Subunit arrangement in the dodecameric chloroplast small heat shock protein Hsp21

Unfolding proteins are prevented from irreversible aggregation by small heat shock proteins (sHsps) through interactions that depend on a dynamic equilibrium between sHsp subunits and sHsp oligomers. A chloroplast-localized sHsp, Hsp21, provides protection to client proteins to increase plant stress resistance. Structural information is lacking concerning the oligomeric conformation of this sHsp. We here present a structure model of Arabidopsis thaliana Hsp21, obtained by homology modeling, single-particle electron microscopy, and lysine-specific chemical crosslinking. The model shows that the Hsp21 subunits are arranged in two hexameric discs, similar to a cytosolic plant sHsp homolog that has been structurally determined after crystallization. However, the two hexameric discs of Hsp21 are rotated by 25° in relation to each other, suggesting a role for global dynamics in dodecamer function.     

Methionine sulfoxidation of the chloroplast small heat shock protein and conformational changes in the oligomer

The small heat shock proteins (sHsps), which counteract heat and oxidative stress in an unknown way, belong to a protein family of sHsps and alpha-crystallins whose members form large oligomeric complexes. The chloroplast-localized sHsp, Hsp21, contains a conserved methionine-rich sequence, predicted to form an amphipatic helix with the methionines situated along one of its sides. Here, we report how methionine sulfoxidation was detected by mass spectrometry in proteolytically cleaved peptides that were produced from recombinant Arabidopsis thaliana Hsp21, which had been treated with varying concentrations of hydrogen peroxide. Sulfoxidation of the methionine residues in the conserved amphipatic helix coincided with a significant conformational change in the Hsp21 protein oligomer.     

Endoplasmic reticulum stress induces the phosphorylation of small heat shock protein, Hsp27

There are several reports describing participation of small heat shock proteins (sHsps) in cellular protein quality control. In this study, we estimated the endoplasmic reticulum (ER) stress-induced response of Hsp27 and alphaB-crystallin in mammalian cells. Treatment targeting the ER with tunicamycin or thapsigargin induced the phosphorylation of Hsp27 but not of alphaB-crystallin in U373 MG cells, increase being observed after 2-10 h and decline at 24 h. Similar phosphorylation of Hsp27 by ER stress was also observed with U251 MG and HeLa but not in COS cells and could be blocked using SB203580, an inhibitor of p38 MAP kinase. Other protein kinase inhibitors, like G?6983, PD98059, and SP600125, inhibitors of protein kinase C (PKC), p44/42 MAP kinase, and JNK, respectively, were without major influence. Prolonged treatment with tunicamycin but not thapsigargin for 48 h caused the second induction of the phosphorylation of Hsp27 in U251 MG cells. Under these conditions, the intense perinuclear staining of Hsp27, with some features of aggresomes, was observed in 10%-20% of the cells.     

Chaperone‐client interactions between Hsp21 and client proteins monitored in solution by small angle X‐ray scattering and captured by crosslinking mass spectrometry

The small heat shock protein (sHsp) chaperones are important for stress survival, yet the molecular details of how they interact with client proteins are not understood. All sHsps share a folded middle domain to which is appended flexible N‐ and C‐terminal regions varying in length and sequence between different sHsps which, in different ways for different sHsps, mediate recognition of client proteins. In plants there is a chloroplast‐localized sHsp, Hsp21, and a structural model suggests that Hsp21 has a dodecameric arrangement with six N‐terminal arms located on the outside of the dodecamer and six inwardly‐facing. Here, we investigated the interactions between Hsp21 and thermosensitive model substrate client proteins in solution, by small‐angle X‐ray scattering (SAXS) and crosslinking mass spectrometry. The chaperone‐client complexes were monitored and the R_g‐values were found to increase continuously during 20 min at 45°, which could reflect binding of partially unfolded clients to the flexible N‐terminal arms of the Hsp21 dodecamer. No such increase in R_g‐values was observed with a mutational variant of Hsp21, which is mainly dimeric and has reduced chaperone activity. Crosslinking data suggest that the chaperone‐client interactions involve the N‐terminal region in Hsp21 and only certain parts in the client proteins. These parts are peripheral structural elements presumably the first to unfold under destabilizing conditions. We propose that the flexible and hydrophobic N‐terminal arms of Hsp21 can trap and refold early‐unfolding intermediates with or without dodecamer dissociation.     

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.     

