Hsp27 is persistently expressed in zebrafish skeletal and cardiac muscle tissues but dispensable for their morphogenesis

Constitutive expression of Hsp27 has been demonstrated in vertebrate embryos, especially in developing skeletal and cardiac muscle. Results of several previous studies have indicated that Hsp27 could play a role in the development of these tissues. For example, inhibition of Hsp27 expression has been reported to cause defective development of mammalian myoblasts in vitro and frog embryos in vivo. In contrast, transgenic mice lacking Hsp27 develop normally. Here, we examined the distribution of Hsp27 protein in developing and adult zebrafish and effects of suppressing Hsp27 expression using phosphorodiamidate morpholino oligonucleotides (PMO) on zebrafish development. Consistent with our previous analysis of hsp27 messenger RNA expression, we detected the protein Hsp27 in cardiac, smooth, and skeletal muscle of both embryonic and adult zebrafish. However, embryos lacking detectable Hsp27 after injection of antisense hsp27 PMO exhibited comparable heart beat rates to that of control embryos and cardiac morphology was indistinguishable in the presence or absence of Hsp27. Loss of Hsp27 also had no effect on the structure of the skeletal muscle myotomes in the developing embryo. Finally, embryos injected with antisense hsp27 and scrambled control PMO displayed equal motility. We conclude that Hsp27 is dispensable for zebrafish morphogenesis but could play a role in long-term maintenance of heart and muscle tissues. Tucker and Ustyugov contributed equally to this work.     

Heat shock protein 27 regulates neutrophil chemotaxis and exocytosis through two independent mechanisms

The targets of the p38 MAPK pathway responsible for regulation of neutrophil chemotaxis and exocytosis are unknown. One target of this pathway is the actin-binding protein, heat shock protein 27 (Hsp27). Therefore, we tested the hypothesis that Hsp27 mediates p38 MAPK-dependent chemotaxis and exocytosis in human neutrophils through regulation of actin reorganization. Sequestration of Hsp27 by introduction of anti-Hsp27 Ab, but not an isotype Ab, inhibited fMLP-stimulated chemotaxis, increased cortical F-actin in the absence of fMLP stimulation, and inhibited fMLP-stimulated exocytosis. Pretreatment with latrunculin A prevented actin reorganization and the changes in fMLP-stimulated exocytosis induced by Hsp27 sequestration. To determine the role of Hsp27 phosphorylation, wild-type, phosphorylation-resistant, or phosphorylation-mimicking recombinant Hsp27 was introduced into neutrophils by electroporation. The phosphorylation-resistant mutant significantly reduced migration toward fMLP, whereas none of the Hsp27 proteins affected fMLP-stimulated or TNF-alpha-stimulated exocytosis or actin polymerization. Endogenous Hsp27 colocalized with F-actin in unstimulated and fMLP-stimulated neutrophils, whereas phosphorylated Hsp27 showed cytosolic localization in addition to colocalization with F-actin. Our results suggest that Hsp27 regulates neutrophil chemotaxis and exocytosis in an actin-dependent, phosphorylation-independent manner. Phosphorylation of Hsp27 regulates chemotaxis, but not exocytosis, independent of regulation of actin reorganization.     

Heat shock protein 27 is a substrate of cGMP-dependent protein kinase in intact human platelets: phosphorylation-induced actin polymerization caused by HSP27 mutants

