Multilevel structural characteristics for the natural substrate proteins of bacterial small heat shock proteins

Small heat shock proteins (sHSPs) are ubiquitous molecular chaperones that prevent the aggregation of various non‐native proteins and play crucial roles for protein quality control in cells. It is poorly understood what natural substrate proteins, with respect to structural characteristics, are preferentially bound by sHSPs in cells. Here we compared the structural characteristics for the natural substrate proteins of Escherichia coli IbpB and Deinococcus radiodurans Hsp20.2 with the respective bacterial proteome at multiple levels, mainly by using bioinformatics analysis. Data indicate that both IbpB and Hsp20.2 preferentially bind to substrates of high molecular weight or moderate acidity. Surprisingly, their substrates contain abundant charged residues but not abundant hydrophobic residues, thus strongly indicating that ionic interactions other than hydrophobic interactions also play crucial roles for the substrate recognition and binding of sHSPs. Further, secondary structure prediction analysis indicates that the substrates of low percentage of β‐sheets or coils but high percentage of α‐helices are un‐favored by both IbpB and Hsp20.2. In addition, IbpB preferentially interacts with multi‐domain proteins but unfavorably with α + β proteins as revealed by SCOP analysis. Together, our data suggest that bacterial sHSPs, though having broad substrate spectrums, selectively bind to substrates of certain structural features. These structural characteristic elements may substantially participate in the sHSP–substrate interaction and/or increase the aggregation tendency of the substrates, thus making the substrates more preferentially bound by sHSPs.     

Roles and applications of small heat shock proteins in the production of recombinant proteins in Escherichia coli

Proteome profiling of the inclusion body (IB) fraction of recombinant proteins produced in Escherichia coli suggested that two small heat shock proteins, IbpA and IbpB, are the major proteins associated with IBs. In this study, we demonstrate that IbpA and IbpB facilitate the production of recombinant proteins in E. coli and play important roles in protecting recombinant proteins from degradation by cytoplasmic proteases. We examined the cytosolic production, and Tat- or Sec-dependent secretion of the enhanced green fluorescent protein (EGFP) in wild type, ibpAB(-) mutant, and ibpAB-amplified E. coli strains. Analysis of fluorescence histograms and confocal microscopic imaging revealed that over-expression of the ibpA and/or ibpB genes enhanced cytosolic EGFP production whereas knocking out the ibpAB genes enhanced secretory production. This strategy seems to be generally applicable as it was successfully employed for the enhanced cytosolic or secretory production of several other recombinant proteins in E. coli.     

Structural and functional homology between periplasmic bacterial molecular chaperones and small heat shock proteins

Abstract The periplasmic Yersinia pestis molecular chaperone Caf1M belongs to a superfamily of bacterial proteins for one of which (PapD protein of Escherichia coli ) the immunoglobulin-like fold was solved by X-ray analysis. The N-terminal domain of Caf1M was found to share a 20% amino acid sequence identity with an inclusion body-associated protein IbpB of Escherichia coli . One of the regions that was compared, was 32 amino acids long, and displayed more than 40% identity, probability of random coincidence was 1.2 × 10⁻⁴. IbpB is involved in a superfamily of small heat shock proteins which fulfil the function of molecular chaperone. On the basis of the revealed homology, an immunoglobulin-like one-domain model of IbpB three-dimensional structure was designed which could be a prototype conformation of sHsp's. The structure suggested is in good agreement with the known experimental data obtained for different members of sHsp's superfamily.     

Chaperone-like activity of the N-terminal region of a human small heat shock protein and chaperone-functionalized nanoparticles

Small heat shock proteins (sHsps) are molecular chaperones employed to interact with a diverse range of substrates as the first line of defense against cellular protein aggregation. The N-terminal region (NTR) is implicated in defining features of sHsps; notably in their ability to form dynamic and polydisperse oligomers, and chaperone activity. The physiological relevance of oligomerization and chemical-scale mode(s) of chaperone function remain undefined. We present novel chemical tools to investigate chaperone activity and substrate specificity of human HspB1 (B1NTR), through isolation of B1NTR and development of peptide-conjugated gold nanoparticles (AuNPs). We demonstrate that B1NTR exhibits chaperone capacity for some substrates, determined by anti-aggregation assays and size-exclusion chromatography. The importance of protein dynamics and multivalency on chaperone capacity was investigated using B1NTR-conjugated AuNPs, which exhibit concentration-dependent chaperone activity for some substrates. Our results implicate sHsp NTRs in chaperone activity, and demonstrate the therapeutic potential of sHsp-AuNPs in rescuing aberrant protein aggregation.     

