The Ubiquitin Proteasome System in ER Quality Control

描述（由申请人提供）：折叠缺陷蛋白的产生是人类许多遗传性疾病发病机制的基础，然而错误折叠蛋白的独特特征和识别它们的细胞机制在很大程度上仍然未知。该项目的长期目标是确定将内质网中折叠缺陷蛋白的识别与内质网中折叠缺陷蛋白的降解结合在一起的细胞因子，该过程被称为er相关降解（ERAD）。尽管在细胞质泛素-蛋白酶体系统（UPS）与ERAD的联系方面取得了相当大的进展，但基本问题仍未得到解答。其中最重要的是识别蛋白质为ERAD底物的顺式和反式信号的性质，以及将蛋白质从生物合成折叠途径转移到ERAD的分子事件。我的实验室和其他许多人之前的工作已经牢固地确立了UPS在靶向错误折叠的内质网蛋白降解中的核心作用。因此，该项目的直接目标是确定内质网中折叠错误的蛋白质被识别的细胞机制。我们将结合功能基因组学和传统细胞生物学来鉴定E3 Ub连接酶和UPS中协同识别ERAD底物的其他关键元件。为此，提出了三个具体目标。在第一个目标中，我们计划筛选针对人类基因组中所有可能的UPS成分的小发夹干扰RNA （shRNA）文库。该筛选结果将通过一系列分析进行验证，并将详细研究新鉴定的组分与底物和其他ERAD机器的相互作用。初步数据包括确定Hrd1作为E3连接酶降解未组装的ampa型谷氨酸受体亚基，从而验证我们的功能基因组筛选的有效性。因此，第二个目的是阐明促进Hrd1依赖性GluR1亚基降解的顺式和反式作用因子。第三个目标是对第一个目标进行全面的生化和微阵列研究，研究细胞应激反应途径如何对拓扑和结构上不同类型的ERAD底物作出反应。预计这一分析将为细胞如何对生理相关的蛋白质应激作出反应提供新的理解，并将有助于识别直接被ERAD降解的特定底物类别的新成分。

DESCRIPTION (provided by applicant): Production of folding-defective proteins underlies the pathogenesis of many heritable disorders in man, yet the unique features of misfolded proteins and the cellular machinery that recognizes them remain largely unknown. The long-term goal of this project is to identify the cellular factors that couple the recognition of folding-defective proteins in the endoplasmic reticulum to their degradation by a process known as ER-associated degradation (ERAD). Despite considerable progress made in linking the cytoplasmic ubiquitin-proteasome system (UPS) to ERAD, fundamental questions remain unanswered. Foremost amongst these is the nature of the cis and trans signals that identify a protein as an ERAD substrate and the molecular events that divert proteins from the biosynthetic folding pathway to ERAD. Previous work from my lab and many others has firmly established a central role for the UPS in the targeting of misfolded ER proteins for degradation. The immediate goal of this project is thus to identify the cellular machinery by which malfolded proteins in the ER are recognized. We will use a combination of functional genomics and traditional cell biology to identify E3 Ub ligases and other critical elements of the UPS that collaborate in the recognition of ERAD substrates. To this end, three specific aims are proposed. In the first aim we plan to screen libraries of small hairpin interfering RNA (shRNA) directed against all probable UPS components in the human genome. Hits from this screen will be validated by a battery of assays and the interaction of the newly identified components with substrate and other ERAD machinery will be studied in detail. Preliminary data are included identifying Hrd1 as an E3 ligase for degradation of unassembled AMPA-type glutamate receptor subunits, thereby validating the efficacy of our functional genomics screen. Thus, the second aim is to elucidate the cis- and trans- acting factors that promoting Hrd1- dependent degradation of GluR1 subunits. The third aim complements the first by performing a comprehensive biochemical and microarray investigation of how cellular stress response pathways respond to topologically and structurally distinct classes of ERAD substrate. It is anticipated that this analysis will provide new understanding of how cells respond to physiologically relevant protein stress, and will facilitate the identification of new components of that direct specific classes of substrate to degradation by ERAD.