Phosphorylation of heat shock protein 27 (Hsp27) in human platelets by mitogen-activated protein kinase-activated protein kinase (MAPKAP) 2 is associated with signaling events involved in platelet aggregation and regulation of microfilament organization. We now show that Hsp27 is also phosphorylated by cGMP-dependent protein kinase (cGK), a signaling system important for the inhibition of platelet aggregation. Stimulation of washed platelets with 8-para-chlorophenylthio-cGMP, a cGK specific activator, resulted in a time-dependent phosphorylation of Hsp27. This is supported by the ability of cGK to phosphorylate Hsp27 in vitro to an extent comparable with the cGK-mediated phosphorylation of its established substrate vasodilator-stimulated phosphoprotein. Studies with Hsp27 mutants identified threonine 143 as a yet uncharacterized phosphorylation site in Hsp27 specifically targeted by cGK. To test the hypothesis that cGK could inhibit platelet aggregation by phosphorylating Hsp27 and interfering with the MAPKAP kinase phosphorylation of Hsp27, the known MAPKAP kinase 2-phosphorylation sites (Ser15, Ser78, and Ser82) as well as Thr143 were replaced by negatively charged amino acids, which are considered to mimic phosphate groups, and tested in actin polymerization experiments. Mimicry at the MAPKAP kinase 2 phosphorylation sites led to mutants with a stimulating effect on actin polymerization. Mutation of the cGK-specific site Thr143 alone had no effect on actin polymerization, but in the MAPKAP kinase 2 phosphorylation-mimicking mutant, this mutation reduced the stimulation of actin polymerization significantly. These data suggest that phosphorylation of Hsp27 and Hsp27-dependent regulation of actin microfilaments contribute to the inhibitory effects of cGK on platelet function.     

Concomitant inhibition of HSP90, its mitochondrial localized homologue TRAP1 and HSP27 by green tea in pancreatic cancer HPAF-II cells

Pancreatic cancer is a deadly disease characterized by poor prognosis and patient survival. Green tea polyphenols have been shown to exhibit multiple antitumor activities in various cancers, but studies on the pancreatic cancer are very limited. To identify the cellular targets of green tea action, we exposed a green tea extract (GTE) to human pancreatic ductal adenocarcinoma HPAF-II cells and performed two-dimensional gel electrophoresis of the cell lysates. We identified 32 proteins with significantly altered expression levels. These proteins are involved in drug resistance, gene regulation, motility, detoxification and metabolism of cancer cells. In particular, we found GTE inhibited molecular chaperones heat-shock protein 90 (Hsp90), its mitochondrial localized homologue Hsp75 (tumor necrosis factor receptor-associated protein 1, or Trap1) and heat-shock protein 27 (Hsp27) concomitantly. Western blot analysis confirmed the inhibition of Hsp90, Hsp75 and Hsp27 by GTE, but increased phosphorylation of Ser78 of Hsp27. Furthermore, we showed that GTE inhibited Akt activation and the levels of mutant p53 protein, and induced apoptosis and growth suppression of the cells. Our study has identified multiple new molecular targets of GTE and provided further evidence on the anticancer activity of green tea in pancreatic cancer.     

Regulation of contractility by Hsp27 and Hic-5 in rat mesenteric small arteries

The regulation of small artery contractility by vasoconstrictors is important for vascular function, and actin cytoskeleton remodeling is required for contraction. p38 MAPK and tyrosine kinases are implicated in actin polymerization and contraction through heat shock protein 27 (Hsp27) and the cytoskeletal protein paxillin, respectively. We evaluated the roles of downstream targets of p38 MAPK and tyrosine kinases in cytoskeletal reorganization and contraction and whether the two signaling pathways regulate contraction independent of each other. We identified the expression of the paxillin homologue hydrogen peroxide-inducible clone-5 (Hic-5) and showed its activation by norepinephrine (NE) in a Src-dependent manner. Furthermore, we demonstrated a NE-induced interaction of proline-rich tyrosine kinase-2 (PYK2) but not Src or p125 focal adhesion kinase with Hic-5. This interaction was Src dependent, suggesting that Hic-5 was a substrate for PYK2 downstream from Src. The activation of Hic-5 induced its relocalization to the cytosol. The parallel activation of Hsp27 by NE was p38 MAPK dependent and led to its dissociation from actin filaments and translocation from membrane to cytosol and increased actin polymerization. Both Hsp27 and Hic-5 activation resulted in their association within the same time frame as NE-induced contraction, and the inhibition of either p38 MAPK or Src inhibited the interaction between Hsp27 and Hic-5 and the contractile response. Furthermore, combined p38 MAPK and Src inhibition had no greater effect on contraction than individual inhibition, suggesting that the two pathways act through a common mechanism. These data show that NE-induced activation and the association of Hsp27 and Hic-5 are required for the reorganization of the actin cytoskeleton and force development in small arteries.     

