The small heat shock-related protein, HSP20, is a cAMP-dependent protein kinase substrate that is involved in airway smooth muscle relaxation

Activation of the cAMP/cAMP-dependent PKA pathway leads to relaxation of airway smooth muscle (ASM). The purpose of this study was to examine the role of the small heat shock-related protein HSP20 in mediating PKA-dependent ASM relaxation. Human ASM cells were engineered to constitutively express a green fluorescent protein-PKA inhibitory fusion protein (PKI-GFP) or GFP alone. Activation of the cAMP-dependent signaling pathways by isoproterenol (ISO) or forskolin led to increases in the phosphorylation of HSP20 in GFP but not PKI-GFP cells. Forskolin treatment in GFP but not PKI-GFP cells led to a loss of central actin stress fibers and decreases in the number of focal adhesion complexes. This loss of stress fibers was associated with dephosphorylation of the actin-depolymerizing protein cofilin in GFP but not PKI-GFP cells. To confirm that phosphorylated HSP20 plays a role in PKA-induced ASM relaxation, intact strips of bovine ASM were precontracted with serotonin followed by ISO. Activation of the PKA pathway led to relaxation of bovine ASM, which was associated with phosphorylation of HSP20 and dephosphorylation of cofilin. Finally, treatment with phosphopeptide mimetics of HSP20 possessing a protein transduction domain partially relaxed precontracted bovine ASM strips. In summary, ISO-induced phosphorylation of HSP20 or synthetic phosphopeptide analogs of HSP20 decreases phosphorylation of cofilin and disrupts actin in ASM, suggesting that one possible mechanism by which HSP20 mediates ASM relaxation is via regulation of actin filament dynamics.     

The small heat shock-related protein, HSP20, is phosphorylated on serine 16 during cyclic nucleotide-dependent relaxation

The small heat shock-related protein 20 (HSP20) is present in four isoforms in bovine carotid artery smooth muscles. Three of the isoforms are phosphorylated and one is not. Increases in the phosphorylation of two isoforms of HSP20 (isoform 3, pI 5.9; and 8, pI 5.7) are associated with cyclic nucleotide-dependent relaxation of bovine carotid artery smooth muscles. Increases in the phosphorylation of another isoform (isoform 4, pI 6.0) are associated with phorbol ester-induced contraction of bovine carotid artery smooth muscles. In this investigation we determined that isoforms 3 and 8 are phosphorylated on Ser16 of the HSP20 molecule during activation of cAMP-dependent signaling pathways. Phosphorylation state-specific antibodies produced against a peptide containing phosphorylated Ser16 recognized isoforms 3 and 8 but not isoform 4. In human vascular tissue, only isoform 3 is present. Incubation of transiently permeabilized strips of bovine carotid artery smooth muscle with synthetic peptides in which Ser16 is phosphorylated, inhibits contractile responses to high extracellular KCl and to serotonin. These data suggest that phosphorylation of HSP20 on Ser16 modulates cAMP-dependent vasorelaxation.     

Transduction of phosphorylated heat shock-related protein 20, HSP20, prevents vasospasm of human umbilical artery smooth muscle.

Activation of cyclic nucleotide-dependent signaling pathways inhibits agonist-induced contraction of most vascular smooth muscles except human umbilical artery smooth muscle (HUASM). This impaired vasorelaxation may contribute to complications associated with preeclampsia, intrauterine growth restriction, and preterm delivery. Cyclic nucleotide-dependent signaling pathways converge at the phosphorylation of the small heat shock-related protein HSP20, causing relaxation of vascular smooth muscle. We produced recombinant proteins containing a protein transduction domain linked to HSP20 (rTAT-HSP20). Pretreatment of HUASM with in vitro phosphorylated rTAT-HSP20 (rTAT-pHSP20) significantly inhibited serotonin-induced contraction, without a decrease in myosin light chain phosphorylation. rTAT-pHSP20 remained phosphorylated upon transduction into isolated HUASM as demonstrated by two-dimensional gel electrophoresis. Transduction of peptide analogs of phospho-HSP20 containing the phosphorylation site on HSP20 and phosphatase-resistant mimics of the phosphorylation site (S16E) also inhibited HUASM contraction. These data suggest that impaired relaxation of HUASM may result from decreased levels of phosphorylated HSP20. Protein transduction can be used to restore intracellular expression levels and the associated physiological response. Transduction of posttranslationally modified substrate proteins represents a proteomic-based therapeutic approach that may be particularly useful when the expression of downstream substrate proteins is downregulated.     

