ER Associated Degradation of Membrane Proteins in Yeast

描述(申请人提供)：囊性纤维化(CF)是北美高加索人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。大多数致病突变阻碍了这种分泌蛋白的成熟，使CFTR被困在内质网(ER)中，并被蛋白酶体降解。这个过程被称为内质网相关降解(ERAD)，已经确定了从酵母到人的30种ERAD底物，其中许多与特定的疾病有关。Erad底物的选择和靶向是由分子伴侣催化的，但到目前为止，很难确定分子伴侣如何以及具体在哪个步骤影响CFTR的降解。此外，定义膜蛋白，如CFTR，如何被运送到蛋白酶体，以及确定最大ERAD效率所需的未鉴定基因一直是一项挑战。为了克服现有的技术障碍，PI的实验室建立了酵母CFTR表达系统，并表明独特的伴侣在ERAD过程中发挥着不同的作用。为了确定催化ERAD的新因素，进行了微阵列“筛选”，发现了一个以前与ERAD没有联系的伴侣类别，促进了酵母中CFTR的降解。在这些研究的同时，建立了一个体外系统，它概括了酵母膜中CFTR和CFTR同系物的多泛素化。基于这些新的数据和工具，这项赠款申请的目标是确定已知的和新发现的伴侣蛋白在CFTR降解途径中的哪一步发挥作用。并且，将第一次在确定的系统中研究ERAD期间底物去泛素化和蛋白酶体靶向的要求。重要的是，从体外试验中获得的数据将通过对野生型和突变酵母菌株的体内研究来补充。这个项目反映了PI对定义负责内质网中蛋白质生物发生的分子机器的长期兴趣，这一拨款申请构成了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中获得的结果将指导未来在哺乳动物细胞中描绘CFTR成熟途径的努力，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。

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.