Protein folding by the universally conserved GroEL (Hsp60) chaperone machine and

通过普遍保守的 GroEL (Hsp60) 分子伴侣机器进行蛋白质折叠

• 批准号: 8258273
• 负责人: Costa Georgopoulos
• 金额: $ 28.86万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8258273
• 关键词: Address; Alzheimer' s Disease; Amino Acids; Bacteriophage T4; Bacteriophages; Binding; Biochemical; Biological; Biological Process; Capsid; Capsid Proteins; Cessation of life; Complement; Complex; Coupled; Crystallography; Dependence; Distant; Equilibrium; Escherichia coli; Evolution; Family; Fostering; Future; Genetic; Goals; Growth; Head; Health; Homologous Gene; Human; Image; In Vitro; Laboratories; Lead; Learning; Life; Life Style; Mitochondria; Modeling; Molecular Chaperones; Mutation; Nature; Neurodegenerative Disorders; Organism; Parkinson Disease; Poisoning; Proteins; Refractory; Regulation; Relative (related person); Resolution; Role; Series; Specificity; Substrate Specificity; System; Variant; Virulent; X-Ray Crystallography; bactericide; design; genetic selection; insight; member; mutant; novel; overexpression; particle; polypeptide; pressure; protein aggregation; protein folding; research study

DESCRIPTION (provided by applicant): To address the problem of protein folding, organisms have evolved molecular chaperone machines whose primary role is to foster nascent polypeptide folding by limiting aggregation. The goal of this proposal is to understand the mechanism of protein folding by the universally conserved GroEL (Hsp60) chaperone machine. Our experimental system comprises E. coli and its T4-like virulent phages. The E. coli GroEL chaperone machine is essential for bacterial viability and consists of two members, the GroEL (Hsp60) chaperone and the GroES (Hsp10) cochaperone. The T4-like phages are dependent on GroEL, but not GroES, to fold their Gp23 capsid protein, the major component of the phage head, growth-limiting and essential for phage propagation. These phages encode Gp31, a novel GroEL cochaperone that is a distant homolog of GroES and is essential for the correct folding of Gp23. The strict dependence of the phage on the host-encoded GroEL and its own Gp31 cochaperone allows us to examine GroEL/Gp31 in a manner that is not always possible in more complex systems, and is as simple as observing the formation of a plaque. Using this basic life or death genetic system, we have identified Gp39.2, a GroEL chaperone regulator also encoded by the T4-like phages. However, in contrast to Gp31, Gp39.2 is normally dispensable for laboratory T4-like phage growth, becoming absolutely essential for phage propagation only on certain groEL mutant hosts. Interestingly, Gp39.2 overexpression is bactericidal to E. coli, possibly by poisoning the GroEL machine. A series of genetic and biochemical studies are proposed to understand the unique regulation of the GroEL machine by the phage-encoded Gp31 cochaperone and Gp39.2. The genetic experiments include isolation of host and phage mutants and their compensatory mutations. The biochemical experiments include the in vitro folding of Gp23 by wild type and mutant forms of GroEL, Gp31 and Gp39.2. The use of cryo-EM imaging and X-ray crystallography of our GroEL mutants, Gp31/GroEL, GroEL/Gp39.2, and other complexes will help decipher the unique mechanisms by which Gp31 and Gp39.2 enable the GroEL chaperone to fold its Gp23 capsid substrate. Due to the extreme conservation of the Hsp60/Hsp10 machine across biological kingdoms, judged by the fact that the human machine supports E. coli growth, mechanistic insights provided by our simple E. coli/phage system will find immediate application with the Hsp60 machine of higher organisms. PUBLIC HEALTH RELEVANCE: The problem of protein aggregation is universal among all living organisms. In humans it can lead to various neurodegenerative diseases, including Parkinson's and Alzheimer's. The universally conserved proteins called "chaperone machines" evolved to protect organisms from protein aggregation. Thus, our studies with the essential GroEL (Hsp60) chaperone machine in Escherichia coli will find immediate application to the mechanism of action of the essential human Hsp60 homolog, residing in mitochondria.

描述（由申请人提供）：为了解决蛋白质折叠的问题，生物体已经进化出分子伴侣机器，其主要作用是通过限制聚集来促进新生多肽折叠。该建议的目的是了解普遍保守的GroEL（Hsp 60）分子伴侣机器的蛋白质折叠机制。我们的实验系统包括E. coli及其T4类毒力菌株。急诊大肠杆菌GroEL分子伴侣是细菌生存所必需的，由GroEL（Hsp 60）分子伴侣和GroES（Hsp 10）辅助分子伴侣两部分组成。T4样噬菌体依赖于GroEL而不是GroES来折叠它们的Gp 23衣壳蛋白，Gp 23衣壳蛋白是噬菌体头部的主要组分，是生长限制性的并且是噬菌体繁殖所必需的。Gp 31是一种新的GroEL伴侣蛋白，是GroES的远亲同源物，对Gp 23的正确折叠至关重要。噬菌体对宿主编码的GroEL及其自身的Gp 31辅助分子伴侣的严格依赖性使我们能够以在更复杂的系统中并不总是可能的方式来检查GroEL/Gp 31，并且与观察斑块的形成一样简单。使用这种基本的生命或死亡遗传系统，我们已经确定了Gp39.2，一种GroEL伴侣调节剂，也由T4样蛋白编码。然而，与Gp 31相反，Gp39.2通常不适合实验室T4样噬菌体生长，仅在某些groEL突变宿主上成为噬菌体繁殖绝对必需的。有趣的是，Gp39.2过表达对E.大肠杆菌，可能是通过毒害GroEL机器。一系列的遗传和生化研究提出了理解的独特的调节GroEL机的噬菌体编码的Gp 31辅伴侣蛋白和Gp39.2。遗传学实验包括宿主和噬菌体突变体的分离及其补偿突变。生化实验包括Gp 23通过GroEL、Gp 31和Gp39.2的野生型和突变形式的体外折叠。我们的GroEL突变体，Gp 31/GroEL，GroEL/Gp39.2和其他复合物的cryo-EM成像和X射线晶体学的使用将有助于破译Gp 31和Gp39.2使GroEL伴侣蛋白折叠其Gp 23衣壳底物的独特机制。由于Hsp 60/Hsp 10机器在生物界中的极端保守性，从人类机器支持E.大肠杆菌生长，我们的简单的E.大肠杆菌/噬菌体系统将立即应用于高等生物的Hsp 60机器。 公共卫生相关性：蛋白质聚集问题在所有生物体中是普遍存在的。在人类中，它可以导致各种神经退行性疾病，包括帕金森氏症和阿尔茨海默氏症。被称为“伴侣机器”的普遍保守的蛋白质进化以保护生物体免受蛋白质聚集。因此，我们的研究与必要的GroEL（热休克蛋白60）分子伴侣机器在大肠杆菌中将立即应用于基本的人类热休克蛋白60同源物，居住在线粒体的作用机制。