Small molecular weight heat shock-related protein, HSP20, exhibits an anti-platelet activity by inhibiting receptor-mediated calcium influx

We have shown that Hsp20, one of small molecular weight heat shock protein, which is present at a high concentration both in vascular smooth muscle cells and in circulating blood in patient with vascular disease, strongly inhibits platelet aggregation in vitro and ex vivo. To clarify the mechanism, we investigated the effect of Hsp20 on free calcium concentration in human platelet cytoplasm using fura 2. Hsp20 inhibited thrombin-induced calcium influx without affecting calcium release from intracellular calcium stores. The degree of inhibition is well-correlated with that of suppression of thrombin-induced platelet aggregation by this substance. Hsp20 also inhibited the elevation of cytoplasmic free calcium level triggered by collagen, but not that by A-23187. In contrast, Hsp28, another type of small molecular weight Hsp, failed to affect the cytoplasmic free calcium level. These findings suggest that Hsp20 inhibits the receptor-mediated calcium influx of platelets without affecting calcium release from intracellular calcium stores, leading to its anti-platelet activity.     

HSP20, low-molecular-weight heat shock-related protein,acts extracellularly as a regulator of platelet functions: a novel defense mechanism

We previously showed that a dissociated form of a low-molecular-weight heat shock-related protein 20 (HSP20) but not an aggregated form of HSP20 suppresses platelet aggregation. In the present study, we investigated the behavior of HSP20 in response to endothelial injury and the possible mechanism of HSP20 in platelet functions. The levels of HSP20 in vessel wall after endothelial injury were markedly reduced. This observation was supported by the results of Western blotting analysis and immunohistochemical analysis. Additionally, the plasma levels of HSP20 in cardiomyopathic hamsters were markedly elevated. Centrifugation on sucrose density gradients allowed detection mainly of the dissociated form of plasma HSP20 in these hamsters. Human platelets showed specific binding sites for HSP20. Moreover, HSP20 markedly reduced thrombin-induced phosphoinositide hydrolysis by phospholipase C in human platelets. Taken together, our results strongly suggest that HSP20, which immediately responds to pathological events, acts extracellularly as a regulator of platelet functions.     

Reduction in Gh protein expression is associated with cytodifferentiation of vascular smooth muscle cells

G_h, a high molecular weight GTP-binding protein that couples ₁-adrenoceptors in heart and liver to phosphatidylinositol (Ptdlns)-specific phospholipase C (PLC), has recently been shown to be a tissue transglutaminase type 11. Transglutaminases have been suggested to play a role in the maintenance of blood vessel structure, and therefore it is possible that changes in their expression may accompany pathological states which involve phenotypic modulation of smooth muscle. Hence, we investigated the expression of G_h during differentiation of rat aortic smooth muscle cells in culture. G_h content was reduced markedly in cultured smooth muscle cells compared to freshly isolated cells as determined by Western blotting using a G_h-specific monoclonal antibody. In contrast, the level of G_q, a heterotrimeric G-protein that couples ₁-adrenoceptors to PLC, was maintained throughout the culture period. These findings indicate that changes in G, expression accompany phenotypic modulation of vascular smooth muscle cells. These changes in G_h protein expression may be important in the altered responsiveness of vessels in pathological disease states.     

