The Ubiquitin Proteasome System in ER Quality Control

描述（由申请人提供）：该项目的长期目标是阐明分泌途径中的蛋白质在内质网相关降解（ERAD）过程中被泛素-蛋白酶体系统（UPS）识别和破坏的机制。承诺这种命运的底物是多种多样的，包括由于突变或低聚物复合物的低效组装而无法正确折叠的分泌和整体膜蛋白。在目前的资助期内，我们在实现建立一个强大的多功能功能基因组学平台以绘制哺乳动物ERAD系统中功能底物特异性子网络的主要目标方面取得了非凡的进展，并且在开发强大的蛋白质组学工具方面已经超越了这些目标，使我们能够编制哺乳动物ERAD相互作用组的初步地图。本申请旨在扩展这些成果，以确定伴侣和聚糖在管腔内质网质量控制监测中的作用，并全面了解哺乳动物ERAD系统的组成和功能组织。这些目标被定义为三个具体目标。第一个具体目标将采用已建立的生化、细胞生物学和生物物理学方法来确定底物参与ERAD途径的机制以及分子伴侣GRP94在这一过程中的作用。第二个具体目标是在一组拓扑多样化的ERAD底物降解过程中对整个UPS进行大规模的综合功能基因组筛选，并使用信息学和生化工具验证这些组件。第三个具体目标是将ERAD蛋白质组的深度挖掘与基因沉默相结合，构建哺乳动物ERAD网络的综合图谱，以确定网络拓扑结构，并将该图谱覆盖在aim 2中生成的底物特异性功能网络上。这些研究的重点是阐明折叠或有组装缺陷的蛋白质被哺乳动物细胞破坏的分子机制，因此，对人类健康有直接影响。

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the mechanisms by which proteins in the secretory pathway are recognized and destroyed by the ubiquitin-proteasome system (UPS) in a process known as endoplasmic reticulum-associated degradation (ERAD). Substrates committed to this fate are diverse, and include secreted and integral membrane proteins that are unable to fold correctly as a consequence of mutation or inefficient assembly into oligomeric complexes. During the current funding period we have made exceptional progress in achieving our primary aims of establishing a robust and versatile functional genomics platform to map functional substrate-specific sub-networks within the mammalian ERAD system, and have exceeded these aims in developing powerful proteomic tools that have enabled us to compile a preliminary map of the mammalian ERAD interactome. The present application proposes to expand these achievements with the goal of defining the role of chaperones and glycans in luminal ER quality control surveillance and to develop a comprehensive understanding of the composition and functional organization of the ERAD system in mammals. These goals are defined three specific aims. The first specific aim will employ established biochemical, cell biological and biophysical methodology to define the mechanisms by which substrates are committed to ERAD pathway and the role of the molecular chaperone, GRP94, in this process. The second specific aim will conduct a large-scale comprehensive functional genomic screen of the entire UPS in the degradation of a set of topologically diverse ERAD substrates and will validate these components using a battery of informatic and biochemical tools. The third specific aim is to construct a comprehensive map of the mammalian ERAD network by combining in-depth mining of the ERAD proteome with gene silencing to identify network topology, and to overlay this map on the substrate-specific functional network generated in Aim 2. These studies are focused on elucidating the molecular mechanisms by which folding or assembly-defective proteins are destroyed by mammalian cells, and therefore, have direct bearing on human health. PUBLIC HEALTH RELEVANCE: The proposed studies will investigate the process by which misfolded or damaged proteins in the secretory pathway are identified and destroyed. Since mutations which influence protein folding underlie most genetic diseases, understanding the cellular mechanisms that defend against such mutations is critical to our understanding of the molecular basis of genetic disease.