Structural and functional defects caused by point mutations in the alpha-crystallin domain of a bacterial alpha-heat shock protein

The diverse family of alpha-crystallin-type small heat shock proteins (alpha-Hsps or sHsps) is characterised by a central, moderately conserved alpha-crystallin domain. Oligomerisation followed by dissociation of subparticles is thought to be a prerequisite for chaperone function. We demonstrate that HspH, a bacterial alpha-Hsp from the soybean-symbiont Bradyrhizobium japonicum, assembles into dynamic complexes freely exchanging subunits with homologous and heterologous complexes. The importance of the alpha-crystallin domain for oligomerisation and chaperone activity was tested by site-directed mutagenesis of 12 different residues. In contrast to mammalian alpha-Hsps, the majority of these mutations elicited severe structural and functional defects in HspH. The individual exchange of five amino acid residues throughout the alpha-crystallin domain was found to compromise oligomerisation to various degrees. Assembly defects resulting in complexes of reduced size correlated with greatly decreased or abolished chaperone activity, reinforcing that complete oligomerisation is required for functionality. Mutation of a highly conserved glycine (G114) at the C-terminal end of the alpha-crystallin domain specifically impaired chaperone activity without interfering with oligomerisation properties, indicating that this residue is critical for substrate interaction. The structural and functional importance of this and other residues is discussed in the context of a modeled three-dimensional structure of HspH.     

Crystal structures of Xanthomonas small heat shock protein provide a structural basis for an active molecular chaperone oligomer

Small heat shock proteins (sHsps) are ubiquitous low-molecular-weight chaperones that prevent protein aggregation under cellular stresses. sHsps contain a structurally conserved α-crystallin domain (ACD) of about 100 amino acid residues flanked by varied N- and C-terminal extensions and usually exist as oligomers. Oligomerization is important for the biological functions of most sHsps. However, the active oligomeric states of sHsps are not defined yet. We present here crystal structures (up to 1.65 Å resolution) of the sHspA from the plant pathogen Xanthomonas (XaHspA). XaHspA forms closed or open trimers of dimers (hexamers) in crystals but exists predominantly as 36mers in solution as estimated by size-exclusion chromatography. The XaHspA monomer structures mainly consist of α-crystallin domain with disordered N- and C-terminal extensions, indicating that the extensions are flexible and not essential for the formation of dimers and 36mers. Under reducing conditions where α-lactalbumin (LA) unfolds and aggregates, XaHspA 36mers formed complexes with one LA per XaHspA dimer. Based on XaHspA dimer-dimer interactions observed in crystals, we propose that XaHspA 36mers have four possible conformations, but only XaHspA 36merB, which is formed by open hexamers in 12mer-6mer-6mer-12mer with protruding dimers accessible for substrate (unfolding protein) binding, can bind to 18 reduced LA molecules. Together, our results unravel the structural basis of an active sHsp oligomer.     

Expression and biochemical characterization of two small heat shock proteins from the thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7

We expressed and characterized two sHsps, StHsp19.7 and StHsp14.0, from a thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain 7. StHsp19.7 forms a filamentous structure consisting of spherical particles and lacks molecular chaperone activity. Fractionation of Sulfolobus extracts by size exclusion chromatography with immunoblotting indicates that StHsp19.7 exists as a filamentous structure in vivo. On the other hand, StHsp14.0 exists as a spherical oligomer like other sHsps. It showed molecular chaperone activity to protect thermophilic 3-isopropylmalate dehydrogenase (IPMDH) from thermal aggregation at 87 degrees C. StHsp14.0 formed variable-sized complexes with denatured IPMDH at 90 degrees C. Using StHsp14.0 labeled with fluorescence or biotin probe and magnetic separation, subunit exchanges between complexes were demonstrated. This is the first report on the filament formation of sHsp and also the high molecular chaperone activity of thermophilic archaeal sHsps.     

Regulation of apoptotic signal transduction pathways by the heat shock proteins

The study about apoptotic signal transductions has become a project to reveal the molecular mechanisms of apoptosis. Heat shock proteins (hsps), which play an important role in cell growth and apoptosis, have attracted great attentions. A lot of researches have showed there is a hsps superfamily including hsp90, hsp70, hsp60 and hsp27, etc., which regulates the biological behaviors of cells, particularly apoptotic signal transduction in Fas pathway, JNK/SAPK pathway and caspases pathway at different levels, partly by the function of molecular chaperone.     

