The Ubiquitin Proteasome System in Quality Control

描述(申请人提供)：折叠缺陷蛋白质的产生是人类许多遗传性疾病的基础，然而错误折叠蛋白质的独特特征和识别它们的细胞机制在很大程度上仍不清楚。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其通过一种称为内质网相关降解(ERAD)的过程相结合的细胞因素。尽管在将细胞质泛素-蛋白酶体系统(UPS)与ERAD联系起来方面取得了相当大的进展，但基本的问题仍然没有得到回答。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ER蛋白进行降解方面的核心作用。因此，该项目的直接目标是确定内质网中错误折叠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中合作识别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组件的小发夹干扰RNA(ShRNA)文库。来自该屏幕的命中将通过一组检测来验证，并将详细研究新确定的组件与衬底和其他ERAD机制的相互作用。初步数据包括确定Hrd1为E3连接酶，用于降解未组装的AMPA型谷氨酸受体亚单位，从而验证了我们的功能基因组学筛选的有效性。因此，第二个目的是阐明促进Hrd1依赖的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的补充，它对细胞应激反应途径如何响应拓扑和结构上不同类别的ERAD底物进行了全面的生化和微阵列研究。预计这一分析将为细胞如何响应生理上相关的蛋白质应激提供新的理解，并将有助于识别直接导致ERAD降解的特定类型底物的新成分。

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.