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

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。本提案的目标是通过普遍保守的GroEL （Hsp60）伴侣机器来理解蛋白质折叠的机制。我们的实验系统包括大肠杆菌及其t4样毒力噬菌体。大肠杆菌GroEL伴侣机对细菌生存至关重要，由两个成员组成，GroEL （Hsp60）伴侣和GroES （Hsp10）伴侣。t4样噬菌体依赖GroEL而不是GroES来折叠Gp23衣壳蛋白，Gp23衣壳蛋白是噬菌体头的主要成分，是噬菌体生长限制和繁殖所必需的。这些噬菌体编码Gp31， Gp31是一种新型的GroEL伴侣蛋白，是GroES的远同源物，对Gp23的正确折叠至关重要。噬菌体对宿主编码的GroEL及其自身的Gp31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检查GroEL/Gp31，并且像观察斑块的形成一样简单。利用这一基本的生死遗传系统，我们鉴定出了Gp39.2，这是一种由t4样噬菌体编码的GroEL伴侣调节因子。然而，与Gp31相反，Gp39.2对于实验室t4样噬菌体生长通常是必不可少的，只有在某些groEL突变体宿主上才能成为噬菌体繁殖的绝对必要条件。有趣的是，Gp39.2过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和Gp39.2对GroEL机器的独特调控作用，提出了一系列的遗传和生化研究。遗传实验包括分离宿主和噬菌体突变体及其代偿突变体。生化实验包括野生型和突变型GroEL、Gp31和Gp39.2对Gp23的体外折叠。对GroEL突变体Gp31/GroEL， GroEL/Gp39.2和其他复合物的冷冻电镜成像和x射线晶体学的使用将有助于破译Gp31和Gp39.2使GroEL伴侣蛋白折叠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性，根据人类机器支持大肠杆菌生长的事实判断，我们简单的大肠杆菌/噬菌体系统提供的机制见解将立即应用于高等生物的Hsp60机器。

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.