Regulation of apoptotic signal transduction pathways by the heat shock proteins

With progressing recognition of apoptosis in bio-logical and medical sciences, the apoptotic signal transduction has rapidly become a dominant project to reveal the molecular mechanisms of apoptotic process. A lot of researches about apoptotic signal transduction have showed the expression of heat shock proteins was closely correlated with cell growth and differen-tiation, and involved in the regulation of apoptosis in different signal transduction pathways. Here we re-view the effects of hsps…     

果蝇热激蛋白的研究进展

热休克蛋白(heat shock proteins,HSPs)是生物体受到应激刺激时诱导产生的一组保守性蛋白,普遍存在于各种生物体中。近年来,果蝇Drosophila作为生命科学与人类疾病研究的重要模式生物,其热激蛋白的研究取得了许多新的进展。文章对果蝇热激蛋白的类别、热激蛋白基因的表达调控机制、热激蛋白的分子伴侣功能、调节细胞存亡和影响发育及寿命等相关生物学功能进行综述,并对热激蛋白在神经退行性疾病治疗中的应用前景作展望。     

Heterooligomeric complexes of human small heat shock proteins

Oligomeric association of human small heat shock proteins HspB1, HspB5, HspB6 and HspB8 was analyzed by means of size-exclusion chromatography, analytical ultracentrifugation and chemical cross-linking. Wild-type HspB1 and Cys mutants of HspB5, HspB6 and HspB8 containing a single Cys residue in position homologous to that of Cys137 of human HspB1 were able to generate heterodimers cross-linked by disulfide bond. Cross-linked heterodimers between HspB1/HspB5, HspB1/HspB6 and HspB5/HspB6 were easily produced upon mixing, whereas formation of any heterodimers with participation of HspB8 was significantly less efficient. The size of heterooligomers formed by HspB1/HspB6 and HspB5/HspB6 was different from the size of the corresponding homooligomers. Disulfide cross-linked homodimers of small heat shock proteins were unable to participate in heterooligomer formation. Thus, monomers can be involved in subunit exchange leading to heterooligomer formation and restriction of flexibility induced by disulfide cross-linking prevents subunit exchange.     

Detection of oligomerisation and substrate recognition sites of small heat shock proteins by peptide arrays

Small heat shock proteins (sHsps) form large oligomers that are characterised by their dynamic behaviour, e.g., complex disassembly/reassembly and extensive subunit exchange. These processes are interrelated with sHsp/substrate interaction. sHsps bind a broad spectrum of unrelated substrate proteins under denaturing conditions. Detailed knowledge about the binding process and regions critical for sHsp/substrate interaction is missing. In this study, we screened cellulose-bound peptide spot libraries derived from a bacterial sHsp and the model-substrate citrate synthase to detect oligomerisation and substrate interaction sites, respectively. In line with previous results, it was demonstrated that multiple contacts involving the N- and C-terminal extensions and the central alpha-crystallin domain are required for oligomerisation. Incubation of the citrate synthase membrane with sHsps revealed a putative substrate interaction site. A soluble peptide with the sequence RTKYWELIYEDCMDL (CS(191-205)) corresponding to that site inhibited chaperone activity of sHsps, presumably by blocking their substrate-binding sites.     

Specific protein homeostatic functions of small heat‐shock proteins increase lifespan

During aging, oxidized, misfolded, and aggregated proteins accumulate in cells, while the capacity to deal with protein damage declines severely. To cope with the toxicity of damaged proteins, cells rely on protein quality control networks, in particular proteins belonging to the family of heat‐shock proteins (HSPs). As safeguards of the cellular proteome, HSPs assist in protein folding and prevent accumulation of damaged, misfolded proteins. Here, we compared the capacity of all Drosophila melanogaster small HSP family members for their ability to assist in refolding stress‐denatured substrates and/or to prevent aggregation of disease‐associated misfolded proteins. We identified CG14207 as a novel and potent small HSP member that exclusively assisted in HSP70‐dependent refolding of stress‐denatured proteins. Furthermore, we report that HSP67BC, which has no role in protein refolding, was the most effective small HSP preventing toxic protein aggregation in an HSP70‐independent manner. Importantly, overexpression of both CG14207 and HSP67BC in Drosophila leads to a mild increase in lifespan, demonstrating that increased levels of functionally diverse small HSPs can promote longevity in vivo.     

Role of the IXI/V motif in oligomer assembly and function of StHsp14.0, a small heat shock protein from the acidothermophilic archaeon, Sulfolobus tokodaii strain 7

Small heat shock proteins (sHsps) are one of the most ubiquitous molecular chaperones. They are grouped together based on a conserved domain, the alpha-crystallin domain. Generally, sHsps exist as oligomers of 9-40 subunits, and the oligomers undergo reversible temperature-dependent dissociation into smaller species as dimers, which interact with denaturing substrate proteins. Previous studies have shown that the C-terminal region, especially the consensus IXI/V motif, is responsible for oligomer assembly. In this study, we examined deletions or mutations in the C-terminal region on the oligomer assembly and function of StHsp14.0, an sHsp from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7. Mutated StHsp14.0 with C-terminal deletion or replacement of IIe residues in the IXI/V motif to Ala, Ser, or Phe residues could not form large oligomers and lost chaperone activity. StHsp14.0WKW, whose Ile residues in the IXI/V motif are changed to Trp, existed as an oligomer like that of the wild type. However, it dissociates to small oligomers and exhibits chaperone activity at relatively lowered temperature. Replacement of two Ile residues in the motif to relatively small residues, Ala or Ser, also resulted in the change of beta-sheet rich secondary structure and decrease of hydrophobicity. Interestingly, StHsp14.0 mutant with amino acid replacements to Phe kept almost the same secondary structure and relatively high hydrophobicity despite that it could not form an oligomeric structure. The results show that hydrophobicity and size of the amino acids in the IXI/V motif in the C-terminal region are responsible not only for assembly of the oligomer but also for the maintenance of beta-sheet rich secondary structure and hydrophobicity, which are important for the function of sHsp.     

