Dissecting mechanisms of p97-assisted protein degradation by the proteasome in the human system.

蛋白酶体对蛋白质的有效降解对于蛋白质稳态和细胞信号传导是必不可少的。大量的泛素化蛋白酶体底物需要AAA+ ATP酶p97（也称为VCP，或酵母中的Cdc 48）的预先处理，这表明p97展开蛋白质以促进蛋白酶体中的后续降解。p97正被探索为癌症药物靶点，而p97的突变导致神经肌肉变性疾病，称为多系统蛋白质病-1。近年来，在使用生物化学方法来理解底物靶向和穿过p97的中央通道导致底物解折叠方面取得了很大进展。然而，未折叠的蛋白质如何从p97出口孔转移到26 S蛋白酶体尚不清楚，特别是在涉及其他因素的人类系统中。在这个项目中，我们将生化重建p97介导的泛素化底物的解折叠与人类系统中的蛋白酶体降解的耦合，并将这些方法与联合收割机结合，以真实的时间监测该过程的关键方面。此外，建立在自己的结构和生物化学的进步，我们将分析辅助蛋白结合接近p97出口孔，并认为整合底物穿梭的蛋白酶体。我们将在细胞中验证我们的发现，并定义受影响的底物蛋白质组。我们的研究结果将为蛋白质降解中缺失的环节提供直接的机制见解，并使我们能够阐明p97相关和相关疾病的分子基础。

Efficient degradation of proteins by the proteasome is essential for protein homeostasis and cell signaling. Large cohorts of ubiquitylated proteasomal substrates require prior processing by the AAA+ ATPase p97 (also called VCP, or Cdc48 in yeast) suggesting that p97 unfolds proteins to facilitate subsequent degradation in the proteasome. p97 is being explored as a cancer drug target, while mutations in p97 cause neuromuscular degenerative disease, termed multisystem proteinopathy-1. In recent years, great progress has been made in using biochemical approaches to understand substrate targeting and threading to and through the central channel of p97 leading to substrate unfolding. However, how unfolded proteins are then handed over from the p97 exit pore to the 26S proteasome is not understood, particularly in the human system that involves additional factors. In this project, we will biochemically reconstitute the coupling of p97-mediated unfolding of ubiquitylated substrates with proteasomal degradation in the human system and combine these approaches with assays that monitor key aspects of the process in real time. Moreover, building on own structural and biochemical advances, we will analyze accessory proteins that bind close to the p97 exit pore and are believed to integrate substrate shuttling to the proteasome. We will validate our findings in cells and define affected substrate proteomes. Our results will provide direct mechanistic insight into the missing link in protein degradation, and allow us to cast light on the molecular basis of p97-associated and related disease.