Biosynthesis and Processing of Peptide Hormone

描述（由申请人提供）：肽激素前体选择性包装到分泌囊泡中对激素生物合成至关重要。在反式高尔基体网络（TGN）中，前体被分选成新生分泌颗粒，在那里它们被加工以产生生物活性多肽。我们实验室的数据表明，高尔基体结构的维持和含激素囊泡从TGN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，以及肌醇磷脂磷脂酰肌醇（4）磷酸，PI（4）P和磷脂酰肌醇（4，5）二磷酸，PI（4，5）P2的合成。该建议的目标是了解这些脂质调节高尔基体结构，从TGN和激素加工的分泌囊泡出芽的机制。目标1：PI（4）P和PI（4，5）P2在维持内分泌细胞中高尔基体结构和功能中的作用：我们将检验以下假设：在垂体GH 3细胞中，PI（4）P和PI（4，5）P2都需要维持高尔基体结构，而PI（4，5）P2还介导高尔基体后囊泡的释放和向质膜的运输。目标I1：高尔基体PI（4，5）P2合成的调节：令人惊讶的是，高尔基体似乎具有很少的PI（4，5）P2，并且其水平可能部分地受到我们已经鉴定的有效的PI（4，5）P2-5-磷酸酶活性的严格控制。我们将检验PI（4，5）P2合成受特定PI（4）P5-激酶向高尔基体的募集和PI（4，5）P2-5-磷酸酶活性的局部调节的假设。Aim III.为了确定PLD异构体和PA在维持高尔基体结构中的作用：PA参与囊泡运输、信号转导和PI（4，5）P2合成的调节。我们证明了在内分泌细胞中，PLD 1和-2，产生PA，定位于高尔基体的不同区域。我们将确定PLD招聘到高尔基体的机制，并使用新的PA报告蛋白来证明PA本身定位到高尔基体。虽然参与磷脂生物合成的多种酶已被表征，但在内分泌细胞中，对不同的活性如何协调并整合在高尔基体中以控制新生分泌囊泡的形成知之甚少。因此，了解分泌囊泡出芽的机制将有助于合理设计可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的药物。

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.