ClpB/Hsp100 proteins and heat stress tolerance in plants

High-temperature stress can disrupt cellular proteostasis, resulting in the accumulation of insoluble protein aggregates. For survival under stressful conditions, it is important for cells to maintain a pool of native soluble proteins by preventing and/or dissociating these aggregates. Chaperones such as GroEL/GroES (Hsp60/Hsp10) and DnaK/DnaJ/GrpE (Hsp70/Hsp40/nucleotide exchange factor) help cells minimize protein aggregation. Protein disaggregation is accomplished by chaperones belonging to the Caseinolytic Protease (Clp) family of proteins. ClpB/Hsp100 proteins are strikingly ubiquitous and are found in bacteria, yeast and multi-cellular plants. The expression of these proteins is regulated by heat stress (HS) and developmental cues. Bacteria and yeast contain one and two forms of ClpB proteins, respectively. Plants possess multiple forms of these proteins that are localized to different cellular compartments (i.e. cytoplasm/nucleus, chloroplast or mitochondria). Overwhelming evidence suggests that ClpB/Hsp100 proteins play decisive roles in cell adaptation to HS. Mutant bacteria and yeast cells lacking active ClpB/Hsp100 proteins are critically sensitive to high-temperature stress. Likewise, Arabidopsis, maize and rice mutants lacking cytoplasmic ClpB proteins are very sensitive to heat. In this study, we present the structural and functional attributes of plant ClpB forms.     

Bioinformatic analysis of embryo development related small heat shock protein Hsp26 in Artemia species

Artemia embryos can endure extreme temperature,long-term anoxia,desiccation and other wide variety of stressful conditions.How the embryos survive these stresses is a very interesting and unsolved subj...     

Disulfide bonds convert small heat shock protein Hsp16.3 from a chaperone to a non-chaperone: implications for the evolution of cysteine in molecular chaperones

Molecular chaperones mainly function in assisting newly synthesized polypeptide folding and protect non-native proteins from aggregation, with known structural features such as the ability of spontaneous folding/refolding and high conformational flexibility. In this report, we verified the assumption that the lack of disulfide bonds in molecular chaperones is a prerequisite for such unique structural features. Using small heat shock protein (one sub-class of chaperones) Hsp16.3 as a model system, our results show the following: (1) Cysteine-free Hsp16.3 wild type protein can efficiently exhibit chaperone activity and spontaneously refold/reassemble with high conformational flexibility. (2) Whereas Hsp16.3 G89C mutant with inter-subunit disulfide bonds formed seems to lose the nature of chaperone proteins, i.e., under stress conditions, it neither acts as molecular chaperone nor spontaneously refolds/reassembles. Structural analysis indicated that the mutant exists as an unstable molten globule-like state, which incorrectly exposes hydrophobic surfaces and irreversibly tends to form aggregates that can be suppressed by the other molecular chaperone (alpha-crystallin). By contrast, reduction of disulfide bond in the Hsp16.3 G89C mutant can significantly recover its character as a molecular chaperone. In light of these results, we propose that disulfide bonds could severely disturb the structure/function of molecular chaperones like Hsp16.3. Our results might not only provide insights into understanding the structural basis of chaperone upon binding substrates, but also explain the observation that the occurrence of cysteine in molecular chaperones is much lower than that in other protein families, subsequently being helpful to understand the evolution of protein family.     

Enhanced stability of alpha B-crystallin in the presence of small heat shock protein Hsp27

Lens alpha-crystallin, alpha A- and alpha B-crystallin, and Hsp27 are members of the small heat shock protein family. Both alpha A- and alpha B-crystallin are expressed in the lens and serve as structural proteins and as chaperones, but alpha B-crystallin is also expressed in nonlenticular organs where Hsp27, rather than alpha A-crystallin, is expressed along with alpha B-crystallin. It is not known what additional function Hsp27 has besides as a heat shock protein, but it may serve, as alpha A-crystallin does in the lens, to stabilize alpha B-crystallin. In this study, we investigate aspects on conformation and thermal stability for the mixture of Hsp27 and alpha B-crystallin. Size exclusion chromatography, circular dichroism (CD), and light scattering measurements indicated that Hsp27 prevented alpha B-crystallin from heat-induced structural changes and high molecular weight (HMW) aggregation. The results indicate that Hsp27 indeed promotes stability of alpha B-crystallin.     

