ER Associated Degradation of Membrane Proteins in Yeast

酵母中内质网相关的膜蛋白降解

• 批准号: 7088330
• 负责人: JEFFREY L. BRODSKY
• 金额: $ 24.97万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7088330
• 关键词: Saccharomyces cerevisiae; chloride channels; cystic fibrosis; endoplasmic reticulum; genetic strain; membrane proteins; microarray technology; microorganism culture; molecular chaperones; mutant; proteasome; protein degradation; protein folding; protein signal sequence; protein structure function; transfection /expression vector; ubiquitin

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is the most common, inherited lethal disease in Caucasians in North America, and arises from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). The majority of disease-causing mutations block the maturation of this secreted protein, such that CFTR becomes trapped in the endoplasmic reticulum (ER) and is degraded by the proteasome. This process is referred to as ER associated degradation (ERAD), and >30 ERAD substrates from yeast to man have been identified, many of which are linked to specific diseases. ERAD substrate selection and targeting are catalyzed by molecular chaperones, but to date it has been difficult to define how and specifically at which step the chaperones impact CFTR degradation. Moreover, it has been challenging to define how a membrane protein, like CFTR, is delivered to the proteasome, and to identify uncharacterized genes required for maximal ERAD efficiency. To surmount existing technical barriers, the PI's laboratory established a yeast CFTR expression system and showed that unique chaperones play distinct roles during ERAD. To identify novel factors that catalyze ERAD, a micro-array "screen" was performed and a chaperone class with no previous connection to ERAD was found to facilitate CFTR degradation in yeast. In parallel with these studies, an in vitro system was established that recapitulates the polyubiquitination of CFTR and a CFTR homologue in yeast membranes. Based on this new data and tools, the goals of this grant application are to determine at which step in the CFTR degradation pathway known and newly identified chaperones function. And, for the first time, the requirements for substrate de-ubiquitination and proteasome targeting during ERAD will be investigated in a defined system. Importantly, data obtained from the in vitro assay will be complemented through in vivo studies in wild type and mutant yeast strains. This project reflects the PI's long-term interest in defining the molecular machines responsible for protein biogenesis in the ER, and this grant application constitutes the primary focus of ongoing research in the PI's laboratory. Finally, the results obtained from the experiments described in this application will direct future efforts to delineate the CFTR maturation pathway in mammalian cells, an effort that is vital as ongoing chaperone-based therapies to treat CF and other protein conformational diseases are entering clinical trials.

描述（由申请人提供）：囊性纤维化（CF）是北美高加索人中最常见的遗传性致死性疾病，由囊性纤维化跨膜传导调节因子（CFTR）突变引起。大多数致病突变阻断了这种分泌蛋白的成熟，使得CFTR被困在内质网（ER）中并被蛋白酶体降解。这个过程被称为ER相关降解（ERAD），已经鉴定了从酵母到人类的>30种ERAD底物，其中许多与特定疾病有关。ERAD底物选择和靶向由分子伴侣催化，但迄今为止，难以定义伴侣如何以及具体在哪个步骤影响CFTR降解。此外，定义膜蛋白（如CFTR）如何被递送至蛋白酶体以及鉴定最大ERAD效率所需的未表征基因一直是具有挑战性的。为了克服现有的技术障碍，PI的实验室建立了一个酵母CFTR表达系统，并表明独特的伴侣蛋白在ERAD过程中发挥着独特的作用。为了鉴定催化ERAD的新因子，进行微阵列“筛选”，发现先前与ERAD没有联系的伴侣蛋白类促进酵母中的CFTR降解。在这些研究的同时，建立了一个体外系统，该系统重现了酵母细胞膜中CFTR和CFTR同源物的多聚泛素化。基于这些新的数据和工具，该资助申请的目标是确定已知和新鉴定的伴侣蛋白在CFTR降解途径的哪个步骤起作用。而且，第一次，ERAD过程中底物去泛素化和蛋白酶体靶向的要求将在一个定义的系统中进行研究。重要的是，从体外试验中获得的数据将通过野生型和突变酵母菌株的体内研究进行补充。该项目反映了PI在定义负责ER中蛋白质生物合成的分子机器方面的长期兴趣，并且该资助申请构成了PI实验室正在进行的研究的主要重点。最后，从本申请中描述的实验获得的结果将指导未来的努力来描绘哺乳动物细胞中的CFTR成熟途径，这一努力是至关重要的，因为正在进行的基于伴侣的治疗CF和其他蛋白质构象疾病的疗法正在进入临床试验。