Identification of heat shock protein 90-associated 84-kDa phosphoprotein

In eukaryotic cells, HSP90 is associated with several protein kinases and regulates their activities. HSP90 was also reported to possess an autophosphorylase activity. In this study, we examined in vitro autophosphorylation of HSP90, which was purified from chick muscle. We show that HSP90 was not phosphorylated in vitro, but an 84-kDa protein (p84) was highly phosphorylated. P84 was neither HSP90 nor its degradative product, as it was not detected by an antibody (BF4) specific to HSP90 in denaturing immunoprecipitation and Western blot analysis. Phosphorylation of a protein similar to p84 was also detected with purified human brain and HeLa HSP90, indicating that p84 is present in many different types of cells. P84 appeared to exist as large complexes, as determined by HPLC and native gel electrophoresis. Native immunoprecipitation using anti-HSP90 (BF4)-conjugated Affi-gel revealed that this phosphoprotein is specifically associated with HSP90. The interaction of p84 and HSP90 was not affected by p84 phosphorylation. In addition, p84 phosphorylation was prevented by the presence of divalent cations such as Mg(2+) and Mn(2+). In contrast, p84 phosphorylation was significantly activated by addition of exogenous Ca(2+) between 100 and 500 microM, although it was blocked by higher concentrations (>1 mM) of Ca(2+). HSP90, but not p84, could be phosphorylated by casein kinase II. Finally, p84 phosphorylation was specifically prevented by hemin, but not by other kinase inhibitors, indicating that p84 phosphorylation may be regulated by heme-regulated protein kinase.     

中国辣椒热激蛋白HSP70基因家族分析

以中国辣椒(Capsicum chinense Jacq.)基因组数据为基础, 采用生物信息学方法对中国辣椒HSP70基因家族进行全基因组鉴定分析.结果显示, 中国辣椒全基因共鉴定得到20个HSP70基因, 编码蛋白序列长度为516～854 aa, 分子量大小为56.21～94.26 kD.系统进化分析结果表明, 中国...     

Role of the heat shock protein family in bone metabolism

Heat shock proteins (HSPs) are a family of proteins produced by cells in response to exposure to stressful conditions. In addition to their role as chaperones, they also play an important role in the cardiovascular, immune, and other systems. Normal bone tissue is maintained by bone metabolism, particularly by the balance between osteoblasts and osteoclasts, which are physiologically regulated by multiple hormones and cytokines. In recent years, studies have reported the vital role of HSPs in bone metabolism. However, the conclusions remain largely controversial, and the exact mechanisms are still unclear, so a review and analyses of previous studies are of importance. This article reviews the current understanding of the roles and effects of HSPs on bone cells (osteoblasts, osteoclasts, and osteocytes), in relation to bone metabolism.     

Characterization of the hydrophobic region of heat shock protein 90

The modes of binding of heat shock protein 90 with phenyl-Sepharose, myristoylated AE-cellulose, and monomyristoylated lysozyme were studied to characterize a hydrophobic region(s) on the surface of the heat shock protein 90 molecule and the following results were obtained. (1) The binding of heat shock protein 90 with phenyl-Sepharose was inhibited by the addition of 30% ethylene glycol. This indicates that the binding involves a hydrophobic interaction. (2) The binding was strengthened by the addition of 10 mM Mg2+, Ca2+, Sr2+, and Ba2+ ions, but not by K+ or Na+ ions. (3) The binding of hsp 90 with phenyl-Sepharose decreased initially and then increased as the temperature was increased from 0 to 50 degrees C, with a minimum at around 35 degrees C. (4) Lowering the pH stimulated the binding of hsp 90 with phenyl-Sepharose. (5) Heat shock protein 90 bound to myristoylated AE-cellulose, which has aliphatic hydrophobic residues, but not to acetylated AE-cellulose. (6) Heat shock protein 90 bound to monomyristoylated lysozyme, but not to control unmodified lysozyme. Based on these results, the possible function of the hydrophobic region(s) of heat shock protein 90 in the interaction with hydrophobic proteins is discussed.     

