Characterization and Sorting of Zymogen Granule Proteins

酶原颗粒蛋白的表征和分选

• 批准号: 6436202
• 负责人: ANSON W LOWE
• 金额: $ 28.67万
• 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 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6436202
• 关键词: acinar cell; chimeric proteins; electron microscopy; genetically modified animals; glycosylphosphatidylinositols; intracellular membranes; intracellular transport; laboratory mouse; laboratory rat; membrane biogenesis; membrane proteins; pancreas; pancreatitis; phosphorylases; protein binding; protein biosynthesis; protein isoforms; protein structure function; protein transport; secretion; tissue /cell culture; transfection; vesicle /vacuole; zymogens

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.

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道中。腺泡 细胞负责胰腺的外分泌功能，可以 其特征为极化的分泌上皮细胞。消化酶的分泌量为 也受到乙酰胆碱和胆囊收缩素的调节和刺激。 负责腺泡调节分泌的关键亚细胞器 细胞是酶原颗粒；储存和浓缩的分泌囊泡 消化酶直至刺激分泌。该项目的重点有 酶原颗粒膜蛋白的表征作为一种手段 旨在了解分泌物形成的机制 颗粒和蛋白质靶向受调节的分泌途径。 GP2 是酶原颗粒膜中的主要蛋白质，占 35 颗粒膜蛋白总量的百分比。体外研究已 证明 GP2 能够与其他外分泌调节的聚集 酸性条件下的分泌蛋白旨在模拟反式高尔基体 网络和未成熟的分泌颗粒进行分选。 GP2最初是 通过糖基磷脂酰肌醇键与膜结合， 其本身赋予顶端质膜膜蛋白分选功能。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，很可能 该蛋白质在将消化酶分类到消化道中起着重要作用 酶原颗粒和调节途径。该应用程序的目标是 下一个资助期将定义 GP2 的职能。转基因敲除技术 将用于产生具有 GP2 无效等位基因的小鼠。因为GP2是 特异性表达于胰腺酶原颗粒和外分泌 胰腺直到出生后才具有功能，因此胚胎不太可能 突变将导致致命。因此已经做好了准备工作 使用生化、形态学和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GP2的作用 酶原颗粒的形成。原代胰腺培养物将是 用于研究突变体中受调节分泌途径的完整性。 确定任何由此产生的表型确实是 GP2 null 的次要表型 突变体，已为野生型 GP2 的重建做好了准备 使用腺病毒介导的基因传递进行原代胰腺培养。腺病毒 多种突变 GP2 构建体的表达将用于鉴定 蛋白质中的重要功能域。最后，将进行研究 GP2突变对实验诱发的胰腺炎的影响。这 我们建议生成的模型将提供有关 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。