrane translocation of beta ARK1. G beta gamma binding to these fusion proteins was inhibited by the alpha subunit of Go (Go alpha), indicating that the binding of G beta gamma to G alpha and the PH domain-containing fusion proteins is mutually exclusive. Studies with a series of truncated PH domains derived from the Ras-guanine-nucleotide-releasing factor indicate that the G beta gamma binding domain includes only the C-terminal portion of the PH domain and sequences just distal to this. Protein-protein interactions between G beta gamma and PH domain-containing proteins may play a significant role in cellular signaling analogous to that previously demonstrated for Src homology 2 and 3 domains.     

Bovine and human alpha-crystallins as molecular chaperones: prevention of the inactivation of glutathione reductase by fructation

With no measurable protein synthesis occurring in the centre of the lens, structural proteins and enzymes there will need to be stable for many years, if not decades, in order to maintain lens integrity and function. Recent work has indicated that alpha-crystallin, which is sequentially related to heat shock proteins, has chaperone-like properties in that it is capable of preventing heat-induced aggregation of various proteins, including other crystallins. Thus this universal vertebrate lens protein may contribute to maintenance of lens integrity by protecting other lens proteins from non-enzymic insults or the consequences thereof. We previously showed that the enzyme glutathione reductase was inactivated in a time-dependent manner when incubated with various sugars, suggesting glycation was responsible for this effect. In this paper we confirmed that this was the case. Using this enzyme model system, the inclusion of either bovine or human alpha-crystallin protected against the inactivation of glutathione reductase by fructation. This action was specific, with control proteins displaying no such protection. Use of high performance liquid chromatography supported the fact that alpha-crystallin did not act simply by mopping up free sugar but rather maintained the activity of the modified enzyme. Dose-dependent experiments indicated that human alpha-crystallin was more effective than its bovine counterpart, which might be expected considering the much longer lifespan of humans. The stoichiometry of the protection by both alpha-crystallins indicated that alpha-crystallin with glutathione reductase was not acting like GroEL as a large complex with a hydrophobic pore, but rather that individual subunits may be capable of acting as chaperones.     

Presence of a non-NMDA glutamate receptor subtype in the sympathetic nervous system of neonatal swine

Calcium-dependent protein kinases (CDPKs) in plants are characterized by a four-domain structure including conserved sequences in the catalytic domain, and in the C-terminal calmodulin-like domain. Based on this conservation we have PCR-amplified and isolated a potato cDNA clone (StCPK1) from a library representing an early stage of tuber development. DNA sequence analysis revealed that in the catalytic domain, StCPK1 shares more homology with CDPK-related kinases than with CDPKs; however, like CDPKs, it possesses canonical EF-hands at the calmodulin-like 3' end. StCPK1 exists in a few copies in the potato genome and is abundantly expressed in the sepals of mature flowers. Floral expression of genes homologous to StCPK1 appears to be widespread in the family Solanaceae.     

Multidomain structure and cellulosomal localization of the Clostridium thermocellum cellobiohydrolase CbhA

The nucleotide sequence of the Clostridium thermocellum F7 cbhA gene, coding for the cellobiohydrolase CbhA, has been determined. An open reading frame encoding a protein of 1,230 amino acids was identified. Removal of a putative signal peptide yields a mature protein of 1,203 amino acids with a molecular weight of 135,139. Sequence analysis of CbhA reveals a multidomain structure of unusual complexity consisting of an N-terminal cellulose binding domain (CBD) homologous to CBD family IV, an immunoglobulin-like beta-barrel domain, a catalytic domain homologous to cellulase family E1, a duplicated domain similar to fibronectin type III (Fn3) modules, a CBD homologous to family III, a highly acidic linker region, and a C-terminal dockerin domain. The cellulosomal localization of CbhA was confirmed by Western blot analysis employing polyclonal antibodies raised against a truncated enzymatically active version of CbhA. CbhA was identified as cellulosomal subunit S3 by partial amino acid sequence analysis. Comparison of the multidomain structures indicates striking similarities between CbhA and a group of cellulases from actinomycetes. Average linkage cluster analysis suggests a coevolution of the N-terminal CBD and the catalytic domain and its spread by horizontal gene transfer among gram-positive cellulolytic bacteria.     