Alpha-synuclein has structural and functional similarities to small heat shock proteins

The aggregation and fibrillization of α-synuclein, a major component of Lewy bodies, is a key event in Parkinson’s disease. Although the mechanisms of fibrils formation are largely investigated, physiological function of α-synuclein is not yet clearly elucidated. Here, we showed that C-terminal region of α-synuclein is similar to α-crystalline domain of small heat shock proteins. In our experiments, α-synuclein, like small heat shock proteins, protected cellular proteins from denaturation, and confer Escherichia coli cellular tolerances against thermal- and oxidative-stresses.     

Chaperone function and mechanism of small heat-shock proteins

Small heat-shock proteins （sHSPs） are ubiquitous ATP-independent molecular chaperones that play crucial roles in protein quality control in cells. They are able to prevent the aggregation and/or inactivation of various non-native sub- strate proteins and assist the refolding of these substrates independently or under the help of other ATP-dependent chaperones. Substrate recognition and binding by sHSPs are essential for their chaperone functions. This review focuses on what natural substrate proteins an sHSP pro- tects and how it binds the substrates in cells under fluctuat- ing conditions. It appears that sHSPs of prokaryotes, although being able to bind a wide range of cellular pro- teins, preferentially protect certain classes of functional proteins, such as translation-related proteins and metabolic enzymes, which may well explain why they could increase the resistance of host cells against various stresses. Mechanistically, the sHSPs of prokaryotes appear to possess numerous multi-type substrate-binding residues and are able to hierarchically activate these residues in a temperature-dependent manner, and thus act as tempera- ture-regulated chaperones. The mechanism of hierarchical activation of substrate-binding residues is also discussed regarding its implication for eukaryotic sHSPs.     

Enhanced molecular chaperone activity of the small heat-shock protein alphaB-cystallin following covalent immobilization onto a solid-phase support

The well-characterized small heat-shock protein, alphaB-crystallin, acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation and precipitation. Structural perturbation (e.g., partial unfolding) enhances the in vitro chaperone activity of alphaB-crystallin. Proteins often undergo structural perturbations at the surface of a synthetic material, which may alter their biological activity. This study investigated the activity of alphaB-crystallin when covalently bound to a support surface; alphaB-crystallin was immobilized onto a range of solid material surfaces, and its characteristics and chaperone activity were assessed. Immobilization was achieved via a plasma-deposited thin polymeric interlayer containing aldehyde surface groups and reductive amination, leading to the covalent binding of alphaB-crystallin lysine residues to the surface aldehyde groups via Schiff-base linkages. Immobilized alphaB-crystallin was characterized by X-ray photoelectron spectroscopy, atomic force microscopy, and quartz crystal microgravimetry, which showed that 300 ng cm(-2) (dry mass) of oligomeric alphaB-crystallin was bound to the surface. Immobilized alphaB-crystallin exhibited a significant enhancement (up to 5000-fold, when compared with the equivalent activity of alphaB-crystallin in solution) of its chaperone activity against various proteins undergoing both amorphous and amyloid fibril forms of aggregation. The enhanced molecular chaperone activity of immobilized alphaB-crystallin has potential applications in preventing protein misfolding, including against amyloid disease processes, such as dialysis-related amyloidosis, and for biodiagnostic detection of misfolded proteins.     

Development and tissue-specific distribution of mouse small heat shock protein hsp25

We have investigated the developmental and tissue-specific distribution of the mouse small hsp25 by immunohistology using an antibody that specifically identifies hsp25. Our analysis shows that the relative amount of hsp25 increases during embryogenesis. Through days 13–20 of embryogenesis, hsp25 accumulation is predominant in the various muscle tissues, including the heart, the bladder, and the back muscles. hsp25 is detectable also in neurons of the spinal cord and the purkinje cells. Furthermore analysis of the closely related α, B-crystallin shows that in several tissues, including the bladder, the notochordal sheath and the eye lens both proteins are coexpressed. Our studies demonstrate that mammalian hsp25 accumulation is developmentally regulated during mouse embryogenesis and support the view of an important functional role of small heat shock proteins in normal cell metabolism. © 1993Wiley-Liss, Inc.