FUNCTION OF GAPDS DURING SPERMATOGENESIS & FERTILIZATION

GAPDS 在精子发生过程中的功能

• 批准号: 6589782
• 负责人: Deborah A. O'Brien
• 金额: $ 17.42万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-02 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6589782
• 关键词: aminoacid; cofactor; cooperative study; enzyme activity; enzyme biosynthesis; enzyme mechanism; enzyme substrate; fertility; fertilization; flagellum; gene targeting; genetically modified animals; glyceraldehyde 3 phosphate dehydrogenase; glycolysis; immunoprecipitation; laboratory mouse; phenotype; protein binding; protein localization; protein protein interaction; site directed mutagenesis; sperm capacitation; sperm motility; spermatogenesis; tissue /cell culture; yeast two hybrid system

Description (provided by applicant): Glycolysis is required for mammalian sperm function and fertilization. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS), a unique isozyme expressed only during the late stages of spermatogenesis, appears to serve a pivotal role in regulating this metabolic pathway in spermatozoa. GAPDS has a novel N-terminus that tightly anchors it to the fibrous sheath, a cytoskeletal structure that extends most of the length of the sperm flagellum. GAPDS is more susceptible than somatic GAPD to inhibition by substrate analogs, which induce rapid and reversible male infertility. The long-term goal of this study is to identify mechanisms that regulate sperm glycolysis anc fertilization. The specific aims are to: 1) Determine if localization of GAPDS to the fibrous sheath is required for normal sperm function. These experiments will use knockout and transgenic mice to identify sequences required for GAPDS targeting and binding to this cytoskeletal structure, and to test the hypothesis that anchoring of GAPDS to the fibrous sheath is critical for normal sperm glycolysis, hyperactivated motility, and fertilization; 2) Identify protein interactions that anchor GAPDS to the fibrous sheath. The fibrous sheath, which serves as a scaffold for both glycolytic enzymes and protein kinase A subunits, is likely to be an important regulator of sperm motility. Proteins that interact with GAPDS will be identified by co-immunoprecipitation and yeast two-hybrid assays to better understand the formation and function of protein complexes along the fibrous sheath; 3) Determine if GAPDS plays a role in the assembly of sperm glycolytic enzymes or the phosphorylation cascade that occurs with hyperactivation. Because GAPDS is tightly bound to the fibrous sheath, it is well positioned to participate in the localization of other glycolytic enzymes to the principal piece of the sperm tail and in the regulation of hyperactivated motility. These studies will determine if GAPDS is required for the localization of other glycolytic enzymes to the principal piece, and if initiation of hyperactivated motility alters the activity, solubility, or phosphorylation status of GAPDS; 4) Identify key amino acids that are responsible for the novel enzymatic properties of GAPDS. Molecular modeling studies have identified eight amino acids near the substrate and cofactor binding sites of GAPDS that are different from somatic GAPD. Site-directed mutagenesis will be used to determine the effects of these amino acids on GAPDS activity and inhibition. These studies will determine if the human ortholog of GAPDS is a feasible target for developing highly specific male contraceptives, and if suppression of GAPDS activity by environmental and therapeutic agents could be a cause of infertility in men.

描述（申请人提供）：哺乳动物需要糖酵解 精子功能和受精。甘油醛3-磷酸脱氢酶-S （GAPDS），一种独特的同工酶，只在晚期阶段的表达， 精子发生，似乎在调节这种代谢中起着关键作用。 精子中的途径。GAPDS有一个新的N端， 纤维鞘是一种细胞骨架结构， 精子鞭毛的长度。GAPDS比体细胞GAPD更易受 通过底物类似物的抑制，其诱导快速和可逆的雄性 不孕本研究的长期目标是确定 调节精子糖酵解和受精。具体目标是：1） 确定是否需要将GAPDS定位于纤维鞘， 正常的精子功能这些实验将使用基因敲除和转基因小鼠 为了鉴定GAPDS靶向和结合所需的序列， 细胞骨架结构，并测试的假设，锚定的GAPDS， 纤维鞘对于正常的精子糖酵解、过度活化 运动和受精; 2）鉴定锚的蛋白质相互作用 纤维鞘间隙。作为支架的纤维鞘 对于糖酵解酶和蛋白激酶A亚基，很可能是一种 精子活动力重要调节器。与GAPDS相互作用的蛋白质将 通过免疫共沉淀和酵母双杂交试验进行鉴定， 了解蛋白质复合物的形成和功能，沿着纤维 3）确定GAPDS是否在精子糖酵解的组装中起作用 酶或磷酸化级联反应，发生超活化。 由于GAPDS与纤维鞘紧密结合，因此其位置良好， 参与定位其他糖酵解酶的主要 精子尾部的一部分，并在超激活运动的调节。 这些研究将确定是否需要GAPDS本地化， 其他糖酵解酶的主要部分，如果启动 过度激活的运动改变了活性、溶解性或磷酸化 GAPDS的状态; 4）鉴定负责GAPDS的关键氨基酸。 GAPDS的新的酶性质。分子模拟研究 在底物和辅因子结合位点附近鉴定了8个氨基酸， 与体细胞GAPD不同的GAPDS。定点诱变将是 用于确定这些氨基酸对GAPDS活性的影响， 抑制作用这些研究将确定GAPDS的人类直系同源物是否是一种 开发高度特异性男性避孕药的可行目标，如果 通过环境和治疗剂抑制GAPDS活性， 男性不育的原因。