赖氨酸乙酰化修饰与精子成熟调控

From its generation to maturation, spermatozoon undergoes series of modifications, among which post translational modification of sperm proteins,has been proved to be involved in sperm motility, capacitation, sperm-zona binding and sperm fertilizing capacity. Lysine acetylation, as a hotspot in post translational modification researches,has not been reported regarding its roles and significance in sperm maturation.We have raised highly specific monoclonal antibodies that recognize acetylated Lysine . With these antibodies,we observed dramatic changes of lysine acetylation profiles of mice sperms from caput epididymis to cauda epididymis. Besides, we also determined the lysine acetylayion profiles of a large array of human sperm proteins.These proteins with lysine acetylayion have been correlated with sperm metabolic regulation and cell signaling in bioformatic analysis. In addition, incubation of sperms with inhibitors of acetyltransferases and deacetyltransferases resulted in sperm motility inhibition in vitro, which suggested that lysine acetylation and deacetylayion should play key roles in the regulation of sperm maturation. Therefore, with an aim to further understand the regulatory mechanisms of sperm maturation, we plan to identify key proteins that regulate sperm maturation through lysine acetylation modification and to explore their individual functions and regulation mechanisms in sperm maturation.Through detecting the lysine acetylation of key proteins in fertile and infertile sperm samples, we will preliminarily evaluate the clinic potential of lysine acetylation testing either in diagnosis or treatment.

精子从发生到成熟经历了一系列的修饰作用，其中精子蛋白翻译后修饰已被证实与精子运动、获能、结合透明带及受精能力相关，而赖氨酸乙酰化修饰是近年来翻译后修饰研究的热点，但是其在精子成熟中的作用及意义鲜见报道。在前期工作中，我们成功制备了高特异性的赖氨酸乙酰化单克隆抗体，发现小鼠附睾精子从附睾头到附睾尾乙酰化修饰变化明显，并且我们在人精子上鉴定出大量乙酰化修饰蛋白，而这些蛋白与精子的代谢和信号传导等密切相关，并且乙酰化酶和去乙酰化酶抑制剂对体外培养精子的运动有明显的抑制作用，提示乙酰化修饰可能在精子成熟过程发挥重要的调控作用。本项目拟在上述工作基础上，筛选出精子成熟过程中通过乙酰化修饰发挥重要调控作用的靶蛋白，探讨其乙酰化修饰的具体功能及其在精子成熟中的作用机理，以期加深我们对精子成熟调控机制的了解；并通过收集和检测正常和不育人群的各类精子样本，初步分析赖氨酸乙酰化修饰在临床诊治中的应用前景。

男性不育越来越受到全球广泛关注，然而，人们对赖氨酸乙酰化修饰在精子发育异常中的作用了解不够，我们首次制备了经过严格质控的能识别泛赖氨酸乙酰化修饰的单克隆抗体，通过对人精子发生乙酰化修饰的肽段进行富集，我们建立了人精子乙酰化修饰蛋白与修饰位点谱，乙酰化修饰肽段富集率高达31%，有456个蛋白973个修饰位点被鉴定，其中包括205个未报道过的蛋白和671个新的位点。我们发现人精子上发生乙酰化修饰的蛋白存在XXXKYXXX, XXXKFXXX和 XXXKHXXX三个保守结构域，经注释发现这些修饰蛋白主要定位于线粒体与胞浆组分，与精子的多个代谢过程相关，通过分析获能前后人精子乙酰化修饰差异蛋白发现：这些蛋白主要与精子的获能、精卵识别、精卵融合以及受精相关，提示相关功能蛋白的乙酰化修饰可能在获能等过程发挥重要调控作用。我们还对修饰蛋白与相关疾病和药物进行了生物信息学分析。通过对临床样本分析我们发现在正常人、弱精子症与死精子症人样本中乙酰化转移酶和去乙酰化酶存在规律性差异。同样我们发现了电压依赖性离子通道蛋白发生乙酰化修饰调控，我们挑选几个表达量相对较高的蛋白作为靶标，购买其对应的抑制剂小分子化合物，将抑制剂与正常人的精子在体外共孵育，检测其抑制剂对精子运动指标的影响，结果发现KAT（赖氨酸乙酰转移酶统称）抑制剂能显著抑制精子运动，并且发现KAT抑制剂处理精子后能明显下调VDAC2(K74)的乙酰化修饰程度。.总之通过本课题我们获得了高质量泛赖氨酸乙酰化修饰单克隆抗体，建立了未获能的人精子乙酰化修饰蛋白与修饰位点谱，初步揭示蛋白的乙酰化修饰与精子功能的关系。另外，我们额外筛选了天然产物中具有乙酰化酶抑制活性的单体化合物，发现冬凌草甲素具有广谱的乙酰化酶抑制活性，能有效抑制P300, GCN5, Tip60与pCAF的乙酰化酶活性。