REGULATION OF SPERM FUNCTION BY PROTEIN PRENYLATION

蛋白质异戊烯化对精子功能的调节

• 批准号: 2889560
• 负责人: GARY E OLSON
• 金额: $ 7.6万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2889560
• 关键词: alkyltransferase; cell membrane; cytoplasm; enzyme activity; guanine nucleotide binding protein; hamsters; immunoelectron microscopy; intermolecular interaction; intracellular transport; isoprenoid; membrane biogenesis; membrane proteins; posttranslational modifications; protein transport; sperm; sperm capacitation; sperm motility; spermatogenesis

DESCRIPTION: (Adapted from applicant's description) The sperm plasma membrane is partitioned into domains of distinct molecular composition and function in fertilization. The assembly of different membrane domains is initiated during spermiogenesis and continues during post-testicular development in the epididymis. Spermatids and spermatozoa possess both farnesyltransferase (Ftase), which functions in post-translational protein lipidation, and the signaling protein Ras, a FTase substrate, which requires prenylation for expression of function. The long range goals of this proposal are to define the role of protein prenylation in sperm development and to determine if prenylated proteins are recruited to specific membrane domains and regulate sperm function. Three aims address these goals. Aim 1 is to determine the mechanisms which sequester farnesyltransferase to specific cytoplasmic regions of spermatids. Co- immunoprecipitation analysis will be used to determine if FTase is bound to specific anchoring proteins, and immunoelectron microscopy will be used to identify structural mechanisms which generate the restricted FTase distribution pattern. Aim 2 is to determine if FTase functions in the prenylation and membrane targeting of distinct proteins in spermatids and epididymal spermatozoa. Farnesylated proteins of metabolically labeled spermatids and epididymal spermatozoa will be identified by N-terminus microsequencing and by immunoblotting with antibodies to known prenylated signaling proteins. Aim 3 is to determine if Ras is recruited into domain specific signaling pathways during post-testicular sperm maturation. Immunolocalization and immunoblotting will be utilized to determine if Ras is integrated into specific membrane domains during sperm development, and co-immunoprecipitation experiments will be performed to determine if Ras interacts with domain-specific effector proteins. Inhibitors of Ras protein-protein interactions will be tested for effects on specific sperm functions. These experiments will provide new insights into mechanisms which assemble the sperm plasma membrane and have application to strategies for fertility regulation and/or improvement.

描述：（改编自申请人的描述）精子等离子体 膜被划分为不同分子组成的结构域和 受精的功能。 不同膜域的组装是 在精子发生过程中启动，并在新的后持续 附睾的发展。 精子和精子都有 Farnesylsylansferase（FTase），该酶在翻译后蛋白中起作用 脂质和信号蛋白Ras，一种FTase底物，需要 促函数表达的前化。 远距离目标 提案是定义蛋白质前化在精子发育中的作用 并确定是否募集了特定膜的蛋白质 域并调节精子功能。 三个目标解决了这些目标。 目标1 是确定隔离法尼基转移酶的机制 精子的特异性细胞质区域。 共沉淀 分析将用于确定FTase是否与特定的锚定结合 蛋白质和免疫电子显微镜将用于鉴定结构 产生限制的FTase分布模式的机制。 目标2 是确定FTase在原始和膜靶向中是否功能 精子和附睾精子中不同的蛋白质。 Farnesylate 代谢标记的精子和附子精子的蛋白质将 通过N末端测序和通过免疫印迹来识别 对已知的原始信号蛋白的抗体。 目标3是确定是否 RA被招募到域特定的信号通路 杀菌后精子成熟。 免疫定位和免疫印迹 将利用用于确定RAS是否集成到特定的膜中 精子发育过程中的域和共免疫沉淀实验 将执行以确定RAS是否与域特异性相互作用 效应蛋白。 RAS蛋白 - 蛋白质相互作用的抑制剂将是 测试了对特定精子功能的影响。 这些实验会 提供有关组装精子等离子体的机制的新见解 膜，并适用于生育法规的策略和/或 改进。