Antibodies against 70-kD heat shock cognate protein inhibit mediated nuclear import of karyophilic proteins

Previously, we found that anti-DDDED antibodies strongly inhibited in vivo nuclear transport of nuclear proteins and that these antibodies recognized a protein of 69 kD (p69) from rat liver nuclear envelopes that showed specific binding activities to the nuclear location sequences (NLSs) of nucleoplasmin and SV-40 large T-antigen. Here we identified this protein as the 70-kD heat shock cognate protein (hsc70) based on its mass, isoelectric point, cellular localization, and partial amino acid sequences. Competition studies indicated that the recombinant hsc70 expressed in Escherichia coli binds to transport competent SV-40 T-antigen NLS more strongly than to the point mutated transport incompetent mutant NLS. To investigate the possible involvement of hsc70 in nuclear transport, we examined the effect of anti-hsc70 rabbit antibodies on the nuclear accumulation of karyophilic proteins. When injected into the cytoplasm of tissue culture cells, anti-hsc70 strongly inhibited the nuclear import of nucleoplasmin, SV-40 T-antigen NLS bearing BSA and histone H1. In contrast, anti-hsc70 IgG did not prevent the diffusion of lysozyme or 17.4-kD FITC-dextran into the nuclei. After injection of these antibodies, cells continued RNA synthesis and were viable. These results indicate that hsc70 interacts with NLS-containing proteins in the cytoplasm before their nuclear import.     

Characterization of purified avian 90,000-Da heat shock protein

The so-called 90,000-Da heat shock protein (hsp90) from chicken liver has been purified and physically characterized in the presence of high levels of the serine phosphatase inhibitor fluoride. The protein is an elongated dimer with a molecular weight of 160,000 and a frictional ratio of 1.6. On two-dimensional electrophoresis it exhibits several isoelectric forms lying between pH 5.1 and 5.8. It contains an average of 5.8 mol of covalently bound phosphate per dimer and is thus extensively phosphorylated. Analysis of the ultraviolet spectrum showed the purified protein to be free of nucleotide-containing components. Molybdate has been shown to stabilize complexes between the 90,000-Da heat shock protein and steroid receptors. However, molybdate has no effect on the sedimentation of the purified heat shock protein. Proteins structurally related to hsp90 have been reported to penetrate the endoplasmic reticulum. However, when purified hsp90 was tested using the partition method of Bordier, which distinguishes hydrophilic and lipophilic proteins, it partitioned totally into the aqueous phase.     

Characterization of the heat shock response in cultured sugarcane cells : I. Physiology of the heat shock response and heat shock protein synthesis

Effect of heat shock on the growth of cultured sugarcane cells (Saccharum officinarum L.) was measured. Heat shock (HS) treatment at 36 to 38°C (2 hours) induced the development of maximum thermotolerance to otherwise nonpermissive heat stress at 54°C (7 minutes). Optimum thermotolerance was observed 8 hours after heat shock. Development of thermotolerance was initiated by treatments as short as 30 minutes at 36°C. Temperatures below 36°C or above 40°C failed to induce maximum thermotolerance. In vivo labeling revealed that HS at 32 to 34°C induced several high molecular mass heat shock proteins (HSPs). A complex of 18 kilodalton HSPs required at least 36°C treatment for induction. The majority of the HSPs began to accumulate within 10 minutes, whereas the synthesis of low molecular mass peptides in the 18 kilodalton range became evident 30 minutes after initiation of HS. HS above 38°C resulted in progressively decreased HSP synthesis with inhibition first observed for HSPs larger than 50 kilodaltons. Analysis of two-dimensional gels revealed a complex pattern of label incorporation including the synthesis of four major HSPs in the 18 kilodalton range and continued synthesis of constitutive proteins during HS.     

The heat shock protein 40 family of the malaria parasite Plasmodium falciparum

Few diseases have had such a profound influence on human evolution and history as malaria. Despite intense efforts malaria infection continues to be a major killer. The causative agent of malaria, the unicellular eukaryote Plasmodium, displays a fascinating biology in which ubiquitous cellular concepts are modified to serve the particular needs of the malaria parasite. In this review, we explore how Plasmodium utilizes the heat shock protein 40 system, a chaperone system that ensures correct protein folding under normal and stress conditions. We highlight the peculiarities of the Plasmodium system and discuss whether any components of the system might be exploited for intervention strategies against this debilitating disease.     