Thiolutin inhibits endothelial cell adhesion by perturbing Hsp27 interactions with components of the actin and intermediate filament cytoskeleton

Thiolutin is a dithiole synthesized by Streptomyces sp. that inhibits endothelial cell adhesion and tumor growth. We show here that thiolutin potently inhibits developmental angiogenesis in zebrafish and vascular outgrowth from tissue explants in 3D cultures. Thiolutin is a potent and selective inhibitor of endothelial cell adhesion accompanied by rapid induction of HSPB1 (Hsp27) phosphorylation. The inhibitory effects of thiolutin on endothelial cell adhesion are transient, potentially due to a compensatory increase in Hsp27 protein levels. Accordingly, heat shock induction of Hsp27 limits the anti-adhesive activity of thiolutin. Thiolutin treatment results in loss of actin stress fibers, increased cortical actin as cells retract, and decreased cellular F-actin. Mass spectrometric analysis of Hsp27 binding partners following immunoaffinity purification identified several regulatory components of the actin cytoskeleton that associate with Hsp27 in a thiolutin-sensitive manner including several components of the Arp2/3 complex. Among these, ArpC1a is a direct binding partner of Hsp27. Thiolutin treatment induces peripheral localization of phosphorylated Hsp27 and Arp2/3. Hsp27 also associates with the intermediate filament components vimentin and nestin. Thiolutin treatment specifically ablates Hsp27 interaction with nestin and collapses nestin filaments. These results provide new mechanistic insights into regulation of cell adhesion and cytoskeletal dynamics by Hsp27.     

Induction of heat shock protein 27 in rat embryos exposed to hyperthermia

Previously we reported that eight proteins were reproducibly induced in postimplantation rat embryos exposed to a brief heat shock (43°C, 15 min). The major heat-inducible rat embryo protein has now been identified as heat shock protein 72 (Hsp 72). In addition, the induction of Hsp 72 is temporally correlated with induction of thermotolerance. One of the other rat embryo proteins previously shown to be induced by elevated temperature is a heat shock protein of approximately 27 kilodaltons (Hsp 27). In this report we show that this protein is recognized by an antibody directed against a conserved peptide sequence of Hsp 27. Unlike Hsp 72, Hsp 27 is constitutively expressed in the rat embryo in the absence of any thermal stress; however, the level of Hsp 27 is increased approximately 2–3-fold after thermal stress (43°C, 10 min). Immunohistochemical analysis revealed that the constitutively expressed Hsp 27 is localized primarily to cells of the heart, cells that are uniquely resistant to the cytotoxic effects of hyperthermia. After thermal stress, Hsp 27 is expressed in all tissues of the embryo. Finally, our data show that Hsp 27 exists in the rat embryo as three major isoforms indicative of different phosphorylation states. Furthermore, most Hsp 27 in the heart is phosphorylated, whereas in the rest of the embryo, nonphosphorylated Hsp 27 predominates. After thermal stress, levels of phosphorylated isoforms increase dramatically in nonheart tissues of the embryo. Together, these results suggest that Hsp 27 may play a role in the development of thermotolerance in the postimplantation mammalian embryo. © 1996 Wiley-Liss, Inc.     

