Characterization and Sorting of Zymogen Granule Proteins

描述（由申请人提供）：外分泌胰腺负责 消化酶的合成和分泌到肠中。腺泡 细胞负责胰腺的外分泌功能， 其特征为极化的分泌上皮。消化酶分泌是 也可以调节，并可以用乙酰胆碱和胆囊收缩素刺激。 腺泡内负责调节分泌的关键亚细胞器 细胞是酶原颗粒;一种储存和浓缩的分泌囊泡 消化酶直到分泌被刺激。该项目的重点是 作为一种手段， 为了了解分泌性细胞形成的机制， 颗粒和蛋白质的靶向调节分泌途径。GP2 是酶原颗粒膜上的优势蛋白，占35 %的总颗粒膜蛋白。体外研究 证明GP 2能够与其他外分泌调节的 分泌蛋白在酸性条件下设计模仿反式高尔基体 未成熟的分泌颗粒和未成熟的分泌颗粒。GP 2最初 通过糖基磷脂酰肌醇键与膜结合， 其本身将膜蛋白分选赋予顶端质膜。因为 GP 2表现出与颗粒内的可溶性消化酶结合， 含有顶端质膜的分选决定子， 这种蛋白质在将消化酶分类到 酶原颗粒及其调控途径。此应用程序的目标是 下一个供资期是界定GP 2的职能。转基因敲除技术 将用于产生具有GP 2无效等位基因的小鼠。因为GP 2是 特异性表达于胰腺酶原颗粒和外分泌 胰腺直到出生后才有功能，胚胎不太可能 突变会导致致命的后果因此，已经做好准备， 使用生物化学，形态学和生物化学方法分析所产生的突变小鼠。 生理学方法。电子显微镜将用于研究GP 2的作用 对酶原颗粒形成的影响。原代胰腺培养物将 用于研究突变体中调节分泌途径的完整性。 为了确定任何产生的表型确实继发于GP 2缺失， 突变体，已经制备了野生型GP 2的重建， 使用腺病毒介导的基因递送的原代胰腺培养物。腺病毒 多种突变体GP 2构建体的表达将用于鉴定 蛋白质中的重要功能域。最后，研究将在 GP 2突变对实验性胰腺炎的影响。的 我们提出的模型将为GP 2生物学提供重要信息 也可能为人类急性和慢性胰腺炎提供潜在的模型 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.