Serine/threonine protein phosphatase 5 (PP5) interacts with substrate under heat stress conditions and forms protein complex in Arabidopsis

Protein phosphatase 5 plays a pivotal role in signal transduction in animal and plant cells, and it was previously shown that Arabidopsis protein phosphatase 5 (AtPP5) performs multiple enzymatic activities that are mediated by conformational changes induced by heat shock stress. In addition, transgenic overexpression of AtPP5 gene conferred enhanced heat shock resistance compared with wild-type plant. However, the molecular mechanism underlying this enhanced heat shock tolerance through functional and conformational changes upon heat stress is not clear. In this report, AtPP5 was shown to preferentially interact with its substrate, MDH, under heat stress conditions. In addition, in co-IP analysis, AtPP5 was observed to form a complex with AtHsp90 in Arabidopsis. These results suggest that AtPP5 may enhance thermotolerance via forming multi-chaperone complexes under heat shock conditions in Arabidopsis. Finally, we show that AtPP5 is primarily localized in the cytoplasm of Arabidopsis.     

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.     

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.     

The minimal α‐crystallin domain of Mj Hsp16.5 is functional at non‐heat‐shock conditions

The small heat shock protein (sHSP) from Methanococcus jannaschii (Mj Hsp16.5) forms a monodisperse 24mer and each of its monomer contains two flexible N‐ and C‐terminals and a rigid α‐crystallin domain with an extruding β‐strand exchange loop. The minimal α‐crystallin domain with a β‐sandwich fold is conserved in sHSP family, while the presence of the β‐strand exchange loop is divergent. The function of the β‐strand exchange loop and the minimal α‐crystallin domain of Mj Hsp16.5 need further study. In the present study, we constructed two fragment‐deletion mutants of Mj Hsp16.5, one with both the N‐ and C‐terminals deleted (ΔNΔC) and the other with a further deletion of the β‐strand exchange loop (ΔNΔLΔC). ΔNΔC existed as a dimer in solution. In contrast, the minimal α‐crystallin domain ΔNΔLΔC became polydisperse in solution and exhibited more efficient chaperone‐like activities to prevent amorphous aggregation of insulin B chain and fibril formation of the amyloidogenic peptide dansyl‐SSTSAA‐W than the mutant ΔNΔC and the wild type did. The hydrophobic probe binding experiments indicated that ΔNΔLΔC exposed much more hydrophobic surface than ΔNΔC. Our study also demonstrated that Mj Hsp16.5 used different mechanisms for protecting different substrates. Though Mj Hsp16.5 formed stable complexes with substrates when preventing thermal aggregation, no complexes were detected when preventing aggregation under non‐heat‐shock conditions. Proteins 2014; 82:1156–1167. © 2013 Wiley Periodicals, Inc.     

Spatial pattern of constitutive and heat shock‐induced expression of the small heat shock protein gene family,hsp30, in Xenopus laevis tailbud embryos

We employed whole‐mount in situ hybridization and immunohistochemistry to study the spatial pattern of hsp30 gene expression in normal and heatshocked embryos during Xenopus laevis development. Our findings revealed that hsp30 mRNA accumulation was present constitutively only in the cement gland of early and midtailbud embryos, while hsp30 protein was detected until at least the early tadpole stage. Heat shock‐induced accumulation of hsp30 mRNA and protein was first observed in early and midtailbud embryos with preferential enrichment in the cement gland, somitic region, lens placode, and proctodeum. In contrast, cytoskeletal actin mRNA displayed a more generalized pattern of accumulation which did not change following heat shock. In heat shocked midtailbud embryos the enrichment of hsp30 mRNA in lens placode and somitic region was first detectable after 15 min of a 33°C heatshock. The lowest temperature capable of inducing this pattern was 30°C. Placement of embryos at 22°C following a 1‐h 33°C heat shock resulted in decreased hsp30 mRNA in all regions with time, although enhanced hsp30 mRNA accumulation still persisted in the cement gland after 11 h compared to control. In late tailbud embryos the basic midtailbud pattern of hsp30 mRNA accumulation was enhanced with additional localization to the spinal cord as well as enrichment across the embryo surface. These studies demonstrate that hsp30 gene expression can be detected constitutively in the cement gland of tailbud embryos and that heat shock results in a preferential accumulation of hsp30 mRNA and protein in certain tissues. Dev. Genet. 25:365–374, 1999. © 1999 Wiley‐Liss, Inc.     

