The functions and mechanism of SHSP degradation in Escherichia coli

描述(由申请人提供)：所有生物都会经历环境压力，这可能会导致蛋白质错误折叠。蛋白质质量控制网络有助于保护细胞免受受损蛋白质的影响。这个网络包括防止聚集的蛋白质、解聚酶、蛋白质复性伴侣和蛋白水解酶。当细胞暴露在更高的温度下时，这些蛋白中的许多都会上调，因此被称为热休克蛋白(HSP)。无处不在的小热休克蛋白有助于维持受损的和/或易于聚集的蛋白质处于一种状态，使它们能够与复性伴侣相互作用，避免形成大的不溶性聚集体。贝克实验室的最新数据也表明，大肠杆菌小分子热休克蛋白(IBP)可被Lon和ClpAP蛋白酶降解。这项建议探讨了大肠杆菌sHSPs的功能及其新发现的降解目的和机制。第一个目标描述了如何阐明在大肠杆菌中作为SHSP的强大客户的蛋白质。为了达到这一目标，我将使用coips、westerns、亲和纯化、二维凝胶分析和MS/MS的组合。我将确定在缺乏一种或两种降解这些蛋白质的蛋白酶的菌株中，与IBPS相关的蛋白质增加。有了一大组IBP客户，我们也许能够识别用于分子识别的共同元素。这一分析还将增加我们对蛋白酶如何与蛋白质质量控制网络的其他元素相互作用的理解。在第二个目标中，IBP-客户复合体将被用来剖析sHSPs、受损蛋白质、复性伴侣和蛋白酶之间的相互作用。目标是确定IBP降解过程中IBP结合的客户蛋白的命运。我将使用放射性标记的客户端和体外IBPS来确定客户端是否被重新折叠或降解。还将进行补充的活体实验。这项工作将有助于确定sHSP和sHSP周转在更大的蛋白质质量控制网络中的作用。最后，sHSPs是如何被蛋白酶识别的还是个未知数。最近的研究结果表明，Lon识别sHSPs保守的1-晶体蛋白结构域。利用生物物理技术和定点突变，我将系统地剖析负责识别蛋白酶识别SHSP的分子决定因素。了解高度保守的折叠1-晶体蛋白结构域是否被蛋白酶识别，或者是否识别了一个一级结构基序，将有助于我们理解蛋白酶对SHSP的特异性，并可能突出底物-蛋白酶相互作用的一种不寻常的模式。了解IBP-客户相互作用的基本原理以及IBP-蛋白酶识别可能会导致对导致与蛋白质聚集相关的疾病，最终导致细胞衰老和死亡的机制的新见解。 与公共卫生相关：氧化和高温等环境压力形成的聚集体可能导致细胞活性下降和人类疾病。了解生物系统如何防止大规模蛋白质聚集，以及受损蛋白质是如何循环利用的，可能会导致蛋白质聚集引起的疾病的新疗法，或者为抗生素提供新的靶点，从而使细菌无法在轻微的压力下生存，如发烧。

DESCRIPTION (provided by applicant): All organisms experience environmental stress that can lead to misfolded proteins. The protein quality-control network helps to protect cells from the effects of damaged proteins. This network includes aggregation-prevention proteins, dissaggregases, protein-refolding chaperons, and proteases. Many of these proteins are upregulated when cells are exposed to increased temperatures, and are thus called heat-shock proteins (HSPs). The ubiquitous small HSPs help maintain damaged and/or aggregation-prone proteins in a state that allows them to interact with refolding chaperones and avoid forming large insoluble aggregates. Recent data from the Baker lab has also shown that that E. coli small HSPs (Ibps) are degraded by the proteases Lon and ClpAP. This proposal explores the functions of the E. coli sHSPs and the purpose and mechanism of their newly discovered degradation. The first aim describes how to elucidate proteins that are robust clients of sHSP in E. coli. I will use a combination of coIPs, westerns, affinity purification, 2D gel analysis and MS/MS to achieve this goal. I will identify proteins that show an increase in association with Ibps in strains lacking one or both of the proteases that degrade these proteins. With a large set of Ibp-clients we maybe able to identify common elements that are used for molecular recognition. This analysis will also increase our understanding of how proteases interact with the other elements of the protein quality-control network. In the second aim Ibp-client complexes will be used to dissect the interplay between sHSPs, damaged proteins, refolding chaperons and proteases. The goal is to determine the fate of Ibp-bound client proteins during Ibp degradation. I will determine if the clients are refolded, or degraded using radioactively labeled clients and Ibps in vitro. Complementary in vivo experiments will be done as well. This work will help define the role of sHSPs and sHSP turnover in the larger protein-quality control network. Finally, how sHSPs are recognized by proteases is unknown. Recent results suggest that Lon recognizes the conserved 1-crystallin domain of sHSPs. Using biophysical techniques and site-directed mutagenesis I will systematically dissect the molecular determinants responsible for recognition of the sHSP by proteases. Understanding if the highly conserved folded 1-crystallin domain is recognized by proteases or if a primary structural motif is recognized will help us understand the specificity of proteases for the sHSP and may highlight an unusual mode of substrate-protease interaction. Understanding the basic principles of Ibp-client interactions as well as Ibp-protease recognition may lead to new insights into the mechanisms that lead to the diseases associated with protein aggregation and ultimately, cellular aging and death. PUBLIC HEALTH RELEVANCE: Formation of aggregates from environmental stresses such as oxidation and high temperature can lead decreased viability of cells and disease in humans. Understanding how biological systems prevent massive protein aggregation and how damaged proteins are recycled may lead to new treatments of disease caused by protein aggregates, or provide new targets for antibiotics, so that bacteria cannot survive mild stresses, such as fever.