MECHANISM OF REGULATION OF HEAT SHOCK GENE EXPRESSION

高温和其他压力，包括暴露在各种各样的有毒化学物质中，会激活/增强编码所谓热休克或应激蛋白（Hsps）的一小群基因的表达。许多热休克蛋白是已知的分子伴侣。这种应激反应的先前激活使细胞、组织和器官对随后的应激反应更具抵抗力，而这种反应的阻断导致应激敏感性增加。应激反应的激活也被证明可以预防或减轻缺血和再灌注损伤、化疗药物毒性和炎症反应，以及增强免疫系统的反应性。应激反应最上游的特异性调节因子是热休克转录因子（HSF），即脊椎动物细胞中的HSF1。我们之前的工作表明，在非应激的哺乳动物细胞中，HSF1与Hsp90形成动态复合物，不能结合DNA。HSF1的激活是一个由两个可区分的步骤组成的过程。当细胞受到压力时，蛋白质展开的速度急剧增加，非天然蛋白质积累。这些非天然蛋白与HSF1竞争Hsp90结合，未络合的HSF1迅速同三聚体化。三聚化伴随着获得dna结合活性和HSF1的核重新定位。在一个比三聚体化反应稍慢的反应中，三聚体HSF1被转化为活性转录因子。间接证据表明，这第二种反应涉及HSF1中某些关键丝氨酸/苏氨酸残基的应激调节磷酸化，或由应激调节磷酸化触发。在压力事件之后，活跃的HSF1被循环利用。本研究旨在阐明三聚体HSF1转化为转录调控因子的未知机制，以及激活HSF1失活并返回非三聚体状态的机制。在衰老细胞和表达朊病毒的细胞中都注意到缺乏功能性应激反应。在这个项目中获得的机械知识将应用于一个系统的检查，旨在发现老化和朊病毒感染细胞中有缺陷的应激反应的原因。

Heat and other stress, including exposure to a wide variety of noxious chemicals, activate/enhance expression of a small group of genes encoding so-called heat shock or stress proteins (Hsps). Many Hsps are known molecular chaperones. Prior activation of this stress response renders cells, tissues and organs more resistant to a subsequent stress, and blockage of the response results in increased stress susceptibility. Activation of the stress response has also been shown to prevent or mitigate ischemia and reperfusion injury, toxicity of chemotherapeutics and inflammatory responses as well as to enhance the responsiveness of the immune system. The most upstream, specific regulator of the stress response is a heat shock transcription factor (HSF), HSF1 in vertebrate cells. Our previous work showed that, in the unstressed mammalian cell, HSF1 forms a dynamic complex with Hsp90 and is incapable of binding DNA. Activation of HSF1 is a process that comprises two distinguishable steps. When the cell is stressed, the rate of protein unfolding dramatically increases, and nonnative proteins accumulate. These nonnative proteins compete with HSF1 for Hsp90 binding, and uncomplexed HSF1 rapidly homotrimerizes. Trimerization is accompanied by acquisition of DNA-binding activity and nuclear relocation of HSF1. In a reaction that is somewhat slower than the trimerization reaction, trimeric HSF1 is converted to the active transcription factor. Indirect evidence suggests that this second reaction involves or is triggered by stress-regulated phosphorylation of certain critical Ser/Thr residues in HSF1. Subsequent to a stressful event, active HSF1 is recycled. This proposal is elucidate the unknown mechanisms by which trimeric HSF1 is converted to the transcriptionally competent factor, and by which active HSF1 is deactivated and returned to the inactive, nontrimeric state. The absence of a functional stress response was noted in aging cells as well as in cells expressing prions. The mechanistic knowledge gained in this project will be applied to a systematic examination aimed at discovering the reason(s) underlying the defective stress response in aging and prion- infected cells.