Platelet adhesion to collagen activates a phosphoprotein complex of heat-shock proteins and protein phosphatase 1

Rapid activation of blood platelets is required for effective haemostasis, with shape change, aggregation, secretion of granule contents and cell adhesion occurring in seconds or even milliseconds. Signal-transduction events, evidenced by changes in protein phosphorylation and calcium levels, also take place in this time domain. We have now shown that platelet adhesion to collagen via the alpha 2 beta 1 integrin under arterial shear forces initiated the rapid dephosphorylation of a 67 kDa protein "band" which contained the 70 kDa constitutive heat-shock protein, hsc70. Immunoprecipitation with hsc70 antibodies revealed a large phosphoprotein complex in resting platelets and adhesion caused dissociation of the complex along with dephosphorylation of hsc70. The complex also contained the hsp90 heat-shock protein, protein phosphatase IC, alpha, delta and M subunits, and some 7-8 unidentified phosphoproteins. The data suggest that heat-shock proteins and protein phosphatases are actively involved in integrin-mediated platelet adhesion.     

Dynamic O-GlcNAcylation of the small heat shock protein alpha B-crystallin

alphaB-Crystallin, originally described as a structural lens protein, is now known to be a member of the small heat shock protein family and is expressed in a number of nonlens tissues. This highly conserved 20 kDa protein aggregates with homologous proteins, including alphaA-crystallin and the small heat shock protein HSP28, to form large heteromeric complexes. Recently, Roquemore et al. (1992) have established that both phosphorylated and unphosphorylated forms of lens alphaB-crystallin are modified with O-linked N-acetylglucosamine, a dynamic posttranslational modification abundant on nuclear and cytoplasmic proteins. In this paper, we have identified the major site of O-GlcNAcylation on lens alphaB as Thr 170. We have further shown that this modification is not restricted to lens alphaB-crystallin but occurs on alphaB isolated from rat heart tissue and human astroglioma cells. Two-dimensional electrophoresis of rat heart alphaB-crystallin revealed two O-GlcNAcylated forms with mobilities corresponding to the unphosphorylated form (alphaB2) and an unidentified, slightly more acidic form. Phosphorylated alphaB-crystallin (alphaB1) was not detected in the rat heart preparation. The major O-GlcNAcylation site on alphaB-crystallins from rat heart also appears to be at Thr 170. Metabolic pulse-chase labeling studies of U373-MG astroglioma cells indicated that turnover of the carbohydrate on alphaB-crystallin is not static but proceeds many-fold more rapidly than turnover of the protein backbone itself, consistent with a regulatory role for O-GlcNAc on this small heat shock protein.     

The J domain of simian virus 40 large T antigen is required to functionally inactivate RB family proteins

Transformation by simian virus 40 large T antigen (TAg) is dependent on the inactivation of cellular tumor suppressors. Transformation minimally requires the following three domains: (i) a C-terminal domain that mediates binding to p53; (ii) the LXCXE domain (residues 103 to 107), necessary for binding to the retinoblastoma tumor suppressor protein, pRB, and the related p107 and p130; and (iii) an N-terminal domain that is homologous to the J domain of DnaJ molecular chaperone proteins. We have previously demonstrated that the N-terminal J domain of TAg affects the RB-related proteins by perturbing the phosphorylation status of p107 and p130 and promoting the degradation of p130 and that this domain is required for transformation of cells that express either p107 or p130. In this work, we demonstrate that the J domain of TAg is required to inactivate the ability of each member of the pRB family to induce a G1 arrest in Saos-2 cells. Furthermore, the J domain is required to override the repression of E2F activity mediated by p130 and pRB and to disrupt p130-E2F DNA binding complexes. These results imply that while the LXCXE domain serves as a binding site for the RB-related proteins, the J domain plays an important role in inactivating their function.     

Identification of residues in the first domain of human Fc alpha receptor essential for interaction with IgA

The FcR family contains multiple receptors for Igs, of which the most distantly related ( approximately 20%) is the IgA receptor (human Fc alpha R), being more homologous ( approximately 35%) to another family of killer-inhibitory receptor-related immunoreceptors with a 19q13.4 chromosomal location in humans. This study of the Fc alpha R demonstrated that, like several IgG receptors, Fc alpha R is a low affinity receptor for Ab (Ka approximately 106 M-1). Rapid dissociation of the rsFc alpha R:IgA complex (t1/2 approximately 25 s) suggests that monomer IgA would bind transiently to cellular Fc alpha Rs, while IgA immune complexes could bind avidly. Mutagenesis of histidyl 85 and arginyl 82, in the FG loop of domain 1, demonstrated that these residues were essential for the IgA-binding activity of Fc alpha R, while arginyl 87 makes a minor contribution to the binding activity of the receptor. This site is unusual among the Fc receptors (Fc gamma RII, Fc gamma RIII, and Fc epsilon RI), in which the ligand binding site is in domain 2 rather than domain 1, but like Fc alpha R, the FG loop comprises part of the ligand binding site. The putative F and G strands flanking the Fc alpha R ligand binding site are highly homologous in the other killer-inhibitory receptor-related immunoreceptors, suggesting they comprise a conserved structural element on which divergent FG loops are presented and participate in the specific ligand interactions of each of these receptors.     

