CHIP interacts with heat shock factor 1 during heat stress

Heat shock factor 1 (HSF1) is a major transactivator of heat shock genes in response to stress and mediates cell protection against various harmful conditions. In this study, we identified the interaction of CHIP (carboxyl terminus of the heat shock cognate protein 70-interacting protein) with the N-terminus of HSF1. Using GST full-down assay, we found that CHIP directly interacts with C-terminal deleted HSF1 (a.a. 1-290) but not with full-length HSF1 under non-stressed conditions. Interestingly, interaction of CHIP with full-length HSF1 was induced by heat shock treatment. The structural change of HSF1 was observed under heat stressed conditions by CD spectra. These observations demonstrate the direct interaction between HSF1 and CHIP and this interaction requires conformational change of HSF1 by heat stress.     

热激蛋白Hsp72促进热休克下HuR对p21CIP1的调控作用

RNA结合蛋白HuR可以结合并调控靶标mRNA稳定性与翻译,但影响HuR 结合活性的因素有待探讨。本研究从蛋白质-蛋白质相互作用角度对影响HuR 与RNA结合活性的因素做了探讨。结果发现,热激蛋白Hsp72在细胞浆与HuR相互作用并促进HuR与p21 (KIP1) 3′UTR（3′非翻译区）的结合; 热休克下Hsp72总蛋白质及细胞浆蛋白质水平上调、但HuR总蛋白质及细胞浆蛋白质水平不变|热休克下HuR与p21 3′UTR的相互作用加强、p21蛋白及mRNA水平上调。上述结果提示,Hsp72可通过与HuR相互作用促进后者与p21 mRNA的结合,进而加强热休克下HuR对p21的表达的促进作用。这些结果为进一步解析HuR的生物学作用机制提供了实验依据。     

HspBP1, a homologue of the yeast Fes1 and Sls1 proteins,is an Hsc70 nucleotide exchange factor

The yeast FES1 and SLS1 genes encode conserved nucleotide exchange factors that act on the cytoplasmic and endoplasmic reticulum luminal Hsp70s, Ssa1p and BiP, respectively. We report here that mammalian HspBP1 is homologous to Fes1p and that HspBP1 promotes nucleotide dissociation from both Ssa1p and mammalian Hsc70. In contrast, Fes1p inefficiently strips nucleotide from mammalian Hsc70, and unlike HspBP1 does not inhibit chaperone-mediated protein refolding in vitro. Together, our data indicate that HspBP1 is a member of this new class of nucleotide exchange factors that exhibit varying degrees of compartment and species specificity.     

Effect of heat shock,pretreatment and hsp70 copy number on wing development in Drosophila melanogaster

Naturally occurring heat shock (HS) during pupation induces abnormal wing development in Drosophila; we examined factors affecting the severity of this induction. The proportion of HS-surviving adults with abnormal wings varied with HS duration and intensity, and with the pupal age or stage at HS administration. Pretreatment (PT), mild hyperthermia delivered before HS, usually protected development against HS. Gradual heating resembling natural thermal regimes also protected wing development against thermal disruption. Because of the roles of the wings in flight and courtship and in view of natural thermal regimes that Drosophila experience, both HS-induction of wing abnormalities and its abatement by PT may have marked effects on Drosophila fitness in nature. Because PT is associated with expression of heat-inducible molecular chaperones such as Hsp70 in Drosophila, we compared thermal disruption of wing development among hsp70 mutants as well as among strains naturally varying in Hsp70 levels. Contrary to expectations, lines or strains with increased Hsp70 levels were no more resistant to HS-disruption of wing development than counterparts with lower Hsp70 levels. In fact, wing development was more resistant to HS in hsp70 deletion strains than control strains. We suggest that, while high Hsp70 levels may aid cells in surviving hyperthermia, high levels may also overly stimulate or inhibit numerous signalling pathways involved in cell proliferation, maturation and programmed death, resulting in developmental failure.     

