Hsp104 and proteostasis yeast

在这个项目中，我们将继续努力阐明被困在不可逆聚集体中的蛋白质是如何被AAA+ atp酶Hsp104溶解和折叠的，并与其他分子伴侣合作。这一发现将有助于在分子水平上对植物和微生物的胁迫生态进行基本理解，并为蛋白质稳态网络的工程设计提供基础知识，从而创造出用于提高作物产量、生物燃料合成和生物制药蛋白质生产的坚固生物体。分子伴侣的标志性特性是它们能够防止由于蛋白质折叠压力（如热量）而导致的未折叠蛋白质聚集。分子伴侣通常识别暴露在错误折叠蛋白质中的疏水氨基酸残基，保护它们免受引起聚集的非特异性相互作用。这项提议的重点是Hsp104，一种真正非传统的分子伴侣。而不是阻止聚集，Hsp104能够分散聚集和恢复蛋白质的功能，否则不可挽回地被困。通过提出的实验，我们将确定Hsp104如何在物理和功能上与蛋白质静止网络的其他成员相互作用。为了了解Hsp104的生物学，我们将进行筛选，以确定依赖于Hsp104重塑的蛋白质和途径。具体目标是：1。我们将通过结合位点定向诱变、细菌Hsp70与细菌同源物ClpB之间底物交换的生化实验、Hsp70与Hsp104结合及其对核苷酸交换和底物释放的影响来确定Hsp104/Hsp70相互作用在蛋白质分解中的作用。我们将确定Hsp104结合不同底物的重要位点。我们使用多肽来确定Hsp104中含有芳香残基的多肽至少存在两个结合位点，并在酵母朊病毒蛋白Sup35上确定了一个区域，该区域是通过Hsp04过表达固化[PSI+]朊病毒所必需的。我们将使用具有可光激活的交联氨基酸衍生物的肽，以替代与Hsp104结合的肽中的芳香残基，以高分辨率绘制氨基酸结合位点。有证据表明，Sup35直接结合到Hsp104的n端结构域，这也是朊病毒固化所必需的。我们将进行实验来验证这一想法，并使用核磁共振波谱或晶体学绘制结合表面。我们将在非应激条件下确定Hsp104的靶点。当我们表达一种工程化的Hsp104，将底物重定向到细菌蛋白酶中进行降解时，酵母的生长速度明显减慢，并具有轻度拉长的表型。这一观察结果表明，Hsp104具有细胞靶点，当其降解时，会导致极化生长和/或细胞周期的缺陷。我们建议使用合成基因阵列进行筛选，以确定可能受hsp104依赖性降解影响的途径。此外，我们将在蛋白酶缺乏的ClpP低聚物中捕获Hsp104靶点。我们将确定Hsp104的缺失是否会影响酵母中选择候选基因的功能或定位（例如，当Hsp104缺失时，它们可能倾向于聚集）或它们参与的途径的组成部分。一份底物清单将用于深入了解哪些类型的分子会受到hsp104依赖性重塑的影响，这些蛋白质之间的共性将帮助我们更多地了解底物选择的结构决定因素。

In this project we will pursue an ongoing effort to elucidate how proteins trapped in irreversible aggregates are solubilized and refolded by the AAA+ ATPase Hsp104 in cooperation with other molecular chaperones. The findings will have implications for the basic understanding the stress ecology of plants and microbes at the molecular level, as well as providing fundamental knowledge that could lead to the engineering of protein homeostasis networks and thereby create rugged organisms for enhanced crop yield, biofuel synthesis, and biopharmaceutical protein production.The hallmark property of molecular chaperones is their ability to prevent the aggregation of unfolded proteins that result from protein folding stresses like heat. Molecular chaperones generally recognize hydrophobic amino acid residues exposed in misfolded proteins, shielding them from non-specific interactions that cause aggregation. The focus of this proposal is Hsp104, a truly unconventional molecular chaperone. Rather than preventing aggregation, Hsp104 is able to disperse aggregates and restore function to proteins that are otherwise irretrievably trapped. Through the proposed experiments we will establish how Hsp104 interacts, both physically and functionally, with other members of a proteostasis network. To understand the biology of Hsp104 we will conduct a screen to identify proteins and pathways that are dependent on remodelling by Hsp104.The specific aims are: 1. We will determine the role of the Hsp104/Hsp70 interaction in protein disaggregation by using a combination of site-directed mutagenesis, biochemical assays for substrate exchange between bacterial Hsp70 and ClpB the bacterial orthologue of Hsp104, and analysis of Hsp70 binding to Hsp104 and its influence on nucleotide exchange and substrate release.2. We will identify sites important for the binding of distinct substrates by Hsp104. We have used peptides to determine the existence of at least two binding sites in Hsp104 for peptides containing aromatic residues and to identify a region on the yeast prion protein Sup35 that is required for the curing of the [PSI+] prion by Hsp04 overexpression. We will map the amino acid binding site with high resolution using peptides with a photoactivatable crosslinking amino acid derivative that substitutes for aromatic residues in peptide binding to Hsp104. Evidence suggests that Sup35 binds directly to the N-terminal domain of Hsp104, which is also required for prion curing. We will conduct experiments to test this idea and map the binding surface using NMR spectroscopy or crystallography.3. We will identify targets of Hsp104 under non-stress conditions. When we express an engineered an Hsp104 that redirects substrates to a bacterial protease for degradation, yeast grow significantly more slowly and have a mildly elongated phenotype. This observation suggests that Hsp104 has cellular targets that, when degraded, result in defects in polrarized growth and/or cell cycle. We propose to conduct a screen using the synthetic gene array to identify pathways that may be affected by Hsp104-dependent degradation. Furthermore, we will trap Hsp104 targets inside a protease deficient ClpP oligomer. We will determine if deletion of Hsp104 affects the function or localization of select candidates in yeast (they may, for example, tend to aggregate when Hsp104 is absent) or the components of pathways in which they participate. A list of substrates will be used to gain insight into what types of molecules are subject to Hsp104-dependent remodeling and commonalities among these proteins will help us learn more about the structural determinants of substrate selection.