Identification and cellular localization of the actin-binding protein ABP-120 from Entamoeba histolytica

Several actin-binding proteins participate in the morphological changes that occur during amoeboid movement. The gene encoding one of these proteins, the gelation factor ABP-120, was identified and characterized from trophozoites of Entamoeba histolytica. The sequence contains 2574 nucleotides, with an open reading frame of 858 amino acids, giving a protein of 93 kDa belonging to the spectrin family. The N-terminal domain of ABP-120 from E. histolytica revealed a consensus site for actin binding homologous to the actin-binding sites of ABP-120 of Dictyostelium discoideum, alpha-actinin and spectrin. Analysis of the central domain revealed the presence of four repeats of a 73-amino-acid motif constituting 31% of the protein. In addition, a stretch of 105 amino acids was highly divergent when compared with the C-terminal domain of D. discoideum ABP-120. This sequence showed short motifs that are homologous to microtubule-binding domains. We found that ABP-120 from E. histolytica binds to F-actin. In addition, upon motility of the parasite, this protein localized in the pseudopod and the uroid region, implying a role for ABP-120 in movement and capping of surface receptors in E. histolytica.     

Higher glycation of beta L- and beta S-crystallins in the anterior lens cortex and maximum glycation of gamma-crystallins in the bovine lens nucleus, demonstrated by frozen sectioning, isoelectric focusing and lectin staining

The aim of the current study was to demonstrate glycation of beta L-, beta S- and gamma-crystallins in the young bovine lens. To establish which of the crystallins are glycated and where they are located in the lens, we carried out microsectioning of the lens, followed by isoelectric focusing (IEF). Four bovine lenses of 1.183 +/- 0.070 years were frozen-sectioned into equator and 11 layers. Water-soluble crystallins were separated by IEF and stained: (1) with Coomassie brilliant blue for proteins; (2) with the lectin concanavalin A, followed by horseradish peroxidase and diaminobenzidine, for glycated proteins. Experiments were performed with crystallins and proteins in native form, in the absence of denaturants. The crystallins were separated by IEF into alpha-crystallins of high molecular weight (HM), alpha L-, beta H-, beta L-, beta S- and gamma-crystallins. In the lectin staining experiments, only HM, beta L-, beta S- and gamma-crystallins were positive, whereas the alpha L- and beta H-crystallins were negative. Contrary to the glycated gamma-crystallins in the lens nucleus, the beta S- and beta L-crystallins were predominantly glycated in the anterior cortex and to a somewhat lower extent also in the posterior cortical regions. The degree of glycation (total densitometric readings of lectin-stained bands/Coomassie-blue-stained bands) is as follows: total gamma-crystallins 2.44, beta S-crystallins 0.77 and beta L-crystallins 0.28. Though glycation in the bovine lens is very low, lectin staining is sufficiently sensitive to detect the various glycated crystallins. The degree of glycation of gamma-crystallins was 3 times higher than that of beta S-crystallins and 9 times higher than that of beta L-crystallins.     

Reduced chaperone-like activity of alpha A(ins)-crystallin, an alternative splicing product containing a large insert peptide

alpha-Crystallin is a multimeric protein complex which is constitutively expressed at high levels in the vertebrate eye lens, where it serves a structural role, and at low levels in several non-lenticular tissues. Like other members of the small heat shock protein family, alpha-crystallin has a chaperone-like activity in suppressing nonspecific aggregation of denaturing proteins in vitro. Apart from the major alpha A- and alpha B-subunits, alpha-crystallin of rodents contains an additional minor subunit resulting from alternative splicing, alpha A(ins)-crystallin. This polypeptide is identical to normal alpha A-crystallin except for an insert peptide of 23 residues. To explore the structural and functional consequences of this insertion, we have expressed rat alpha A- and alpha A(ins)-crystallin in Escherichia coli. The multimeric particles formed by alpha A(ins) are larger and more disperse than those of alpha A, but they are native-like and display a similar thermostability and morphology, as revealed by gel permeation chromatography, tryptophan fluorescence measurements, and electron microscopy. However, as compared with alpha A, the alpha A(ins)-particles display a diminished chaperone-like activity in the protection of heat-induced aggregation of beta low-crystallin. Our experiments indicate that alpha A(ins)-multimers have a 3-4-fold reduced substrate binding capacity, which might be correlated to their increased particle size and to a shielding of binding sites by the insert peptides. The structure-function relationship of the natural mutant alpha A(ins)-crystallin may shed light on the mechanism of chaperone-like activity displayed by all small heat shock proteins.