Biosynthesis and Processing of Peptide Hormone

描述（由申请人提供）：选择性地将肽激素前体包装到分泌囊泡中是激素生物合成所必需的。在反式高尔基网络（TGN）中，前体被分类成新生的分泌颗粒，在那里它们被加工成生物活性多肽。我们实验室的数据表明，高尔基结构的维持和TGN中含有激素的囊泡的释放需要磷脂酸（PA），磷脂酶D （PLD）介导的磷脂酰胆碱水解的产物，以及肌醇磷脂磷脂(4)磷酸磷脂酰肌醇(4)P和磷脂酰肌醇（4,5）二磷酸，PI(4,5)P2的合成。本提案的目的是了解这些脂质调节高尔基结构的机制，从TGN分泌囊泡的萌芽和激素加工。目的1:PI(4)P和PI(4,5)P2在维持内分泌细胞高尔基结构和功能中的作用：我们将验证垂体GH3细胞中PI(4)P和PI(4,5)P2是维持高尔基结构所必需的，而PI(4,5)P2还介导高尔基后囊泡的释放和向质膜的运输。目标1：高尔基体PI(4,5)P2合成的调控：令人惊讶的是，高尔基体似乎只有很少的PI(4,5)P2，其水平可能受到我们已经确定的强效Pl(4,5)P2-5磷酸酶活性的严格控制。我们将验证PI(4,5)P2的合成是通过特定PI(4) p5激酶募集到高尔基体和Pl(4,5)P2-5磷酸酶的活性来局部调节的假设。第三目标。为了确定PLD异构体和PA在维持高尔基结构中的作用：PA参与了囊泡运输、信号转导和PI(4,5)P2合成的调节。我们在内分泌细胞中证实产生PA的PLD1和-2位于高尔基体的不同区域。我们将确定PLD募集到高尔基体的机制，并使用新的PA报告蛋白来证明PA本身定位于高尔基体。虽然参与磷脂生物合成的多种酶已经被表征，但在内分泌细胞中，人们对不同的活性如何在高尔基体中协调和整合以控制新生分泌囊泡的形成知之甚少。因此，了解分泌囊泡萌芽的机制将有助于合理设计药物，用于增强或减少包括糖尿病在内的各种病理条件下的激素分泌。

DESCRIPTION (provided by applicant): The selective packaging of peptide hormone precursors into secretory vesicles is essential for hormone biosynthesis. In the trans Golgi network (TGN), the precursors are sorted into nascent secretory granules where they are processed to generate bioactive polypeptides. Data from our laboratory has demonstrated that the maintenance of Golgi structure and release of hormone containing vesicles from the TGN requires phosphatidic acid (PA), the product of phospholipase D (PLD)-mediated phosphatidylcholine hydrolysis, as well as synthesis of the inositol phospholipids phosphatidylinositol (4) phosphate, PI(4)P, and phosphatidylinositol (4,5) bisphosphate, PI(4,5)P2. The goal of this proposal is to understand the mechanisms whereby these lipids regulate Golgi structure, budding of secretory vesicles from the TGN and hormone processing. Aim 1: The role of PI(4)P and PI(4,5)P2 in maintaining Golgi structure and function in endocrine cells: We will test the hypothesis that in pituitary GH3 cells both PI(4)P and PI(4,5)P2 are required to maintain Golgi architecture whereas Pi(4,5)P2 also mediates release of post-Golgi vesicles and trafficking to the plasma membrane. Aim I1: The Regulation of Golgi PI(4,5)P2 synthesis: Surprisingly, the Golgi apparatus appears to possess little PI(4,5)P2 and its level maybe tightly controlled, in part, by a potent Pl(4,5)P2-5-phosphatase activity that we have identified. We will test the hypothesis that PI(4,5)P2 synthesis is regulated locally by recruitment of specific PI(4)P 5-kinases to the Golgi apparatus and by the activity of the Pl(4,5)P2-5-phosphatase. Aim III. To determine the role of PLD isoforms and PA in maintaining Golgi structure: PA has been implicated in vesicle trafficking, signal transduction and regulation of PI(4,5)P2 synthesis. We demonstrated in endocrine cells that PLD1 and -2, which generate PA, localize to different regions of the Golgi apparatus. We will determine the mechanism of PLD recruitment to the Golgi apparatus and use novel PA reporter proteins to demonstrate PA itself localizes to the Golgi apparatus. Although multiple enzymes involved in phospholipid biosynthesis have been characterized, in endocrine cells relatively little is known whereby the different activities are coordinated and integrated in the Golgi apparatus to control the formation of nascent secretory vesicles. Consequently, understanding the mechanism of secretory vesicle budding will facilitate the rational design of drugs that could be used to enhance or diminish hormone secretion in a variety of pathological conditions including diabetes.