HSP25, a small heat shock protein associated with dense bodies and M-lines of body wall muscle in Caenorhabditis elegans

HSP25, a previously uncharacterized member of the alpha-crystallin family of small heat shock proteins in Caenorhabditis elegans, has been examined using biochemical and immunological techniques. HSP25 is the second largest of 16 identifiable small heat shock proteins in the nematode and is expressed at all developmental stages under normal growth conditions. Recombinant HSP25 produced in Escherichia coli exists predominantly as small oligomers (dimers to tetramers) and possesses chaperone activity against citrate synthase in vitro. In C. elegans, HSP25 is localized to dense bodies and M-lines in body wall muscle, to the lining of the pharynx, and to the junctions between cells of the spermathecal wall. Affinity chromatography of nematode extracts on a column of immobilized HSP25 resulted in specific binding of vinculin and alpha-actinin but not actin, as revealed by Western blotting. These results suggest a role for HSP25 in the organization or maintenance of the myofilament lattice and adherens junctions in C. elegans.     

Comparison of the small heat shock proteins alphaB-crystallin, MKBP, HSP25, HSP20, and cvHSP in heart and skeletal muscle

Seven members of the small heat shock protein (sHSP) family are exceptional with respect to their constitutive high abundance in muscle tissue. It has been suggested that sHSPs displaying chaperone-like properties may stabilize myofibrillar proteins during stress conditions and prevent them from loss of function. In the present study five sHSPs (alphaB-crystallin, MKBP, HSP25, HSP20, and cvHSP) were investigated with respect to similarities and differences of their expression in heart and skeletal muscle under normal and ischemic conditions. In ischemic heart and skeletal muscle these five sHSPs translocated from cytosol to the Z-/I-area of myofibrils. Myofibrillar binding of all sHSPs was very tight and resisted for the most part extraction with 1 M NaSCN or 1 M urea. MKBP and HSP20 became extracted by 1 M NaSCN to a significant extent indicating that these two sHSPs may bind partially to actin-associated proteins which were completely extracted by this treatment. Ultrastructural localization of alphaB-crystallin showed diffuse distribution of immunogold label throughout the entire I-band in skeletal muscle fibers whereas in cardiomyocytes alphaB-crystallin was preferentially located at the N-line position of the I-band. These observations indicate different myofibrillar binding sites of alphaB-crystallin in cardiomyocytes versus skeletal muscle fibers. Further differences of the properties of sHSPs could be observed regarding fiber type distribution of sHSPs. Thus sHSPs form a complex stress-response system in striated muscle tissue with some common as well as some distinct functions in different muscle types.     

In vivo modifications of the maize mitochondrial small heat stress protein, HSP22

A maize (Zea mays L.) small heat shock protein (HSP), HSP22, was previously shown to accumulate to high levels in mitochondria during heat stress. Here we have purified native HSP22 and resolved the protein into three peaks using reverse phase high performance liquid chromatography. Mass spectrometry (MS) of the first two peaks revealed the presence of two HSP22 forms in each peak which differed in mass by 80 daltons (Da), indicative of a monophosphorylation. Phosphorylation of HSP22 by [gamma-(32)P]ATP was also observed in mitochondria labeled in vitro, but not when purified native HSP22 was similarly used, demonstrating that HSP22 does not autophosphorylate, implicating a kinase involvement in vivo. Collisionally induced dissociation tandem MS (CID MS/MS) identified Ser(59) as the phosphorylated residue. We have also observed forms of HSP22 that result from alternative intron splicing. The two HSP22 proteins in the first peak were approximately 57 Da larger than the two HSP22 proteins in the second peak. MS analysis revealed that the +57-Da forms have an additional Gly residue directly N-terminal of the expected Asp(84), which had been converted to an Asn residue. These results are the first demonstrations of phosphorylation and alternative intron splicing of a plant small HSP.     

