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

描述(申请人提供)：为了解决蛋白质折叠的问题，生物进化出了分子伴侣机器，其主要作用是通过限制聚集来促进新生多肽折叠。本提案的目的是了解普遍保守的GroEL(Hsp60)伴侣机器折叠蛋白质的机制。我们的实验系统由大肠杆菌及其类似T4的强毒噬菌体组成。大肠杆菌GroEL伴侣对细菌的生存至关重要，它由两个成员组成，即GroEL(Hsp60)伴侣和Groes(Hsp10)辅助伴侣。T4类噬菌体依赖于GroEL而不是GroE来折叠它们的Gp23衣壳蛋白，Gp23衣壳蛋白是噬菌体头部的主要成分，限制生长，对噬菌体繁殖至关重要。这些噬菌体编码Gp31，这是一种新的GroEL辅伴侣，是Groes的远程同源物，对于Gp23的正确折叠是必不可少的。噬菌体对宿主编码的GroEL及其自身的Gp31辅伴侣的严格依赖使我们能够以一种在更复杂的系统中并不总是可能的方式来检查GroEL/Gp31，并且就像观察菌斑的形成一样简单。利用这个基本的生死遗传系统，我们已经鉴定出Gp39.2，一个GroEL伴侣调节因子，也由T4样噬菌体编码。然而，与Gp31相比，Gp39.2对于实验室T4样噬菌体的生长通常是必不可少的，只有在某些GroEL突变宿主上才成为噬菌体繁殖的绝对必需。有趣的是，Gp39.2的过度表达对大肠杆菌具有杀菌作用，可能是通过毒害GroEL机器。为了了解噬菌体编码的Gp31辅伴侣和Gp39.2对GroEL机器的独特调控，我们提出了一系列的遗传和生化研究。基因实验包括分离寄主和噬菌体突变体及其补偿突变。生化实验包括用野生型和突变型GroEL、Gp31和Gp39.2体外折叠Gp23。使用我们的GroEL突变体Gp31/GroEL、GroEL/Gp39.2和其他复合体的冷冻-EM成像和X射线结晶学将有助于破译Gp31和Gp39.2使GroEL伴侣能够折叠其Gp23衣壳底物的独特机制。由于HSP60/HSP10机器在生物界的极端保守，根据人类机器支持大肠杆菌生长的事实，我们简单的大肠杆菌/噬菌体系统提供的机械洞察力将立即应用于高等生物的HSP60机器。 与公共卫生相关：蛋白质聚集的问题在所有生物中都是普遍存在的。在人类中，它会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。这种普遍保守的蛋白质被称为“伴侣机器”，进化出来是为了保护生物免受蛋白质聚集的影响。因此，我们在大肠杆菌中使用Essential GroEL(Hsp60)伴侣机器的研究将立即应用于位于线粒体中的必要的人类Hsp60同源物的作用机制。

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.