Regulation of the Heat Shock Response in E. Coli

大肠杆菌热激反应的调节

• 批准号: 6867630
• 负责人: CAROL Anne GROSS
• 金额: $ 46.2万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6867630
• 关键词: Escherichia coli; bacterial genetics; bacterial proteins; biological signal transduction; chemical structure function; computer program /software; endopeptidases; enzyme activity; enzyme mechanism; gene expression; genetic mapping; genetic transcription; heat shock proteins; high throughput technology; molecular biology; molecular chaperones; oligonucleotides; polymerase chain reaction; protein degradation; protein folding; western blottings

DESCRIPTION (provided by applicant): Effective mechanisms to combat stress are vital to all living organisms. We study the fundamental design principles of the signal transduction cascades that sense temperature stress in E. coli. Interestingly, cells compartmentalize their stress responses. The cytoplasmic heat shock response (HSR), controlled by sigma32, regulates expression of the universally conserved heat shock proteins (hsps). The envelope stress response (ESR), controlled by sigmaE, regulates expression of proteins required to maintain envelope integrity. The proposed studies will define the cellular inputs that negatively regulate the HSR and define the protease cascade that regulates the ESR by: 1. Determining how chaperone-mediated inputs to the Hsr are partitioned between GroEL/S and DnaK/J and the features of sigma32 making it amenable to chaperone mediated inactivation. 2. Testing whether FtsH protease plays a regulatory role in the HSR by determining when changes in substrate occupancy of FtsH alter sigma32 activity. 3. Finding and characterizing the factor necessary to obtain rapid degradation of sigma32 by FtsH protease in vitro. 4. Determining how RseB facilitates sequential degradation of RseA, the sigmaE-specific antisigma by preventing inappropriate cleavage of intact RseA by YaeL, the second protease in the pathway. 5. Testing whether a DegS-YaeL machine mediates concerted degradation of RseA. 6. Identifying the proteases that complete RseA degradation and demonstrating their function in vitro. 7. Identifying additional regulatory inputs to the ESR to understand the broader role of this response and whether any signals work at different steps in the protease cascade. Given the universality of these responses and the increasing evidence that many organisms choose similar general solutions to deal with stress, our studies of these signal transduction cascades have broad applicability to the understanding of the successful design of such circuits in all organisms. In addition, the proteases carrying out these responses, which are most easily studied in E. coli, are poorly understood, but widely distributed, often mediating stress responses in organisms ranging from microbial pathogens to man. Finally, studies on the heat shock response has direct medical relevance as T cells reactive to heat shock proteins may provide a first line of defense against infection, heat shock proteins are often up-regulated in cancer and the increasing variety of protein folding diseases makes it imperative to understand how the protein folding state of the cell is maintained.

描述（由申请人提供）：对抗压力的有效机制对所有生物体都至关重要。我们研究了E.杆菌有趣的是，细胞将它们的压力反应区分开来。细胞质热休克反应（HSR）由sigma 32控制，调节普遍保守的热休克蛋白（hsps）的表达。由sigmaE控制的包膜应激反应（ESR）调节维持包膜完整性所需的蛋白质的表达。拟议的研究将定义负调节HSR的细胞输入，并定义调节ESR的蛋白酶级联反应：1。确定分子伴侣介导的Hsr输入如何在GroEL/S和DnaK/J之间分配，以及σ 32的特征使其适合分子伴侣介导的失活。2.通过测定FtsH底物占有率的变化何时改变σ 32活性来测试FtsH蛋白酶是否在HSR中起调节作用。3.发现并表征在体外通过FtsH蛋白酶获得快速降解σ 32所必需的因素。4.确定RseB如何通过防止YaeL（途径中的第二种蛋白酶）对完整RseA的不适当切割来促进RseA（σ E特异性抗σ）的顺序降解。5.测试DegS-YaeL机器是否介导RseA的协同降解。6.鉴定完成RseA降解的蛋白酶并在体外证明其功能。7.确定ESR的其他调节输入，以了解这种反应的更广泛作用，以及是否有任何信号在蛋白酶级联反应的不同步骤中起作用。鉴于这些反应的普遍性和越来越多的证据表明，许多生物体选择类似的一般解决方案来应对压力，我们对这些信号转导级联的研究具有广泛的适用性，以了解所有生物体中这种电路的成功设计。此外，进行这些反应的蛋白酶，这是最容易在大肠杆菌中研究。最后，对热休克反应的研究具有直接的医学相关性，因为对热休克蛋白反应的T细胞可以提供对抗感染的第一道防线，热休克蛋白通常是由于蛋白质在癌症中的调节以及蛋白质折叠疾病的种类不断增加，因此必须了解细胞的蛋白质折叠状态是如何维持的。