The Hsp27 gene is not required for Drosophila development but its activity is associated with starvation resistance

Heat shock proteins are induced under stress conditions and they act as molecular chaperones to refold denatured polypeptides. Stress resistances including thermotolerance generally are correlated with levels of the heat shock proteins. We investigated a fruit fly gene encoding a small heat shock protein, Hsp27, to determine if it functions in stress response of Drosophila melanogaster. A knockout Hsp27 allele was generated. Flies homozygous for this allele were viable, without obvious defects, and fertile, indicating Hsp27 is not essential for development. In stress-response tests, loss of the Hsp27 gene caused no defects in resistance to heat shock or oxidative treatments. However, a significant reduction in starvation resistance was associated with the genotype without a functional Hsp27 gene. The data suggest that the Drosophila HSP27 protein acts as a chaperone to provide cellular stress resistance, although its function may be limited to a subset of the stress response such as the starvation resistance.     

The Yeast AAA+ Chaperone Hsp104 Is Part of a Network That Links the Actin Cytoskeleton with the Inheritance of Damaged Proteins

The yeast AAA⁺ chaperone Hsp104 is essential for the development of thermotolerance and for the inheritance of prions. Recently, Hsp104, together with the actin cytoskeleton, has been implicated in the asymmetric distribution of carbonylated proteins. Here, we investigated the interplay between Hsp104 and actin by using a dominant-negative variant of Hsp104 (HAP/ClpP) that degrades substrate proteins instead of remodeling them. Coexpression of HAP/ClpP causes defects in morphology and the actin cytoskeleton. Taking a candidate approach, we identified Spa2, a member of the polarisome complex, as an Hsp104 substrate. Furthermore, we provided genetic evidence that links Spa2 and Hsp104 to Hof1, a member of the cytokinesis machinery. Spa2 and Hof1 knockout cells are affected in the asymmetric distribution of damaged proteins, suggesting that Hsp104, Spa2, and Hof1 are members of a network controlling the inheritance of carbonylated proteins.The ensemble of molecular chaperones and proteases constitutes the cellular system that repairs and eliminates misfolded proteins. The activity of this system ensures not only the recovery of cells from protein-damaging stress conditions, but also the maintenance of protein homeostasis under normal growth conditions. The concomitant involvement of members of the Hsp70 and Hsp90 chaperone families in stress-related, regulatory, and housekeeping functions allows the integration of environmental stimuli into regulatory networks (4, 24, 39, 40). However, it has remained unclear whether other chaperones are also involved in regulatory processes.One chaperone which so far has been connected only to stress-related protein quality functions is the oligomeric AAA⁺ chaperone Hsp104 of Saccharomyces cerevisiae. Hsp104 is essential for the development of thermotolerance by reactivating aggregated proteins after severe stress conditions and for prion propagation by severing prion fibrils (31). Yeast cells, when grown at 30°C, harbor approximately 5,000 copies of Hsp104 hexamers per cell, a number that is minor compared to other cytosolic chaperone machineries (e.g., Hsp70 and Hsp90) that are involved in general protein-folding events (10). The known cellular functions of Hsp104, however, cannot provide a rationale for the determined Hsp104 levels, since protein aggregation is hardly detectable in yeast cells at 30°C even in mutant cells lacking Hsp104 function. Furthermore, yeast prions occur de novo at a very low rate of 10⁻⁶ per cell. In consequence, both well-characterized Hsp104 activities are barely required at 30°C, suggesting that Hsp104 has additional, so far unknown housekeeping functions. On the other hand, an S. cerevisiae hsp104 knockout exhibits no obvious phenotype at 30°C (27), giving no clues to a potential involvement of Hsp104 in other cellular processes.Recently, Hsp104 was demonstrated to influence the asymmetric distribution of oxidatively damaged (carbonylated) proteins (8). It remained unclear whether the role of Hsp104 in this process relies on its known activities in protein quality control or on an unknown involvement in other cellular processes. Here, we provide evidence that Hsp104 is part of a network that controls the inheritance of damaged proteins under physiological growth conditions.     

