Molecular genetics of Intracellular Protein Transport

细胞内蛋白质运输的分子遗传学

• 批准号: 7174781
• 负责人: Chris Alan Kaiser
• 金额: $ 33.16万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7174781
• 关键词: Address; Binding; Biochemical; Biochemical Genetics; Biochemistry; Biological Assay; Biological Process; Caenorhabditis elegans; Cell physiology; Cells; Chemistry; Class; Condition; Coupled; Cysteine; Diagnosis; Disulfides; Endoplasmic Reticulum; Enzymes; Eukaryota; Eukaryotic Cell; Extracellular Protein; Extracellular Space; Gene Proteins; Gene Silencing; Generations; Genetic; Genetic Screening; Genomics; Giardia; Giardia lamblia; Glutathione; Isoenzymes; Laboratories; Life; Link; Mammalian Cell; Membrane; Methods; Molecular Genetics; Object Attachment; Organism; Orthologous Gene; Oxidants; Oxidases; Oxidation-Reduction; Pathway interactions; Peptides; Process; Protein Complex Subunit; Protein Disulfide Isomerase; Protein Family; Proteins; Research; Roentgen Rays; Saccharomyces cerevisiae; Source; Specificity; Sulfhydryl Compounds; Thinking; Work; Yeasts; cDNA Library; crosslink; design; disulfide bond; glutathione transporter; in vitro Assay; insight; interest; intracellular protein transport; oxidation; parallel processing; paralogous gene; polypeptide; protein folding; protein structure; research study; secretory protein

DESCRIPTION (provided by applicant): The endoplasmic reticulum (ER) is the compartment where membrane and secretory proteins are modified, folded, and assembled. Part of the folding process for most extracellular proteins includes formation of disulfide bonds which can add stability to folded polypeptides and can link subunits of protein complexes. The formation of disulfide bonds requires both enzymes for the generation of disulfide bonds in the lumen of the ER as well as a pathway for the transfer of these bonds to substrate proteins. Dr. Kaiser's research group proposes a combination of biochemical, structural, and genetic experiments in the yeast S. cerevisiae to give fundamental insight into the mechanisms of disulfide bond formation in living cells. Previous work in Dr. Kaiser's laboratory has delineated the core pathway in the ER for protein disulfide bond formation in which a luminal oxidase Ero1p (or Erv2p) transfers a disulfide bond to protein disulfide isomerase (PDI) which in turn transfers its disulfide bond to a substrate protein. Structural studies of Ero1p and Erv2p reveal that although these proteins are not similar in sequence, they nevertheless share key structural features giving insight into the mechanisms by which disulfide bonds are generated and transferred from one protein to another. In this application, Dr. Kaiser proposes to determine how disulfide bonds are selectively transferred from Ero1p to PDI. Additional experiments are designed to understand how the functions of ER oxidases are integrated into the redox biochemistry of the cell. Studies of disulfide bond formation in yeast will be extended to understand interesting disulfide bond forming processes in other organisms including G. lamblia and C. elegans. 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 providing new opportunities to control the assembly and secret on of extracellular proteins.

描述(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。大多数胞外蛋白质的部分折叠过程包括形成二硫键，这可以增加折叠的多肽的稳定性，并可以连接蛋白质复合体的亚单位。二硫键的形成既需要在内质网管腔中产生二硫键的酶，也需要将这些键转移到底物蛋白的途径。Kaiser博士的研究小组建议对酿酒酵母进行生化、结构和遗传实验的组合，以深入了解活细胞中二硫键形成的机制。Kaiser博士的实验室以前的工作已经描绘了内质网中形成蛋白质二硫键的核心途径，其中鲁米那氧化酶Ero1p(或Erv2p)将二硫键转移到蛋白质二硫键异构酶(PDI)，后者又将其二硫键转移到底物蛋白质上。Ero1p和Erv2p的结构研究表明，虽然这些蛋白质在序列上不相似，但它们有共同的关键结构特征，从而深入了解了二硫键的产生和从一种蛋白质转移到另一种蛋白质的机制。在这项应用中，Kaiser博士建议确定二硫键是如何选择性地从Ero1p转移到PdI的。此外，还设计了其他实验，以了解ER氧化酶的功能如何整合到细胞的氧化还原生化中。酵母中二硫键形成的研究将被扩展到理解其他生物中有趣的二硫键形成过程，包括革兰氏杆菌和线虫。 在酿酒酵母中，将有可能应用发育良好的遗传有机体的全部力量来发现负责内质网中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使理解哺乳动物细胞中的平行过程成为可能，为诊断哺乳动物细胞内质网中功能异常的折叠开辟了道路，并为控制胞外蛋白的组装和分泌提供了新的机会。