Function of the Six ATPases in the Yeast 19S Particle

描述（由申请人提供）：真核生物中泛素/蛋白酶体系统的阐明极大地促进了我们对细胞如何工作的理解。然而，26 S蛋白酶体功能的许多机制特征仍不清楚。一个关键问题涉及在蛋白酶体的19 S调节颗粒中发现的六种AAA ATP酶的功能。尽管这些蛋白质在蛋白酶体功能中的重要性，但很少有研究直接涉及ATP酶活性在蛋白酶体功能中的特定作用。该提案的目标是应用一种新的“化学遗传学”方法，使我们能够系统地推断19 S中每个ATP酶的分子功能。抑制剂是通过用大体积取代基化学修饰嘌呤衍生物来产生的，以补充靶ATP酶的ATP结合位点的合理设计的扩大。虽然“类似物敏感性”ATP酶可以结合正常ATP或ATP类似物，但野生型ATP酶在ATP类似物存在下不受影响，从而允许在体外和体内测定中特异性抑制给定的ATP酶。最初，我们将描述酵母19 S ATP酶Rpt 1和Rpt 5在蛋白酶体功能中的作用，最终目标是描述所有六种ATP酶。

DESCRIPTION (provided by applicant): The elucidation of the ubiquitin/proteasome system in eukaryotes has greatly contributed to our understanding of how cells work. However, many mechanistic features of how 26S proteasomes function remain unclear. One key question involves the function of the six AAA ATPases found in the 19S regulatory particle of the proteasome. Despite the importance of these proteins in proteasome function, very few studies have directly addressed the specific roles of ATPase activity in proteasome function. The goal of this proposal is to apply a novel 'chemical genetics' approach that will enable us to systematically deduce the molecular function of each ATPase in the 19S. Inhibitors are generated by chemically modifying purine derivatives with bulky substituents to complement a rationally designed enlargement of the ATP-binding sites of the target ATPases. While the 'analogue-sensitive' ATPases can bind to either normal ATP or the ATP analogue, wild-type ATPases are unaffected in the presence of ATP analogue, allowing specific inhibition of a given ATPase in both in vitro and in vivo assays. Initially we will characterize the roles of the yeast 19S ATPases Rpt1 and Rpt5 in proteasome function, with the goal of eventually characterizing all six ATPases.