Hsp110 protein chaperone function in yeast

描述（由申请人提供）：在人类中，蛋白质折叠缺陷可导致神经退行性疾病，如阿尔茨海默氏症、帕金森病和亨廷顿病以及使人衰弱的共济失调。70 kD热休克蛋白（Hsp70）是一个普遍存在的蛋白质伴侣蛋白家族，参与蛋白质生物发生、修复和降解等蛋白质稳态的各个方面。Hsp70的活性受两种辅助因子的控制；含J结构域的蛋白激活ATP水解步骤，导致高亲和力底物结合，而核苷酸交换因子（nef）促进ADP解离和多肽释放。从酵母到人类的真核生物表达三种结构多样的胞质nef家族（Hsp110（酵母中的Sse1/2）， HspBP1 （Fes1）和Bag-domain (Snl1)），它们具有基本相同的生化功能。这些蛋白对hsp70依赖性活动的独特贡献在任何生物系统中都是未知的。本提案的主要目标是确定这些nef如何与细胞质Hsp70伴侣蛋白合作介导真核细胞中的蛋白质静止。我们假设，虽然nef具有刺激Hsp70活性的共同能力，但它们与细胞质Hsp70的Ssa和Ssb家族相互作用以促进蛋白质折叠和修复的差异。此外，我们假设Bag同源Snl1同时招募Hsp70和核糖体，以促进内质网（ER）的蛋白质生物生成。提出了三条调查路线来检验这些假设。在第一个目标中，我们将确定表面上主要的NEF Hsp110如何整合到细胞质NEF网络中，以促进蛋白质的生物发生和修复。这一目标的一个主要组成部分将是遗传和细胞生物学实验，以评估Hsp110底物结合域对Hsp70依赖性蛋白折叠的贡献。与已发表的Fes1在体外结合Ssa和Ssb的结果相反，我们已经获得了Fes1在体内仅与Ssa相互作用的初步证据。在Aim 2中，我们将解决这些相互矛盾的发现，并确定Fes1-Hsp70特异性的机制。最后，我们发现内质网膜相关的Snl1除了与Hsp70结合外，还与核糖体结合。目的3将侧重于确定Hsp70 NEF对翻译机制的膜募集的生理意义。这些研究首次通过遗传、生化和基因组方法对胞质Hsp70 nef进行了全面分析。由于Hsp70伴侣网络的这些和其他组成部分在真核生物中高度保守，该结果将直接适用于理解Hsp70 nef在人类细胞中蛋白质生物发生和质量控制中的作用。

DESCRIPTION (provided by applicant): In humans, defects in protein folding can lead to neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's disease and debilitating forms of ataxia. The 70 kD heat-shock proteins (Hsp70) are a ubiquitous family of protein chaperones involved in all aspects of protein homeostasis including protein biogenesis, repair and degradation. Hsp70 activity is governed by two types of cofactors; J domain-containing proteins activate the ATP hydrolysis step leading to high affinity substrate binding, while nucleotide exchange factors (NEFs) promote ADP dissociation and polypeptide release. Eukaryotes from yeast to humans express three recently described families of structurally diverse cytosolic NEFs (Hsp110 (Sse1/2 in yeast), HspBP1 (Fes1), and Bag-domain (Snl1)) that perform essentially the same biochemical function. The distinct contributions these proteins make to Hsp70-dependent activities are unknown in any biological system. The primary goal of this proposal is to determine how these NEFs partner with cytosolic Hsp70 chaperones to mediate proteostasis in eukaryotic cells. We hypothesize that while the NEFs share a common ability to stimulate Hsp70 activity, they differentially interact with the Ssa and Ssb families of cytosolic Hsp70 to promote protein folding and repair. In addition, we hypothesize that the Bag homolog Snl1 simultaneously recruits Hsp70 and the ribosome to promote protein biogenesis at the endoplasmic reticulum (ER). Three lines of investigation are proposed to test these hypotheses. In the first aim, we will determine how the apparently principal NEF Hsp110 is integrated into the cytosolic NEF network to promote protein biogenesis and repair. A major component of this aim will be genetic and cell biological experiments to assess the contribution of the Hsp110 substrate binding domain to Hsp70- dependent protein folding in vivo. In contrast to published results that Fes1 binds Ssa and Ssb in vitro, we have obtained preliminary evidence that Fes1 interacts solely with Ssa in vivo. In Aim 2 we will resolve these conflicting findings and determine the mechanism of Fes1-Hsp70 specificity. Lastly, we have discovered that the ER membrane-associated Snl1 binds the ribosome in addition to Hsp70. Aim 3 will be focused on determining the physiological significance of membrane recruitment of the translation machinery by an Hsp70 NEF. These studies represent the first comprehensive analysis of cytosolic Hsp70 NEFs by genetic, biochemical, and genomic approaches. Due to the high degree of conservation of these and other components of the Hsp70 chaperone network in eukaryotes, the results will be directly applicable to understanding the roles of Hsp70 NEFs in protein biogenesis and quality control in human cells.