Hsp104 and proteostasis yeast

在这个项目中，我们将继续努力，以阐明如何被困在不可逆的聚集蛋白质溶解和重折叠的AAA+ ATP酶Hsp 104与其他分子伴侣的合作。这些发现将在分子水平上对植物和微生物的胁迫生态学的基本理解产生影响，并提供可能导致蛋白质稳态网络工程的基础知识，从而创造出坚固的生物体，以提高作物产量，生物燃料合成和生物制药蛋白质生产。分子伴侣的标志性特性是它们能够防止由蛋白质折叠应力（如热）引起的未折叠蛋白质的聚集。分子伴侣通常识别错误折叠蛋白质中暴露的疏水氨基酸残基，保护它们免受引起聚集的非特异性相互作用。这项建议的重点是热休克蛋白104，一个真正的非传统分子伴侣。而不是防止聚集，热休克蛋白104是能够分散聚集体和恢复功能的蛋白质，否则无法挽回的陷阱。* 通过拟议的实验，我们将建立如何热休克蛋白104相互作用，在物理和功能上，与蛋白质稳定网络的其他成员。为了了解Hsp 104的生物学，我们将进行筛选，以确定依赖于Hsp 104重塑的蛋白质和途径。具体目标是：*1.我们将通过结合使用定点诱变、细菌Hsp 70和ClpB（Hsp 104的细菌直向同源物）之间底物交换的生化测定以及Hsp 70与Hsp 104结合及其对核苷酸交换和底物释放的影响的分析，来确定Hsp 104/Hsp 70相互作用在蛋白质解聚中的作用。2.我们将确定网站的重要性，不同的底物的结合热休克蛋白104。我们已经使用肽来确定Hsp 104中存在至少两个含有芳香族残基的肽的结合位点，并确定了酵母朊病毒蛋白Sup 35上的一个区域，该区域是通过Hsp 04过表达来治愈[PSI+]朊病毒所需的。我们将使用具有可光活化交联氨基酸衍生物的肽以高分辨率绘制氨基酸结合位点，所述可光活化交联氨基酸衍生物取代与Hsp 104结合的肽中的芳香族残基。有证据表明Sup 35直接与Hsp 104的N-末端结构域结合，这也是朊病毒治疗所必需的。我们将进行实验来测试这个想法，并使用NMR光谱学或晶体学绘制结合表面。3.我们将在非应激条件下确定Hsp 104的靶点。当我们表达一种工程化的热休克蛋白104，它将底物重定向到细菌蛋白酶进行降解时，酵母的生长速度明显更慢，并具有轻度延长的表型。这一观察结果表明，Hsp 104具有细胞靶点，当降解时，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Hsp 104依赖性降解影响的途径。此外，我们将在蛋白酶缺陷型ClpP寡聚体内捕获Hsp 104靶标。我们将确定Hsp 104的缺失是否会影响酵母中选定候选物的功能或定位（例如，当Hsp 104不存在时，它们可能倾向于聚集）或它们参与的途径的组分。底物的列表将用于深入了解哪些类型的分子受到热休克蛋白104依赖性重塑和这些蛋白质之间的共性将帮助我们了解更多关于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.