Characterization and Sorting of Zymogen Granule Proteins

描述(申请人提供)：外分泌胰腺负责 消化酶的合成和分泌进入肠道。《腺泡》 细胞负责胰腺的外分泌功能，可以 以极化的分泌上皮为特征的。消化酶的分泌是 也受乙酰胆碱和缩胆素的调节和刺激。 腺泡中负责调节分泌的关键亚细胞器 细胞是酵素原颗粒；储存和浓缩的分泌性小泡 消化酶，直到分泌被刺激。这个项目的重点是 以酶原颗粒膜蛋白的表征为手段 通向理解分泌物形成的机制 颗粒和蛋白质靶向调节的分泌途径。GP2 是酶原颗粒膜上的主要蛋白质，占35% 占总颗粒膜蛋白的百分比。体外研究表明 证明了GP2能够与其他外分泌调节因子聚集 酸性条件下的分泌蛋白质，旨在模拟反式高尔基体 出现分选的网状结构和未成熟的分泌颗粒。GP2最初是 通过糖基磷脂酰肌醇连接到膜上，通过 它本身将膜蛋白分选给顶端质膜。因为 GP2与颗粒内的可溶性消化酶结合， 包含顶端质膜的分选决定因素，它很可能 这种蛋白质在将消化酶分选到 酶原颗粒及其调控途径。这个应用程序的目标是 下一个资金期是定义GP2的S函数。转基因基因敲除技术 将被用来制造一只带有GP2零等位基因的小鼠。因为GP2是 在胰酶原颗粒和外分泌中特异表达 胰腺直到出生后才有功能，胚胎不太可能 这种突变将导致致命的后果。因此，已经做好了准备 对产生的突变小鼠进行生化、形态和 生理学方法。将用电子显微镜研究GP2的S作用 关于酵素原颗粒的形成。原代胰腺培养将是 用于研究突变体中调节的分泌途径的完整性。 以确定任何由此产生的表型确实次于GP2空 突变体，野生型GP2的重组已做好准备 用腺病毒介导的基因传递技术进行原代胰腺培养。腺病毒 各种突变的GP2结构的表达将被用来识别 蛋白质中的重要功能结构域。最后，将对以下方面进行研究 GP2基因突变在实验性胰腺炎中的作用这个 我们建议建立的模型将提供有关GP2生物学的重要信息 也可能为人类急性和慢性胰腺疾病提供潜在的模型。 疾病。

DESCRIPTION (provided by applicant): The exocrine pancreas is responsible for the synthesis and secretion of digestive enzymes into the intestine. The acinar cell is responsible for the pancreas' exocrine functions and can be characterized as a polarized secretory epithelia. Digestive enzyme secretion is also regulated and can be stimulated with acetylcholine and cholecystokinin. The key subcellular organelle responsible for regulated secretion in the acinar cell is the zymogen granule; a secretory vesicle that stores and concentrates digestive enzymes until secretion is stimulated. The focus of this project has been the characterization of zymogen granule membrane proteins as a means toward understanding the mechanisms underlying the formation of secretory granules and the targeting of proteins to the regulated secretory pathway. GP2 is the dominant protein in the zymogen granule membrane and accounts for 35 percent of the total granule membrane protein. In vitro studies have demonstrated that GP2 is able to aggregate with other exocrine regulated secretory proteins in acidic conditions designed to mimic the trans-Golgi network and immature secretory granule where sorting occurs. GP2 is initially bound to the membrane through a glycosylphosphotidylinositol linkage, which by itself confers membrane protein sorting to the apical plasma membrane. Because GP2 exhibits binding to the soluble digestive enzymes within the granule and contains a sorting determinant for the apical plasma membrane, it is likely that the protein plays a significant role in sorting digestive enzymes into the zymogen granule and the regulated pathway. The goal of this application for the next funding period is to define GP2's function. Transgenic knockout techniques will be employed to produce a mouse with a GP2 null allele. Because GP2 is specifically expressed in the pancreatic zymogen granule and the exocrine pancreas is not functional until after birth, it is unlikely that an embryonic lethal will result from the mutation. Thus preparations have been made to analyze the resultant mutant mice using biochemical, morphological, and physiological approaches. Electron microscopy will be used to study GP2's role on the formation of the zymogen granule. Primary pancreatic cultures will be used to study the integrity of the regulated secretory pathway in the mutants. To establish that any resultant phenotypes are truly secondary to the GP2 null mutant, preparations have been made for the reconstitution of wild-type GP2 in primary pancreatic cultures using adenovirus mediated gene delivery. Adenovirus expression of a variety of mutant GP2 constructs will be used to identify important functional domains in the protein. Last, studies will be performed on the effects of the GP2 mutation in experimentally induced pancreatitis. The model we propose to generate will provide important information on GP2 biology and may also provide potential models for human acute and chronic pancreatic diseases.