HSP22, a new member of the small heat shock protein superfamily, interacts with mimic of phosphorylated HSP27 ((3D)HSP27)

Most of the members of the superfamily of mammalian small heat shock or stress proteins are abundant in muscles where they play a role in muscle function and maintenance of muscle integrity. One member of this protein superfamily, human HSP27, is rapidly phosphorylated on three serine residues (Ser(15), Ser(78), and Ser(82)) during cellular response to a number of extracellular factors. To understand better the role of HSP27, we performed a yeast two-hybrid screen of a human heart cDNA library for HSP27-interacting proteins. By using the triple aspartate mutant, a mimic of phosphorylated HSP27, as "bait" construct, a protein with a molecular mass of 21.6 kDa was identified as an HSP27-binding protein. Sequence analysis revealed that this new protein shares an overall sequence identity of 33% with human HSP27. This protein also contains the alpha-crystallin domain in its C-terminal half, a hallmark of the superfamily of small stress proteins. Thus, the new protein itself is a member of this protein superfamily, and consequently we designated it HSP22. According to the two-hybrid data, HSP22 interacts preferentially with the triple aspartate form of HSP27 as compared with wild-type HSP27. HSP22 is expressed predominantly in muscles. In vitro, HSP22 is phosphorylated by protein kinase C (at residues Ser(14) and Thr(63)) and by p44 mitogen-activated protein kinase (at residues Ser(27) and Thr(87)) but not by MAPKAPK-2.     

Priming effect of exogenous ABA on heat stress tolerance in rice seedlings is associated with the upregulation of antioxidative defense capability and heat shock-related genes

Plant Growth Regulation - Heat stress is a major restrictive factor that suppresses rice production. In this study, we investigated the potential priming effect of exogenous abscisic acid (ABA) on...     

Critical role for heat shock protein 20 (HSP20) in migration of malarial sporozoites

Plasmodium sporozoites, single cell eukaryotic pathogens, use their own actin/myosin-based motor machinery for life cycle progression, which includes forward locomotion, penetration of cellular barriers, and invasion of target cells. To display fast gliding motility, the parasite uses a high turnover of actin polymerization and adhesion sites. Paradoxically, only a few classic actin regulatory proteins appear to be encoded in the Plasmodium genome. Small heat shock proteins have been associated with cytoskeleton modulation in various biological processes. In this study, we identify HSP20 as a novel player in Plasmodium motility and provide molecular genetics evidence for a critical role of a small heat shock protein in cell traction and motility. We demonstrate that HSP20 ablation profoundly affects sporozoite-substrate adhesion, which translates into aberrant speed and directionality in vitro. Loss of HSP20 function impairs migration in the host, an important sporozoite trait required to find a blood vessel and reach the liver after being deposited in the skin by the mosquito. Our study also shows that fast locomotion of sporozoites is crucial during natural malaria transmission.     

Role of small heat shock protein HSP25 in radioresistance and glutathione-redox cycle

Expression of heat shock proteins (HSPs) has been shown to protect mammalian cells exposed to a variety of stress stimuli. Among various HSPs, small HSPs from diverse species were shown to protect cells against oxidative stress. Here, we show that the overexpression of the mouse small hsp gene, hsp25, provides protection against ionizing radiation. Our results demonstrate that the radiation survival of the L929 cells stably transfected with hsp25 was enhanced compared with that of the parental or vector transfected control, L25#1 cells. Our results also demonstrate that the radiation-induced apoptosis was reduced in HSP25 overexpressors. A detailed analysis of glutathione composition of those clones that overexpressed HSP25 revealed the increases of the glutathione pool, which primarily resulted from the increase of reduced glutathione. Our data suggest that higher content of GSH in HSP25 overexpressors was because of a faster reduction of oxidized glutathione (GSSG) to GSH rather than an increased de novo synthesis of GSH. The activities of glutathione reductase (GRd) and glutathione peroxidase (GPx) were greater in HSP25 overexpressors but the activity of gamma-glutamylcysteine synthetase was similar between the transfectants and the control cells. Consistent with our view, a steady state ratio of the GSH/GSSG was greater in the transfectants in comparison with the control L25#1 cells. A difference in the relative ratio became more significant after exposure to the ionizing radiation. To our knowledge, this study provides the first experimental evidence in support of the hypothesis that small HSP plays a key role in radioresistance by modulating the metabolism of glutathione. Based on the results obtained from the current investigation, we propose that HSP25 helps facilitate the glutathione-redox cycle and therefore, enhances glutathione utilization and maintains the cellular glutathione pool in favor of the reduced states.     

