Molecular Genetics of Intracellular Protein Transfer

描述（由申请人提供）：内质网（ER）是膜和分泌蛋白被修饰、折叠和组装的隔室。大多数细胞外蛋白质折叠的关键步骤包括二硫键的形成，这可以增加折叠多肽的稳定性，并可以将蛋白质复合物的亚基连接在一起。该项目之前的工作已经描绘了真核细胞中蛋白质二硫键形成的核心途径，其中氧化酶Ero1p产生的二硫键随后被转移到蛋白质二硫异构酶（PDI）上，PDI反过来将二硫键转移到底物蛋白质上。通过对酿酒酵母的生化、结构和遗传实验的结合，我们已经对这一途径中每个步骤的分子机制有了基本的了解。在这个应用中，我们现在专注于了解蛋白质氧化途径如何整合到细胞的氧化还原生物化学中。该提案的目的包括：了解Pdi1p和相关蛋白如何调节Ero1p活性，研究由过度活跃的Ero1p活性引发的内质网转运的新阻断，以及阐明内质网中与二硫化碳形成耦合的新电子传递链。公共卫生相关性：在酿酒酵母中，将有可能应用一个发育良好的遗传有机体的全部力量来揭示内质网中负责蛋白质折叠的基因和蛋白质。对酿酒酵母中这些途径的详细了解将有助于理解哺乳动物细胞中的平行过程，为诊断哺乳动物细胞内质网折叠功能障碍以及二硫键形成途径产生的活性氧可能产生的有害影响开辟道路。

DESCRIPTION (provided by applicant): The endoplasmic reticulum (ER) is the compartment where membrane and secretory proteins are modified, folded, and assembled. A key step in the folding of most extracellular proteins includes formation of disulfide bonds, which can add stability to folded polypeptides and can link together subunits of protein complexes. Previous work on this project has delineated the core pathway for protein disulfide bond formation in eukaryotic cells in which disulfide bonds generated by an oxidase, Ero1p, are then transferred to protein disulfide isomerase (PDI), which in turn transfers a disulfide bond to substrate proteins. Through a combination of biochemical, structural, and genetic experiments in the yeast S. cerevisiae we have gained fundamental insight into the molecular mechanisms that underlie each of the steps in this pathway. In this application we now focus on understanding how the protein oxidation pathway is integrated into the redox biochemistry of the cell. Aims of the proposal include: understanding how Pdi1p and related proteins regulate Ero1p activity, investigation of a novel block in ER translocation instigated by hyperactive Ero1p activity, and elucidation of a new electron transport chain in the ER coupled to disulfide bon formation. PUBLIC HEALTH RELEVANCE: In S. cerevisiae it will be possible to apply the full power of a well developed genetic organism to uncover the genes and proteins responsible protein folding in the ER. A detailed understanding of these pathways in S. cerevisiae will make it possible to understand parallel processes in mammalian cells, opening the way to diagnose dysfunctional folding in the ER of mammalian cells and possible detrimental effects of reactive oxygen species generated by the disulfide bond formation pathway.