Protein Kinase G Regulation of Granulosa Cell Viability

蛋白激酶 G 对颗粒细胞活力的调节

• 批准号: 7027071
• 负责人: JOHN J PELUSO
• 金额: $ 7.23万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-05 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7027071
• 关键词: apoptosis; cGMP dependent protein kinase; cysteine endopeptidases; enzyme activity; enzyme mechanism; female; fluorescence microscopy; granulosa cell; hydrogen transporting ATP synthase; immunoprecipitation; laboratory rat; mitochondria; ovary; phosphorylation; protein localization; protein protein interaction; proteomics; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): Granulosa cell apoptosis is a major physiological event within the mammalian ovary that affects ovarian steroidogenesis, the number of ovarian follicles that ovulate and the rate at which follicles are depleted. Thus, understanding the mechanism that regulates granulosa cell apoptosis could provide insights into premature ovarian failure and certain types of "unexplained" infertility. In this grant application, we demonstrate that PKG [protein kinase G] is involved in maintaining granulosa cell viability. The mechanism through which PKG mediates its anti-apoptotic action is unknown and will be the focus of this grant application. A review of the literature indicates that there are at least 20 known PKG targets that could mediate its action. These potential PKG targets include such diverse proteins as ion channels, ion pumps, kinases, heat shock proteins, receptors and transcription factors. In our initial grant application, we proposed to determine which of these potential PKG targets are actually involved in regulating granulosa cell viability by utilizing a proteomic approach. We have now completed our initial proteomic screen. This screen revealed two potential proteins that could be involved in regulating granulosa cell apoptosis, 14-3-3 sigma and ATP synthase beta precursor. Other studies have shown that 14-3-3 sigma binds ATP synthase beta precursor and in mammalian cells, 14-3-3 directs proteins to the mitochondria. Once within the mitochondria, the presequence of ATP synthase beta is removed and the ATP synthase beta is activated. We propose that PKG dependent phosphorylation of both 14-3-3 sigma and ATP synthase beta precursor is essential for their interaction and the subsequent delivery of ATP synthase beta precursor to the mitochondria. The localization of ATP synthase beta to the mitochondria would allow for a continuous synthesis of ATP, which is required for cell viability. We now propose to test this hypothesis using biochemical, genetic, imaging and cell biological techniques.

描述（由申请人提供）：颗粒细胞凋亡是影响卵巢类固醇生成的哺乳动物卵巢中的一个主要生理事件，排卵的卵巢卵泡数量以及耗尽卵泡的速率。因此，了解调节颗粒细胞细胞凋亡的机制可以为早产卵巢衰竭和某些类型的“无法解释的”不育症提供见解。 在此赠款应用中，我们证明了PKG [蛋白激酶G]参与维持颗粒细胞活力。 PKG介导其抗凋亡作用的机制尚不清楚，这将是该赠款应用的重点。对文献的综述表明，至少有20个已知的PKG目标可以调节其作用。这些潜在的PKG靶标包括不同的蛋白质，例如离子通道，离子泵，激酶，热激蛋白，受体和转录因子。在我们最初的赠款应用中，我们建议通过使用蛋白质组学方法来确定这些潜在的PKG靶标实际上参与了调节颗粒细胞活力的调节。现在，我们已经完成了初始蛋白质组学屏幕。该屏幕揭示了可能参与调节颗粒细胞凋亡的两种潜在蛋白质，即14-3-3 Sigma和ATP合酶β前体。其他研究表明，14-3-3 Sigma结合ATP合酶β前体和哺乳动物细胞中，14-3-3将蛋白质引导到线粒体上。一旦在线粒体内，就可以去除ATP合酶β的率，并激活ATP合酶β。我们提出，14-3-3 Sigma和ATP合酶β前体的PKG依赖性磷酸化对于它们的相互作用和随后的ATP合酶β前体向线粒体至关重要。 ATP合酶β在线粒体中的定位将允许对ATP的连续合成，这是细胞活力所必需的。现在，我们建议使用生化，遗传，成像和细胞生物学技术检验这一假设。