The Ubiquitin Proteasome System in ER Quality Control

描述（由申请人提供）：本项目的长期目标是阐明分泌途径中的蛋白质在被称为内质网相关降解（ERAD）的过程中被泛素-蛋白酶体系统（UPS）识别和破坏的机制。致力于这种命运的底物是多样的，并且包括由于突变或低效组装成寡聚复合物而不能正确折叠的分泌的和整合的膜蛋白。在目前的资助期间，我们在实现我们的主要目标，建立一个强大的和多功能的功能基因组学平台，映射功能底物特定的子网络在哺乳动物ERAD系统中取得了非凡的进展，并已超过这些目标，在开发强大的蛋白质组学工具，使我们能够编译的哺乳动物ERAD相互作用组的初步地图。本申请提出扩展这些成就，目标是定义伴侣蛋白和聚糖在管腔ER质量控制监测中的作用，并发展对哺乳动物中ERAD系统的组成和功能组织的全面理解。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物化学，细胞生物学和生物物理学方法来定义底物被提交给ERAD途径的机制和分子伴侣GRP 94在该过程中的作用。第二个具体目标将进行大规模的全面功能基因组筛选的整个UPS在一组拓扑结构不同的ERAD基板的降解，并将验证这些组件使用电池的信息和生化工具。第三个具体的目标是构建一个全面的地图哺乳动物ERAD网络相结合的ERAD蛋白质组的深入挖掘与基因沉默，以确定网络拓扑结构，并覆盖此地图上的目标2中产生的基板特异性功能网络。这些研究的重点是阐明折叠或组装缺陷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.