Hsp110 protein chaperone function in yeast

Hsp110 蛋白伴侣在酵母中的功能

• 批准号: 7090987
• 负责人: KEVIN ANTHONY MORANO
• 金额: $ 25.26万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7090987
• 关键词: SDS polyacrylamide gel electrophoresis; adenosinetriphosphatase; biological signal transduction; corticosteroid receptors; enzyme activity; fungal proteins; heat shock proteins; immunoprecipitation; mitogen activated protein kinase; molecular chaperones; protein folding; protein protein interaction; protein purification; protein structure function

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.

描述（由申请人提供）：热休克蛋白（Hsps）在细胞生物学中起双重作用；它们是抵御细胞毒性应激的第一道防线，并且在正常生长条件下紧密地整合到信号传导和调节途径中。热休克蛋白的一个子集，包括Hsp70和Hsp90，作为分子伴侣，可能在底物蛋白生命周期的多个阶段都需要，从生物发生、定位、稳定到激活和降解。蛋白伴侣蛋白在许多病理生理条件下被诱导，包括缺血和肿瘤发生，并且已知可以促进癌蛋白（如v-src激酶和p53肿瘤抑制因子）的稳定性和活性。Hsp110类伴侣蛋白是一种鲜为人知的Hsp70亲缘蛋白，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同型。我们的长期目标是阐明这个伴侣家族的细胞作用。烘焙酵母Hsp110同源物由SSE1和SSE2基因编码，对其功能知之甚少。我们发现Sse1在体内以异源二聚体的形式与胞质Hsp70s Ssa和Ssb存在，并且Sse1是Hsp90伴侣系统信号转导活性所必需的。因此，我们假设Sse1以及哺乳动物Hsp110伴侣可能主要作为Hsp70活性的调节剂起作用。本研究旨在通过提出两个具体问题来了解Hsp110伴侣的细胞作用机制：1)Sse1如何与酵母Hsp70 Ssa1合作，2)Sse1如何参与与Hsp90的信号转导。在第一个目标中，我们将使用纯化的伴侣蛋白确定Sse1对Hsp70的相互作用位点和调控，最终破译Sse1对体外蛋白折叠的影响。我们将用体内实验来补充这些实验，以确定Sse1对ssa1依赖性过程的贡献。在第二个目标中，我们将确定Sse1在Hsp90底物折叠周期中的作用阶段，以及使用模型客户蛋白糖皮质激素受体对底物成熟的具体影响。最后，我们将应用这些发现来了解Sse1如何与Hsp90合作，通过调节细胞完整性通路中Slt2 MAP激酶的信号来实现热休克存活。这些在酵母中的研究将作为预测Hsp110在哺乳动物细胞中可能促进哪些过程的模型。