The role of Hsp27 and actin in the regulation of movement in human cancer cells responding to heat shock

Human heat shock 27-kDa protein 1 (HSPB1)/heat shock protein (Hsp) 27 is a small heat shock protein which is thought to have several roles within the cell. One of these roles includes regulating actin filament dynamics in cell movement, since Hsp27 has previously been found to inhibit actin polymerization in vitro. In this study, the role of Hsp27 in regulating actin filament dynamics is further investigated. Hsp27 protein levels were reduced using siRNA in SW480 cells, a human colon cancer cell line. An in vitro wound closure assay showed that cells with knocked down Hsp27 levels were unable to close wounds, indicating that this protein is involved in regulating cell motility. Immunoprecipitation pull down assays were done, to observe if and when Hsp27 and actin are in the same complex within the cell, before and after heat shock. At all time points tested, Hsp27 and actin were present in the same cell lysate fraction. Lastly, indirect immunostaining was done before and after heat shock to evaluate Hsp27 and actin interaction in cells. Hsp27 and actin showed colocalization before heat shock, little association 3 h after heat shock, and increased association 24 h after heat shock. Cytoprotection was observed as early as 3 h after heat shock, yet cells were still able to move. These results show that Hsp27 and actin are in the same complex in cells and that Hsp27 is important for cell motility. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular cloning of bovine cardiac muscle heat-shock protein 70 kDa and its phosphorylation by cAMP-dependent protein kinase in vitro

The 70-kDa heat-shock protein (Hsp70) has been cloned and sequenced from bovine cardiac muscle. On the basis of sequence features, the gene corresponds to the cytoplasmic form of Hsp70. This cardiac Hsp70 cDNA clone has an open reading frame of 1926 bp coding for 641 amino acids and a predicted molecular mass of 70.25 kDa. Comparison of the amino acid sequence revealed an extensive sequence identity with other species of Hsp70. Escherichia coli expressed cardiac Hsp70 stimulated a 2-fold increase in calcineurin (CaN) activity. Notably, we observed that Hsp70 directly interacts with CaN using a pull-down assay. Furthermore, expressed cardiac-specific Hsp70 was phosphorylated in vitro by cAMP-dependent protein kinase. Phosphorylation resulted in the incorporation of 0.1 mol of phosphate per mol of Hsp70. The phosphorylated Hsp70 was unable to activate the phosphatase activity of CaN. This is the first demonstration that Hsp70 is phosphorylated by cAMP-dependent protein kinase and provides an on/off switch for the regulation of CaN signaling by Hsp70.     

Hsp27 silencing coordinately inhibits proliferation and promotes Fas-induced apoptosis by regulating the PEA-15 molecular switch

Heat shock protein 27 (Hsp27) is emerging as a promising therapeutic target for treatment of various cancers. Although the role of Hsp27 in protection from stress-induced intrinsic cell death has been relatively well studied, its role in Fas (death domain containing member of the tumor necrosis factor receptor superfamily)-induced apoptosis and cell proliferation remains underappreciated. Here, we show that Hsp27 silencing induces dual coordinated effects, resulting in inhibition of cell proliferation and sensitization of cells to Fas-induced apoptosis through regulation of PEA-15 (15-kDa phospho-enriched protein in astrocytes). We demonstrate that Hsp27 silencing suppresses proliferation by causing PEA-15 to bind and sequester extracellular signal-regulated kinase (ERK), resulting in reduced translocation of ERK to the nucleus. Concurrently, Hsp27 silencing promotes Fas-induced apoptosis by inducing PEA-15 to release Fas-associating protein with a novel death domain (FADD), thus allowing FADD to participate in death receptor signaling. Conversely, Hsp27 overexpression promotes cell proliferation and suppresses Fas-induced apoptosis. Furthermore, we show that Hsp27 regulation of PEA-15 activity occurs in an Akt-dependent manner. Significantly, Hsp27 silencing in a panel of phosphatase and tensin homolog on chromosome 10 (PTEN) wild-type or null cell lines, and in LNCaP cells that inducibly express PTEN, resulted in selective growth inhibition of PTEN-deficient cancer cells. These data identify a dual coordinated role of Hsp27 in cell proliferation and Fas-induced apoptosis via Akt and PEA-15, and indicate that improved clinical responses to Hsp27-targeted therapy may be achieved by stratifying patient populations based on tumor PTEN expression.     

