The functions and mechanism of SHSP degradation in Escherichia coli

大肠杆菌降解SHSP的功能和机制

• 批准号: 8132915
• 负责人: Ellen Frances Vieux
• 金额: $ 5.13万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2012-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8132915
• 关键词: Affinity Chromatography; Antibiotics; Bacteria; Beryllium; Binding; Biological Assay; Cell Aging; Cell Survival; Cells; Cessation of life; Client; Co-Immunoprecipitations; Complex; Crystallins; Data; Dependence; Disease; Elements; Escherichia coli; Fever; Gel; Goals; Heat shock proteins; High temperature of physical object; Human; In Vitro; Label; Lead; Life Cycle Stages; Molecular; Molecular Chaperones; Monitor; Organism; Peptide Hydrolases; Prevention; Protein Binding; Proteins; Proteolysis; Quality Control; Reagent; Recycling; Role; Site-Directed Mutagenesis; Specificity; Stress; Structure; Techniques; Temperature; Work; biological systems; endopeptidase La; experience; in vivo; insight; molecular recognition; oxidation; oxidative damage; prevent; protein aggregate; protein aggregation; protein degradation; protein misfolding; public health relevance; research study

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.

描述（由申请方提供）：所有生物体都经历可能导致错误折叠蛋白质的环境压力。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。该网络包括聚集预防蛋白、解聚气体、蛋白质重折叠伴侣和蛋白酶。当细胞暴露于升高的温度时，这些蛋白质中的许多被上调，因此被称为热休克蛋白（HSP）。普遍存在的小HSP有助于维持受损和/或易于聚集的蛋白质处于允许它们与重折叠分子伴侣相互作用并避免形成大的不溶性聚集体的状态。贝克实验室最近的数据也表明，E.大肠杆菌小热休克蛋白（Ibp）被蛋白酶Lon和ClpAP降解。本文探讨了E. coli sHSPs及其新发现的降解目的和机制。第一个目标描述了如何在E.杆菌我将使用coIP，western，亲和纯化，2D凝胶分析和MS/MS的组合来实现这一目标。我将鉴定在缺乏降解这些蛋白质的蛋白酶中的一种或两种的菌株中显示与Ibp相关的蛋白质。有了大量的IBP客户端，我们也许能够识别用于分子识别的共同元素。这种分析也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-客户端复合物将用于剖析sHSPs，受损蛋白质，重折叠伴侣和蛋白酶之间的相互作用。目标是确定在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确定客户端是否被重折叠或降解。还将进行补充体内实验。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，sHSP如何被蛋白酶识别尚不清楚。最近的研究结果表明，Lon识别sHSPs的保守1-晶体蛋白结构域。使用生物物理技术和定点突变，我将系统地剖析负责识别的sHSP蛋白酶的分子决定因素。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，或者是否被一级结构基序识别，将有助于我们了解蛋白酶对sHSP的特异性，并可能突出底物-蛋白酶相互作用的不寻常模式。了解Ibp-客户端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并最终导致细胞衰老和死亡的机制的新见解。 公共卫生关系：由环境应力如氧化和高温形成的聚集体可导致人类细胞活力降低和疾病。了解生物系统如何防止大量蛋白质聚集以及受损蛋白质如何回收利用，可能会导致蛋白质聚集体引起的疾病的新疗法，或为抗生素提供新的靶点，使细菌无法在发烧等轻微压力下存活。