Hsp110 protein chaperone function in yeast

描述(申请人提供)：热休克蛋白(HSPs)在细胞生物学中具有双重作用；它们是抵御细胞毒性应激的第一道防线，在正常生长条件下紧密整合到信号和调节通路中。热休克蛋白的一部分，包括热休克蛋白70和热休克蛋白90，作为分子伴侣，在底物蛋白质生命周期的多个阶段，从生物发生、定位和稳定，到激活和降解，都可能是必需的。蛋白伴侣在包括缺血和肿瘤发生在内的多种病理生理条件下被诱导，并被认为促进了癌蛋白的稳定性和活性，如v-src激酶和p53肿瘤抑制因子。Hsp110类伴侣蛋白是一个知之甚少的Hsp70家族，存在于所有真核生物中，在人类中具有未知功能的组织特异性亚型。我们的长期目标是阐明这个伴侣蛋白家族的细胞角色。面包师的酵母Hsp110同源基因由SSE1和SSE2基因编码，对它们的功能知之甚少。我们发现Sse1在体内以异二聚体的形式存在，与胞浆中的Hsp70s SSA和SSB一起存在，并且Sse1是Hsp90分子伴侣系统信号转导活性所必需的。因此，我们假设Sse1，进而延伸到哺乳动物的Hsp110伴侣，可能主要作为Hsp70活性的调节器发挥作用。这项建议试图通过提出两个具体的问题来从机制上理解Hsp110伴侣蛋白的细胞作用：1)Sse1如何与酵母Hsp70 Ssa1合作运作；2)Sse1如何参与Hsp90的信号转导？在第一个目标中，我们将利用纯化的伴侣蛋白来确定Sse1与Hsp70的相互作用部位和调节，最终破译Sse1在体外对蛋白质折叠的影响。我们将用体内实验来补充这些实验，以确定Sse1对Ssa1依赖过程的贡献。在第二个目标中，我们将利用模型客户蛋白糖皮质激素受体来确定Sse1在Hsp90底物折叠周期中的作用阶段，以及对底物成熟的特定影响。最后，我们将应用这些发现来理解Sse1如何与Hsp90合作，通过调节细胞完整性途径中的SLt2 MAP激酶来调节热休克生存所需的信号。酵母中的这些研究路线将作为预测Hsp110可能促进哺乳动物细胞哪些过程的模型。

DESCRIPTION (provided by applicant): Heat shock proteins (Hsps) play dual roles in cellular biology; they are the first line of defense against cytotoxic stresses, and are tightly integrated into signaling and regulatory pathways under normal growth conditions. A subset of Hsps, including Hsp70 and Hsp90, act as molecular chaperones, and may be required at multiple stages during a substrate protein's lifetime, ranging from biogenesis, localization, and stability, to activation and degradation. Protein chaperones are induced during numerous pathophysiological conditions including ischemia and tumorigenesis, and are known to facilitate stability and activity of oncoproteins such as v-src kinase and the p53 tumor suppressor. The Hsp110 class of chaperones is a poorly understood Hsp70 relative and is present in all eukaryotes, with tissue-specific isoforms of unknown function in humans. Our long-term objective is to elucidate the cellular roles of this chaperone family. The baker's yeast Hsp110 homolog is encoded by the SSE1 and SSE2 genes, and little is known about their function. We have discovered that Sse1 exists as a heterodimer in vivo with the cytosolic Hsp70s Ssa and Ssb, and that Sse1 is required for signal transduction activity of the Hsp90 chaperone system. We therefore hypothesize that Sse1, and by extension the mammalian Hsp110 chaperone, may function primarily as a modulator of Hsp70 activity. This proposal seeks to gain a mechanistic understanding of the cellular roles of Hsp110 chaperones by asking two specific questions: 1) How does Sse1 operate in partnership with the yeast Hsp70 Ssa1 and 2) How does Sse1 participate in signal transduction with Hsp90? In the first aim we will determine interaction sites and regulation of Hsp70 by Sse1 using purified chaperones, ultimately deciphering effects of Sse1 on protein folding in vitro. We will complement these experiments with in vivo assays to determine the contribution of Sse1 to Ssa1-dependent processes. In the second aim, we will determine both the stage at which Sse1 acts in the Hsp90 substrate folding cycle, and specific effects on substrate maturation using the model client protein glucocorticoid receptor. Finally we will apply these findings to understand how Sse1 in collaboration with Hsp90 is required for heat shock survival by modulating signaling through the Slt2 MAP kinase in the cell integrity pathway. These lines of investigation in yeast will serve as a model for predicting which processes Hsp110 may facilitate in mammalian cells.