CHARACTERIZATION AND SORTING OF ZYMOGEN GRANULE PROTEINS

酶原颗粒蛋白的表征和分选

• 批准号: 2016402
• 负责人: ANSON W LOWE
• 金额: $ 17.51万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-02-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2016402
• 关键词: acinar cell; cellular polarity; chimeric proteins; granule; intracellular transport; laboratory rabbit; laboratory rat; membrane biogenesis; membrane proteins; pancreas; protein biosynthesis; protein isoforms; protein structure; protein structure function; protein transport; tissue /cell culture; transfection; transferrin receptor; vesicle /vacuole; zymogens

DESCRIPTION: The exocrine pancreas represents an excellent system to study epithelial polarity, protein sorting, and regulated secretion. This application builds on the initial characterization of two zymogen granule membrane proteins, VAMP and GP2. VAMP (vesicle associated membrane protein) represents a family of integral membrane proteins that are essential to the vesicular transport of proteins. Studies supported by the initial First Award established that a protein similar to VAMP-2 found in synaptic vesicles is also present in the pancreatic zymogen granule. Further studies of VAMP, however, suggested a novel isoform is present in the pancreas. A cDNA clone for a new VAMP family member, currently named VAMP-3 has been isolated and the nucleotide sequence determined. A specific aim of this proposal is to characterize the subcellular the subcellular distribution of the VAMP-3 protein. Whether the subcellular distribution of VAMP-3 is distinct from the three other mammalian VAMPs previously characterized will be determined. Approaches to be used include subcellular fractionation, pulse chase studies, and microscopy. VAMP-3 exhibits a large intravesicular domain that differs from the other known mammalian VAMP isoforms. A potential function of this unique domain includes a role in VAMP sorting. In vitro mutagenesis will be used to generate chimeric proteins with the human transferrin receptor to determine whether the VAMP-3 unique domain can serve as a sorting signal. GP2 is the major membrane protein in the zymogen granule. Although its function is currently unclear, it is likely to be important in exocrine secretion. Recent data indicates that GP2 is processed intracellularly by endoproteolytic cleavage at the amino terminal end. Based on the hypotheses that intracellular processing regulates GP2 function, the sites for GP2 processing will be identified and mutated. These GP2 processing mutants will then be used in in vivo and in vitro studies to study GP2 function. Whether processing affects the ability of GP2 to bind to itself or to other zymogens will be determined. The role of protein processing in GP2 sorting will also be studied. Characterization of GP2 processing will lead to a better understanding of its function, sorting, and potential role in human diseases such as chronic pancreatitis.

描述：胰腺外分泌是一个极好的研究系统 上皮极性、蛋白质分选和调节分泌。 这 应用建立在两种酶原颗粒的初步表征基础上 膜蛋白、VAMP 和 GP2。 VAMP（囊泡相关膜蛋白）代表一个整合蛋白家族 膜蛋白对于蛋白质的囊泡运输至关重要。 最初的一等奖支持的研究表明，蛋白质 与突触小泡中发现的 VAMP-2 类似，也存在于 胰酶原颗粒。 然而，对 VAMP 的进一步研究表明 胰腺中存在新的亚型。 新 VAMP 的 cDNA 克隆 家族成员，目前命名为 VAMP-3 已被分离，其核苷酸 顺序确定。 该提案的一个具体目标是描述 亚细胞 VAMP-3 蛋白的亚细胞分布。 无论是 VAMP-3 的亚细胞分布与其他三种不同 先前表征的哺乳动物 VAMP 将被确定。 接近 使用的方法包括亚细胞分离、脉冲追踪研究，以及 显微镜。 VAMP-3 表现出与其他蛋白不同的大囊泡内结构域 已知的哺乳动物 VAMP 亚型。 这个独特域的潜在功能 包括 VAMP 排序中的作用。 体外诱变将用于 生成与人转铁蛋白受体的嵌合蛋白以确定 VAMP-3独特结构域是否可以作为分选信号。 GP2 是酶原颗粒中的主要膜蛋白。 虽然其 目前功能尚不清楚，可能在外分泌中很重要 分泌。 最近的数据表明 GP2 在细胞内被加工 氨基末端的内蛋白水解裂解。 基于假设 细胞内加工调节 GP2 功能，即 GP2 的位点 处理将被识别和变异。 这些 GP2 加工突变体 然后将用于体内和体外研究以研究 GP2 功能。 加工是否影响 GP2 与其自身或其他物质结合的能力 将测定酶原。 蛋白质加工在 GP2 分选中的作用 也将被研究。 GP2 处理的表征将导致 更好地了解其功能、分类以及在人类中的潜在作用 慢性胰腺炎等疾病。