心肌特异性高表达热休克蛋白27转基因鼠建立

目的 建立人热休克蛋白27（heat shock protein 27, Hsp27）基因在小鼠心肌特异性表达的转基因鼠模型。 方法 将人心肌Hsp27cDNA插入含有心肌特异性表达启动子αMHC的pBSⅡ-SK+载体中,限制性内切酶EcoRI酶切、纯化后,获得含有αMHC启动子－Hsp27cDNA－HGH polyA的线性DNA片段,以显微注射法将目的基因导入受精卵, PCR筛选基因型,Western Blot鉴定转移基因的表达和表达的组织特异性。结果和结论 共获得两个转基因系小鼠,均呈心肌组织特异性高表达。     

Downregulation of the Hsp90 system causes defects in muscle cells of Caenorhabditis elegans

The ATP-dependent molecular chaperone Hsp90 is required for the activation of a variety of client proteins involved in various cellular processes. Despite the abundance of known client proteins, functions of Hsp90 in the organismal context are not fully explored. In Caenorhabditis elegans, Hsp90 (DAF-21) has been implicated in the regulation of the stress-resistant dauer state, in chemosensing and in gonad formation. In a C. elegans strain carrying a DAF-21 mutation with a lower ATP turnover, we observed motility defects. Similarly, a reduction of DAF-21 levels in wild type nematodes leads to reduced motility and induction of the muscular stress response. Furthermore, aggregates of the myosin MYO-3 are visible in muscle cells, if DAF-21 is depleted, implying a role of Hsp90 in the maintenance of muscle cell functionality. Similar defects can also be observed upon knockdown of the Hsp90-cochaperone UNC-45. In life nematodes YFP-DAF-21 localizes to the I-band and the M-line of the muscular ultrastructure, but the protein is not stably attached there. The Hsp90-cofactor UNC-45-CFP contrarily can be found in all bands of the nematode muscle ultrastructure and stably associates with the UNC-54 containing A-band. Thus, despite the physical interaction between DAF-21 and UNC-45, apparently the two proteins are not always localized to the same muscular structures. While UNC-45 can stably bind to myofilaments in the muscular ultrastructure, Hsp90 (DAF-21) appears to participate in the maintenance of muscle structures as a transiently associated diffusible factor.     

Cloning, characterization, and heat stress-induced redistribution of a protein homologous to human hsp27 in the zebrafish Danio rerio

Hsp27 is a small heat shock protein (shsp) regulating stress tolerance and increasingly thought to play roles in tissue homeostasis and differentiation. The zebrafish Danio rerio is an important model for the study of developmental processes, but little is known regarding shsps in this animal. Here, we report the sequence, expression, regulation, and function of a zebrafish protein (zfHsp27) homologous to human Hsp27. zfHsp27 contains three conserved phosphorylatable serines and a cysteine important for regulation of apoptosis, but it lacks much of a C-terminal tail domain and shows low homology in two putative actin interacting domains that are features of mammalian Hsp27. zfHsp27 mRNA is most abundant in adult skeletal muscle and heart and is upregulated during early embryogenesis. zfHsp27 expressed in mammalian fibroblasts was phosphorylated in response to heat stress and anisomycin, and this phosphorylation was prevented by treatment with SB202190, an inhibitor of p38 MAPK. Expression of zfHsp27 and human Hsp27 in mammalian fibroblasts promoted a similar degree of tolerance to heat stress. zfHsp27 fusion proteins entered the nucleus and associated with the cytoskeleton of heat stressed cells in vitro and in zebrafish embryos. These results reveal conservation in regulation and function of mammalian and teleost Hsp27 proteins and define zebrafish as a new model for the study of Hsp27 function.     