Expression of the small heat shock protein (hsp) 27 in human astrocytomas correlates with histologic grades and tumor growth fractions

Summary 1. Cellular expression and distribution of the stress response small heat shock protein 27 (hsp27) in 39 high-grade astrocytomas (27 glioblastoma multiformes, 12 anaplastic astrocytomas) and in 27 low-grade astrocytomas (grade I–II) were analyzed immunohistochemically.2. The correlation between hsp27 expression and tumor growth fractions of the astrocytomas was examined following Ki-67 immunostaining.3. The hsp27 staining was cell cytoplasmic. The hsp27 immunopositive rate was significantly higher in high-grade astrocytomas; the rates were 74% for glioblastomas, 58% for anaplastic astrocytomas, and 37% for low-grade astrocytomas. The small and large tumor cells, especially in glioblastomas, multinucleated tumor giant cells, tumor cells in the pseudopalisading and necrotic areas, cells of the microvascular endothelial proliferations, and tumor vascular smooth muscles were usually hsp27 positive. The mean percentage of hsp27-positive cells was significantly higher in the glioblastomas alone and in the combined high-grade astrocytomas, compared to the low-grade, and in recurrent rather than in primary high-grade astrocytomas.4. The high-grade astrocytomas had a highly statistical significant Ki-67 labeling index. The Ki-67 labeling indices were significantly higher in the hsp27-positive than the hsp27-negative astrocytomas, irrespective of the histological grade. In the high-grade astrocytomas with a Ki-67 labeling index of five and above, 81% of those tumors were hsp27 positive.5. Thus, a large number of human astrocytomas express hsp27, and hsp27 expression correlates with histological grades of astrocytoma and with tumor growth fractions. This being the case, hsp27 is likely to have a role in the growth of human astrocytomas.     

Identification of bis-ANS binding sites in Mycobacterium tuberculosis small heat shock protein Hsp16.3: evidences for a two-step substrate-binding mechanism

Small heat shock proteins (sHSPs), as one important subclass of molecular chaperones, are able to specifically bind to denatured substrate proteins rather than to native proteins, of which their substrate-binding sites are far from clear. Our previous study showed an overlapping nature of the sites for both hydrophobic probe 1,1'-Bi(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) binding and substrate binding in Mycobacterium tuberculosis Hsp16.3 [X. Fu, H. Zhang, X. Zhang, Y. Cao, W. Jiao, C. Liu, Y. Song, A. Abulimiti, Z. Chang, A dual role for the N-terminal region of M. tuberculosis Hsp16.3 in self-oligomerization and binding denaturing substrate proteins, J. Biol. Chem. 280 (2005) 6337-6348]. In this work, two bis-ANS binding sites in Hsp16.3 were identified by a combined use of reverse phase HPLC, mass spectroscopy and N-terminal protein sequencing. One site is in the N-terminal region and the other one in the N-terminus of alpha-crystallin domain, both of which are similar to those identified so far in sHSPs. However, accumulating data suggest that these two sites differentially function in binding substrate proteins. With regard to this difference, we proposed a two-step mechanism by which Hsp16.3 binds substrate proteins, i.e., substrate proteins are recognized and initially captured by the N-terminal region that is exposed in the dissociated Hsp16.3 oligomers, and then the captured substrate proteins are further stabilized in the complex by the subsequent binding of the N-terminus of alpha-crystallin domain.     

Biochemical characterization of small heat shock protein HspB8 (Hsp22)–Bag3 interaction

Interaction of human Bag3 with small heat shock proteins HspB6, HspB8 and its K141E mutant was analyzed by different biochemical methods. The data of size-exclusion chromatography indicate that the wild type HspB8 forms tight complexes with Bag3. K141E mutant of HspB8 and especially HspB6 weaker interact with Bag3. The data of chemical crosslinking and analytical ultracentrifugation indicate that in vitro the stoichiometry of complexes formed by HspB8 and Bag3 is variable and is dependent on concentration of protein partners. Interaction of Bag3 and HspB8 is accompanied by increase of thermal stability measured by intrinsic tryptophan fluorescence and increased resistance to limited chymotrypsinolysis. The data of size-exclusion chromatography, analytical ultracentrifugation and limited proteolysis indicate that Bag3 belongs to the group of intrinsically disordered proteins. It is supposed that having unordered structure Bag3 might weakly interact with different small heat shock proteins which recognize unfolded proteins and this interaction is especially strong with intrinsically disordered HspB8. The complexes formed by Bag3 and HspB8 might have variable stoichiometry and can participate in different processes including clearing of the cell from improperly folded proteins.