Intracellular trafficking of heat shock factor 2

HSF2 is an enigmatic member of the heat shock factor family, identified in the homeotherm classes of birds and mammals. We report the characterization of HSF2 from an evolutionary ancient vertebrate, the fish rainbow trout (rtHSF2). rtHSF2 appears closely related to its mammalian counterparts at structural and functional levels. The conservation of the distinctive features of HSF2 from fish to human suggests that it should ensure important biological functions, not redundant with those of HSF1. Proteasome inhibition, reported as a potent stimulator of HSF2, leads to the stabilization and to a striking nuclear trafficking of rtHSF2-GFP fusion protein. Upon treatment with the proteasome inhibitor MG132, rtHSF2-GFP accumulates into PML nuclear bodies (NBs) independently of its sumoylation and, if expressed at moderate level, moves to nucleoli. The translocation of rtHSF2-GFP from NBs to nucleoli is greatly favored by overexpression of the heat shock protein Hsp70. The mammalian counterpart mouse HSF2 (mHSF2) also exhibited changes in intracellular distribution upon MG132 treatment. mHSF2 partitioned between a juxtanuclear area that we characterized as an aggresome and the nucleoli. These relocalizations are likely to reflect common structural changes of mouse and trout HSF2 upon activation.     

Evidence for bacterial origin of heat shock RNA-1

The heat shock RNA-1 (HSR1) is a noncoding RNA (ncRNA) reported to be involved in mammalian heat shock response. HSR1 was shown to significantly stimulate the heat-shock factor 1 (HSF1) trimerization and DNA binding. The hamster HSR1 sequence was reported to consist of 604 nucleotides (nt) plus a poly(A) tail and to have only a 4-nt difference with the human HSR1. In this study, we present highly convincing evidence for bacterial origin of the HSR1. No HSR1 sequence was found by exhaustive sequence similarity searches of the publicly available eukaryotic nucleotide sequence databases at the NCBI, including the expressed sequence tags, genome survey sequences, and high-throughput genomic sequences divisions of GenBank, as well as the Trace Archive database of whole genome shotgun sequences, and genome assemblies. Instead, a putative open reading frame (ORF) of HSR1 revealed strong similarity to the amino-terminal region of bacterial chloride channel proteins. Furthermore, the 5′ flanking region of the putative HSR1 ORF showed similarity to the 5′ upstream regions of the bacterial protein genes. We propose that the HSR1 was derived from a bacterial genome fragment either by horizontal gene transfer or by bacterial infection of the cells. The most probable source organism of the HSR1 is a species belonging to the order Burkholderiales.     

Radiosensitization of wildtype p53 cancer cells by the MDM2-inhibitor PXN727 is associated with altered heat shock protein 70 (Hsp70) levels

The oncoprotein MDM2 (murine double minute 2) is often overexpressed in human tumors and thereby attenuates the function of the tumor suppressor p53. In this study, we investigated the effects of the novel MDM2-inhibitor PXN727 on p53 activation, cell proliferation, cell cycle distribution and radiosensitivity. Since the localization of heat shock protein 70 (Hsp70) exerts different effects on radioresistance of tumor cells, we investigated the impact of PXN727 on intracellular, membrane, and secreted Hsp70 levels. We could show that PXN727 exerts its effects on wildtype p53 (HCT116 p53^+/+, A549) but not p53 depleted (HCT116 p53^−/−) or mutated (FaDu) tumor cells. PXN727 activates p53, induces the expression of p21, reduces the proportion of cells in the radioresistant S-phase and induces senescence. Radiosensitivity was significantly increased by PXN727 in HCT116 p53^+/+ tumor cells. Furthermore, PXN727 causes a downregulation of Hsp70 membrane expression and an upregulated secretion of Hsp70 in wildtype p53 tumor cells. Our data suggest that re-activation of p53 by MDM2-inhibition modulates Hsp70 membrane expression and secretion which might contribute to the radiosensitizing effect of the MDM2-inhibitor PXN727.     

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.