CHARACTERIZATION AND SORTING OF ZYMOGEN GRANULE PROTEINS

描述：外分泌胰腺是一个很好的研究系统。 上皮极性、蛋白质分选和调节分泌。这 应用建立在两种酵素原颗粒的初步表征上 膜蛋白、VAMP和GP2。 VAMP(囊泡相关膜蛋白)代表了一个完整的家族 对蛋白质的囊泡运输至关重要的膜蛋白。 最初的第一个奖项支持的研究确定了一种蛋白质 与VAMP-2类似，在突触小泡中也存在 胰酶原颗粒。然而，对VAMP的进一步研究表明， 新的异构体存在于胰腺中。一种新的VAMP的cDNA克隆 家族成员，目前命名为VAMP-3已被分离和核苷酸 顺序已确定。这项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 之前描述的哺乳动物鞋面将被确定。实现以下目标的方法 包括亚细胞分离、脉冲追逐研究和 显微镜。 VAMP-3显示了一个不同于其他蛋白的大的囊泡内结构域 已知的哺乳动物VAMP亚型。这一独特领域的潜在功能 包括VAMP排序中的角色。体外诱变技术将用于 产生与人转铁蛋白受体的嵌合蛋白以确定 VAMP-3唯一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。尽管它的 目前功能尚不清楚，它可能在外分泌中起重要作用 分泌物。最近的数据表明，GP2是由 氨基末端的内切蛋白裂解。基于假设 细胞内处理调节GP2的功能，即GP2的位置 加工过程将被识别和突变。这些处理GP2的突变体 然后将用于体内和体外研究，以研究GP2的功能。 处理是否会影响GP2与自身或他人绑定的能力 酵母菌将会被确定。蛋白质加工在GP2分选中的作用 也将进行研究。GP2处理的表征将导致 更好地理解它的功能、分类和在人类中的潜在作用 慢性胰腺炎等疾病。

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.