同型半胱酸化修饰失活ATR/CHK1通路在神经管畸形发生机制中的作用

Neural tube defects (NTDs) are severe congenital anomaly initiated from the abnormal process of neural tube closure. A plethora of evidence shows that the initiation of NTDs is due to DNA damage. Homocysteine (Hcy), a metabolite of folate pathway, plays an important role in NTDs. The unknown underlying mechanism of Hcy causes that periconceptional supplement of folic acid could not completely protect newborns from NTDs. Elevated hcy concentration increases the level of protein lysine homocysteinylation. However, the effect of protein homocysteinylation on NTDs remains unclear. In the current study, we generate specific polyclonal antibody of homocysteinylated lysine and identify the cell-wide homocysteinylated proteins. Then we identify NTDs related proteins by bioinformatics method. Given ATM and Rad3-related protein (ATR) is a master regulator of DNA damage response and maitains embryo development, we therefore investigate the influence of homocysteinylation on ATR function and its pathway. To further elucidate the pathogenic role of Hcy in NTDs, we explored its regulatory effect on DNA damage and cell biological behaviour via homocysteinylation in vitro and in vivo. This study will reveal novel pathological mechanisms of Hcy-K and shed lights on new intervening strategies of NTDs.

神经管畸形（NTDs）是由于胚胎发育早期神经管化障碍引起的中枢神经系统出生缺陷，细胞DNA损伤参与其发生与发展。叶酸代谢途径产物—同型半胱氨酸（Hcy）与NTDs发生密切相关，其致病机理不清导致围孕期增补叶酸不能完全预防子代NTDs的发生。Hcy浓度的增加会引起细胞内蛋白质赖氨酸同型半胱氨酸化修饰（Hcy-K）水平的上升，然而，Hcy-K在NTDs发生中的分子机理有待阐明。本研究将利用抗Hcy-K特异性抗体，借助蛋白质修饰谱检测平台，在全细胞水平富集并检测Hcy-K修饰底物及位点。在此基础上，通过生物信息学方法筛选与NTDs相关的关键蛋白，如ATM和Rad3相关蛋白（ATR）为研究对象，在细胞系及动物模型中探明Hcy通过Hcy-K修饰对ATR功能和通路的调控作用，以及对DNA损伤和细胞生物学行为的影响。本项目的实施将揭示Hcy-K病理机制，为通过降低Hcy-K防治NTDs提供新的理论依据。

神经管畸形（NTDs）是由于胚胎发育早期神经管化障碍引起的中枢神经系统出生缺陷，细胞DNA损伤参与其发生与发展。叶酸代谢途径产物—同型半胱氨酸（Hcy）与NTDs发生密切相关，其致病机理不清导致围孕期增补叶酸不能完全预防子代NTDs的发生。Hcy浓度的增加会引起细胞内蛋白质赖氨酸同型半胱氨酸化修饰（Hcy-K）水平的上升，然而，Hcy-K在NTDs发生中的分子机理有待阐明。本项目以Hcy-K为观察点，在细胞离体实验中，用抗Hcy-K抗体和修饰蛋白组学方法绘制Hcy-K修饰蛋白谱并筛选出与NTDs发生相关的信号通路；蛋白免疫印迹法检验Hcy通过Hcy-K修饰损伤ATR/CHK1通路活性；彗星分析实验观察Hcy-K修饰对细胞DDR功能的影响；流式细胞仪和CCK8法检测Hcy-K修饰对细胞周期、增殖和凋亡的影响。在NTDs标本中，验证“Hcy通过Hcy-K修饰失活ATR/CHK1通路→DDR功能受损，DNA损伤积聚→细胞凋亡→NTDs”，并检验通过抑制MARS干扰Hcy-K修饰对干预子代NTDs发生的有效性。本项目的实施有助于我们认识Hcy引起NTDs的分子机制，不仅会为NTDs的防治提供全新思路，也会为探索其它Hcy相关疾病的发生、发展、预防和治疗带来一个崭新视角。