Twinfilin is required for actin-dependent developmental processes in Drosophila.

The actin cytoskeleton is essential for cellular remodeling and many developmental and morphological processes. Twinfilin is a ubiquitous actin monomer-binding protein whose biological function has remained unclear. We discovered and cloned the Drosophila twinfilin homologue, and show that this protein is ubiquitously expressed in different tissues and developmental stages. A mutation in the twf gene leads to a number of developmental defects, including aberrant bristle morphology. This results from uncontrolled polymerization of actin filaments and misorientation of actin bundles in developing bristles. In wild-type bristles, twinfilin localizes diffusively to cytoplasm and to the ends of actin bundles, and may therefore be involved in localization of actin monomers in cells. We also show that twinfilin and the ADF/cofilin encoding gene twinstar interact genetically in bristle morphogenesis. These results demonstrate that the accurate regulation of size and dynamics of the actin monomer pool by twinfilin is essential for a number of actin-dependent developmental processes in multicellular eukaryotes.     

Some properties of complexes formed by small heat shock proteins with denaturated actin

The effects of a recombinant small heat shock protein with an apparent molecular weight of 27 kDa (Hsp27) and both wild type (Hsp27-wt) and S15D, S78D, S82D mutants (Hsp27-3D), which mimic the naturally occurring phosphorylation of this protein, on the thermal denaturation and aggregation of F-actin were studied. It has been shown that at a Hsp27/actin weight ration of 1/4, both Hsp27-wt and Hsp27-3D do not affect the thermal denaturation of F-actin, but efficiently prevent its further aggregation by forming soluble complexes with denatured actin. Formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. Hsp27-wt is known to exist as a high-molecular-weight oligomer, whereas Hsp27-3D forms only small dimers or tetramers. However, the complexes formed by Hsp27-wt and Hsp27-3D with denatured actin did not differ in their size, as measured by dynamic light scattering, and demonstrated the same hydrodynamic radius of 17–18 nm. On the other hand, the sedimentation coefficients of these complexes differed: they ranged 10–45 S in the case of Hsp27-3D and 18–60 S in case of Hsp27-wt. Thus, the initial oligomeric state of Hsp27 does not significantly affect its capacity to form small soluble complexes with denatured actin.     

Some properties of complexes formed by small heat shock proteins with denatured actin

We applied different methods to analyze the effects of the recombinant wild-type small heat shock protein with an apparent molecular mass of 27 kD (Hsp27-wt) and its S15,78,82D mutant (Hsp27-3D), which mimics the naturally occurring phosphorylation of this protein, on the thermal denaturation and aggregation of F-actin. It has been shown that, at the weight ratio of Hsp27/actin equal to 1/4, both Hsp27-wt and Hsp27-3D do not affect the thermal unfolding of F-actin but effectively prevent the aggregation of F-actin by forming soluble complexes with denatured actin. The formation of these complexes occurs upon heating and accompanies the F-actin thermal denaturation. It is known that Hsp27-wt forms high-molecular-mass oligomers, whereas Hsp27-3D forms small dimers or tetramers. However, the complexes formed by Hsp27-wt and Hsp27-3D with denatured actin did not differ in their size, as measured by dynamic light scattering, and demonstrated the same hydrodynamic radius of 17-18 nm. On the other hand, the sedimentation coefficients of these complexes were distributed within the range 10-45 S in the case of Hsp27-3D and 18-60 S in the case of Hsp27-wt. Thus, the ability of Hsp27 to form soluble complexes with denatured actin does not significantly depend on the initial oligomeric state of Hsp27.