REGULATION OF SPERM FUNCTION BY PROTEIN PRENYLATION

描述：(根据申请者的描述改编)精子血浆 膜被划分为具有不同分子组成的区域和 在受精中的作用。不同膜结构域的组装是 在精子发生过程中开始，在睾丸后持续 附睾体的发育。精子细胞和精子两者兼有 在翻译后蛋白中起作用的法尼基转移酶(Ftase) 脂肪作用，以及信号蛋白RAS，这是一种FTase底物，需要 为表达功能而进行的戊烯基化。这样做的长期目标是 建议确定蛋白质预烯基化在精子发育中的作用 并确定预烯基化的蛋白质是否被招募到特定的膜上 结构域和调节精子功能。有三个目标解决了这些目标。目标1 是为了确定将法尼基转移酶隔离为 精子细胞的特定细胞质区域。免疫共沉淀 将使用分析来确定FTase是否绑定到特定锚定 蛋白质和免疫电子显微镜将被用来鉴定结构 产生限制性FTase分布模式的机制。目标2 是为了确定FTase是否在苯丙基化和膜靶向中起作用 在精子细胞和附睾精子中的不同蛋白质。法尼化 代谢性标记的精子细胞和附睾精子的蛋白质 经N-末端微测序和免疫印迹鉴定 针对已知的预烯基化信号蛋白的抗体。目标3是确定 RAS在过程中被招募到域特定信号通路中 睾丸后精子成熟。免疫定位和免疫印迹 将用于确定RAS是否整合到特定的膜中 精子发育过程中的结构域和免疫共沉淀实验 将执行以确定RAS是否与特定于域的 效应器蛋白。RAS蛋白-蛋白质相互作用的抑制剂将是 测试对特定精子功能的影响。这些实验将 为组装精子浆的机制提供了新的见解 膜并可应用于生育调节和/或战略 进步。

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.