The Ubiquitin Proteasome System in Quality Control

泛素蛋白酶体系统在质量控制中的应用

• 批准号: 7616236
• 负责人: RON R KOPITO
• 金额: $ 25.75万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7616236
• 关键词: AMPA Receptors; Biochemical; Biological Assay; Cells; Cellular Stress Response; Cellular biology; Client; Complement; Complex; Data; Disease; Elements; Endoplasmic Reticulum; Event; Genes; Glutamate Receptor; Goals; Heat shock proteins; Hemorrhage; Human Genome; Integral Membrane Protein; Investigation; Libraries; Ligase; Link; Maps; Mediating; Membrane Proteins; Molecular; Nature; Pathogenesis; Pathway interactions; Process; Production; Proteins; Quality Control; RNA; RNA Interference; RNA library; Recombinant Proteins; Role; Signal Transduction; System; Trans-Activators; Ubiquitin; Work; biological adaptation to stress; functional genomics; man; multicatalytic endopeptidase complex; overexpression; protein complex; protein degradation; protein misfolding; response; small hairpin RNA; stress protein; ubiquitin ligase; ubiquitin-protein ligase

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.

描述（由申请人提供）：折叠缺陷蛋白质的产生是人类许多遗传性疾病发病机制的基础，但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其降解过程（称为ER相关降解（ERAD））相结合的细胞因子。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，但基本问题仍然没有答案。其中最重要的是识别蛋白质作为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。我的实验室和许多其他实验室的先前工作已经牢固地确立了UPS在靶向错误折叠的ER蛋白降解中的核心作用。因此，该项目的直接目标是确定识别ER中错误折叠蛋白质的细胞机制。我们将使用功能基因组学和传统的细胞生物学相结合，以确定E3 Ub连接酶和其他关键要素的UPS，在ERAD基板的识别合作。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组分的小发夹干扰RNA（shRNA）文库。将通过一系列试验验证该筛选的结果，并详细研究新鉴定的组分与底物和其他ERAD机制的相互作用。初步数据包括识别Hrd 1作为E3连接酶的未组装的AMPA型谷氨酸受体亚基的降解，从而验证我们的功能基因组学筛选的功效。因此，第二个目标是阐明促进Hrd 1依赖性GluR 1亚基降解的顺式和反式作用因子。第三个目标是通过进行全面的生物化学和微阵列研究来补充第一个目标，研究细胞应激反应途径如何响应拓扑和结构不同的ERAD底物。预计这种分析将提供新的理解细胞如何响应生理相关的蛋白质压力，并将有助于识别新的成分，直接特定类别的底物降解ERAD。