热休克蛋白与自身免疫病相关研究进展

热休克蛋白(heat shock proteins,HSPs)是动物体内一类热应激蛋白,是机体受到高热或其他理化生物等因素刺激时产生的特殊蛋白质。HSPs与自身免疫病的发生发展有着密切关系,在正常生理状态下,HSPs可以在天然免疫反应和适应性免疫反应中分别发挥作用,以帮助机体应对过激环境;而在异常病理条件下,HSPs可参与多种自身免疫病的发生发展。重点概述了HSP22、HSP27、HSP60、HSP70、HSP90与几种常见自身免疫病的相关作用。     

Characterization and mapping of a highly conserved processed pseudogene and an intron-carrying gene of the heat shock cognate protein 70 (Hsc70) gene family in the rat

A processed pseudogene of the rat Hsc70 gene, Hsc70-ps1, is described, which still presents the open reading frame of the original gene. The pseudogene does not appear to be expressed. It maps to rat Chromosome (Chr) 2. The intron-carrying Hsc70 gene localizes to Chr 8. Hsc70-specific probes detect a large number of more than 20 cross-hybridizing fragments, which show only limited length polymorphism among various inbred rat strains.     

The 70-kD heat shock cognate protein (hsc70) facilitates the nuclear export of the import receptors

Transport receptors of the importin beta family continuously shuttle between the nucleus and cytoplasm. We previously reported that the nuclear export of importin beta involves energy-requiring step(s) in living cells. Here, we show that the in vitro nuclear export of importin beta also requires energy input. Cytosol, depleted of ATP-binding proteins, did not support the sufficient nuclear export of importin beta. Further purification revealed that the active component in the absorbed fraction was a 70-kD heat shock cognate protein (hsc70). The addition of recombinant hsc70, but not an ATPase-deficient hsc70 mutant, to the depleted cytosol restored the export activity. In living cells, depletion of hsc70 caused the significant nuclear accumulation of importin beta. These effects of hsc70 were observed in the nuclear export of importin beta, but also for other import receptors, transportin and importin alpha. These results suggest that hsc70 broadly modulates nucleocytoplasmic transport systems by regulating the nuclear export of receptor proteins.     

Characterization and purification of the small 28,000-dalton mammalian heat shock protein

We describe the biochemical characterization and purification of the small 28,000-dalton heat shock protein (28-kDa protein) of mammalian cells. Metabolic pulse labeling of heat shock-treated cells with either [3H]leucine or H3 32PO4 and analysis of the labeled proteins by two-dimensional gel electrophoresis revealed increased levels of three 28-kDa proteins differing only in their relative isoelectric point. Using both peptide mapping and immunological analysis, we demonstrate that all three proteins are related isoforms, with two of the isoforms containing phosphate. Cell fractionation studies revealed that the 28-kDa protein localizes predominantly within the nuclear pellet very shortly after the heat shock treatment. With increasing times of recovery of the heat-treated cells back at 37 degrees C, the majority of the 28-kDa protein was now observed to fractionate within the soluble fraction of the cells. Both gel filtration and velocity sedimentation studies revealed that the 28-kDA protein exists as a higher order structure with an approximate S20,w value of 10-18 S, a Stokes radius of about 60-70 A, and an estimated native molecular mass of at least 500,000 daltons. We describe a relatively simple and rapid purification of the proteins employing both ion-exchange and gel filtration chromatography.     

A family of related proteins is encoded by the major Drosophila heat shock gene family.

At least four proteins of 70,000 to 75,000 molecular weight (70-75K) were synthesized from mRNA which hybridized with a cloned heat shock gene previously shown to be localized to the 87A and 87C heat shock puff sites. These in vitro-synthesized proteins were indistinguishable from in vivo-synthesized heat shock-induced proteins when analyzed on sodium dodecyl sulfate-polyacrylamide gels. A comparison of the pattern of this group of proteins synthesized in vivo during a 5-min pulse or during continuous labeling indicates that the 72-75K proteins are probably not kinetic precursors to the major 70K heat shock protein. Partial digestion products generated with V8 protease indicated that the 70-75K heat shock proteins are closely related, but that there are clear differences between them. The partial digestion patterns obtained from heat shock proteins from the Kc cell line and from the Oregon R strain of Drosophila melanogaster are very similar. Genetic analysis of the patterns of 70-75K heat shock protein synthesis indicated that the genes encoding at least two of the three 72-75K heat shock proteins are located outside of the major 87A and 87C puff sites.