Discovering the machinery and mechanism of ERAD-M retrotranslocation

发现 ERAD-M 逆转录转位的机制和机制

基本信息

批准号：
9061744
负责人：
Sonya Elina Neal
金额：
$ 5.8万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-01 至 2017-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9061744
关键词：
26S proteasome Aging Alzheimer&apos s Disease Binding Biochemical Biochemical Genetics Biological Assay Biological Process Candidate Disease Gene Cells Cellular Stress Clinical Collection Complex Cytosol Defect Degradation Pathway Dependence Disease Endoplasmic Reticulum Ensure Enzymes Event Excision Gene Proteins Genes Genetic Genetic Screening Goals Health Heart Huntington Disease Hydroxymethylglutaryl-CoA reductase In Vitro Isoenzymes Knowledge Laboratories Libraries Location Maintenance Membrane Membrane Proteins Methods Modeling Nature Parkinson Disease Parkinsonian Disorders Pathway interactions Phase Prions Process Protein translocation Proteins Proteomics Quality Control Regulation Rheumatoid Arthritis Role Route Running Sterols Stress Syndrome Techniques Testing Ubiquitin Ursidae Family Validation Variant Work Yeasts base forward genetics gene discovery genetic approach in vitro Assay in vivo luminal membrane member multicatalytic endopeptidase complex novel novel therapeutics physical property protein degradation protein folding protein misfolding prototype tool

26S proteasome Aging Alzheimer&apos s Disease Binding Biochemical Biochemical Genetics Biological Assay Biological Process Candidate Disease Gene Cells Cellular Stress Clinical Collection Complex Cytosol Defect Degradation Pathway Dependence Disease Endoplasmic Reticulum Ensure Enzymes Event Excision Gene Proteins Genes Genetic Genetic Screening Goals Health Heart Huntington Disease Hydroxymethylglutaryl-CoA reductase In Vitro Isoenzymes Knowledge Laboratories Libraries Location Maintenance Membrane Membrane Proteins Methods Modeling Nature Parkinson Disease Parkinsonian Disorders Pathway interactions Phase Prions Process Protein translocation Proteins Proteomics Quality Control Regulation Rheumatoid Arthritis Role Route Running Sterols Stress Syndrome Techniques Testing Ubiquitin Ursidae Family Validation Variant Work Yeasts base forward genetics gene discovery genetic approach in vitro Assay in vivo luminal membrane member multicatalytic endopeptidase complex novel novel therapeutics physical property protein degradation protein folding protein misfolding prototype tool

展开

项目摘要

DESCRIPTION (provided by applicant): Elimination of misfolded proteins by ER-associated protein degradation (ERAD) ensures that proteins entering the secretory pathway are correctly folded and that ER stress is maintained at acceptable low levels. All variations of ERAD includes a protein translocation process termed retrotranslocation, in which ubiquitinated ERAD subsrates are selectively extracted by dedicated machinery from the ER before degradation by the cytosolic 26S proteasome. The overall goal of this project is to unravel the unknown mechanism of retrotranslocation. In the course of discovering the conserved HRD ERAD pathway, our laboratory has developed a variety of unique tools, techniques and expertise that will be brought to bear on the pressing and open question of retrotranslocation. Specifically, I will 1) perform biochemical and proteomic analyses on the prototypical ERAD substrate HMG-CoA reductase to understand the mechanism of its retrotranslocation from the ER and identify the key proteins involved in this process. Furthermore, I will 2) employ a genetic approach as an independent and complementary approach to identify retrotranslocation factors. They will include running traditional and array-based screens, combined with validation of candidate genes using direct in vivo and in vitro assays. Finally, 3) I will discern the roles of all discoveed factors in each known branch of ERAD. Taken together, these studies will reveal a key, universal and conserved process at the heart of ERAD involved in managing cell stress and a variety of clinical maladies. My studies in yeast will provide a new fundamental knowledge of the retrotranslocation process of ERAD. ERAD has been implicated in diseases such as aging, Alzheimer's disease, Huntington's disease, Parkinson's disease and in normal sterol regulation, my studies are pertinent for understanding how defects in ERAD are associated with these processes and maladies.

描述（由申请人提供）：通过ER相关的蛋白质降解（ERAD）消除错误折叠的蛋白质可确保进入分泌途径的蛋白质正确折叠，并且ER应力保持在可接受的低水平。 ERAD的所有变化包括称为转向转换的蛋白质转运过程，其中泛素化的ERAD补充物是通过胞质26S蛋白酶体从ER中从ER中从ER中选择性提取的。该项目的总体目标是揭示未知的逆转录机制。在发现保守的HRD Erad途径的过程中，我们的实验室开发了各种独特的工具，技术和专业知识，这些工具，技术和专业知识将在紧迫和开放的逆转转移问题上承受。具体而言，我将1）对原型ERAD底物HMG-COA还原酶进行生化和蛋白质组学分析，以了解其从ER逆转转移的机制，并识别此过程中涉及的关键蛋白。此外，我将2）采用一种遗传方法作为一种独立和互补的方法来识别逆转录定位因子。它们将包括运行传统和基于数组的屏幕，并使用直接体内和体外测定法结合验证候选基因。最后，3）我将辨别所有已知分支在Erad的每个已知分支中的作用。综上所述，这些研究将揭示一个关键，普遍和保守的过程，这是涉及控制细胞应激和各种临床疾病的ERAD的核心。我在酵母中的研究将提供有关ERAD逆转录过程的新基本知识。 Erad与衰老，阿尔茨海默氏病，亨廷顿氏病，帕金森氏病以及正常的固醇调节有关，我的研究与了解ERAD中的缺陷如何与这些过程和疾病有关。