Presbyopia and heat: changes associated with aging of the human lens suggest a functional role for the small heat shock protein, alpha-crystallin, in maintaining lens flexibility

Presbyopia, the inability to focus up close, affects everyone by age 50 and is the most common eye condition. It is thought to result from changes to the lens over time making it less flexible. We present evidence that presbyopia may be the result of age-related changes to the proteins of the lens fibre cells. Specifically, we show that there is a progressive decrease in the concentration of the chaperone, α-crystallin, in human lens nuclei with age, as it becomes incorporated into high molecular weight aggregates and insoluble protein. This is accompanied by a large increase in lens stiffness. Stiffness increases even more dramatically after middle age following the disappearance of free soluble α-crystallin from the centre of the lens. These alterations in α-crystallin and aggregated protein in human lenses can be reproduced simply by exposing intact pig lenses to elevated temperatures, for example, 50 °C. In this model system, the same protein changes are also associated with a progressive increase in lens stiffness. These data suggest a functional role for α-crystallin in the human lens acting as a small heat shock protein and helping to maintain lens flexibility. Presbyopia may be the result of a loss of α-crystallin coupled with progressive heat-induced denaturation of structural proteins in the lens during the first five decades of life.     

Phosphorylation of the 105-kDa heat shock proteins, HSP105alpha and HSP105beta, by casein kinase II

The 105-kDa heat shock protein alpha (HSP105alpha) and HSP105beta are mammalian heat shock proteins that belong to the HSP105/HSP110 family. Both HSP105alpha and HSP105beta consist of acidic and basic isoforms. Here we report that the acidic isoforms are serine phosphorylated HSP105alpha or HSP105beta. Furthermore, using an in-gel kinase assay with HSP105alpha or HSP105beta as the substrate, the protein kinase that phosphorylates HSP105alpha and HSP105beta was identified as casein kinase II. Since phosphorylated HSP105alpha is especially prominent in the brain compared to other tissues of mice and rats, the phosphorylation of HSP105alpha by casein kinase II may be biologically significant.     

Effect of NO donors on protein phosphorylation in intact vascular and nonvascular smooth muscles

It is generally well accepted that nitrovasodilator-induced relaxation of vascular smooth muscle involves elevation of cGMP and activation of a specific cGMP-dependent protein kinase [protein kinase G (PKG)]. However, the protein targets of PKG and the underlying mechanisms by which this kinase leads to a relaxant response have not been elucidated. Several types of smooth muscle, including rat myometrium and vas deferens, are not relaxed by sodium nitroprusside, even at concentrations that produce marked elevation of cGMP and activation of PKG. The main objective of our studies was to compare PKG-mediated protein phosphorylation in intact rat aorta, rat myometrium, and rat vas deferens using two-dimensional gel electrophoresis. In intact rat aorta, seven PKG substrates were detected during relaxation of the tissue. None of the PKG substrates identified in the rat aorta appeared to be phosphorylated in the myometrium or vas deferens after administration of various cGMP-elevating agents. Thus the failure of the rat myometrium and rat vas deferens to relax in the face of cGMP elevation and PKG activation may be due to a lack of PKG substrate phosphorylation.