赖氨酸同型半胱氨酸修饰致先天性心脏病的作用及机理

Elevated homocysteine (HCY) levels are an independent risk factor for a series of birth defects, including congenital heart disease (CHD). Maternal folic acid supplements can lower HCY and reduce 40-60 % CHD. This suggests that there are unknown downstream regulators of HCY to induce CHD. HCY is activated by methionine tRNA synthetase (MARS) into homocysteine thiolactone (HTL), which can modify lysine residue and in turn affect protein function. Our previous study found that more than 1,000 proteins can be modified by HTL. HTL modification reflects the levels of HCY and affects development pathways, such as Wnt. These results linked our two previous findings: 1) Gene variants ofseveral folate metabolic enzymes can increase HCY level (Circulation 2012, etc.) and 2) disrupted non-canonical Wnt pathway cause several birth defects such as neural tube defects (NEJM, 2010). Therefore, we conclude that: HTL modification on protein lysine residue, which is regulated by HCY and MARS, is an important causing factor for CHD. The goals of this project are to understand: 1) the HTL modified cellular protein spectrum by using proteomics; 2) the regulatory role of MARS on HTL modification by molecular and cell biology; 3) the link between mutations in MARS and CHD; and 4) the effect of synthesized small molecules on CHD prevention. Those small molecules are synthesized to mimic HTL and/or regulate MARS activity. Implementation of the project can improve our understanding of the causing effects of high HCY on CHD; and provide new ideas in how to prevent CHD.

同型半胱氨酸(HCY)升高是先天性心脏病等出生缺陷的独立风险因子。但是补服叶酸降低HCY只能防止40-60%的先心病，提示HCY下游存在致病的未知调控机制。HCY通过蛋氨酸tRNA合成酶(MARS)激活成同型半胱氨酸硫内酯(HTL)后修饰赖氨酸并影响蛋白功能。我们前期发现超过千数蛋白能被HTL修饰，初步功能研究发现HTL修饰是感知HCY水平并影响Wnt通路的主要机制。这些结果建立了我们前期发现的系列叶酸代谢酶突变升高HCY（Circulation等）和非经典Wnt通路失调致出生缺陷（NEJM）的联系。推论：受HCY及MARS调控的HTL修饰是先心病的重要诱因。本项目将完善蛋白HTL修饰谱，研究MARS调控HTL修饰的机制，鉴定MARS突变与先心病的关联，合成并研究调控MARS活性的小分子对HTL修饰及先心病的干预效果，有望阐明高HCY致先心病新机制，为设计干预新手段提供全新的视角。

同型半胱氨酸(HCY)升高是先天性心脏病的独立风险因子，但补服叶酸降低HCY只能防止部分先心病，提示HCY下游存在致病的未知调控机制。课题组前期工作发现，HCY通过蛋氨酸tRNA合成酶(MARS)激活成同型半胱氨酸硫内酯(HTL)后修饰赖氨酸并影响蛋白功能，推论受HCY及MARS调控的HTL修饰是先心病的重要诱因。为验证该假说而申请本项目。项目组在规模化鉴定HCY修饰谱的基础上，首先证实HCY修饰是广泛存在于细胞内各种蛋白质的翻译后修饰，进一步通过蛋白组学和分子细胞生物学研究发现，高HCY通过提高细胞内超氧化物歧化酶（SOD）的N-同型半胱氨酸修饰（N-Hcy）使之失活，进而升高细胞内的活性氧（ROS）水平，并增加细胞的凋亡。MARS因催化N-Hcy修饰所必须的HTL生成，对SOD的N-Hcy修饰过程发挥调节作用。干扰MARS可钝化Hcy诱导的氧化应激、促进细胞凋亡、抑制细胞增殖等效应；而过表达MARS加剧Hcy诱导细胞凋亡等毒性效应。进一步发现，HCY的结构类似物NAC及AHT竞争性抑制MARS生成HTL，可以降低SOD N-Hcy修饰及活性氧水平，并在神经管畸形（NTD）大鼠模型中证实，HCY类似物可降低NTD的发生。从而初步提示：MARS是高同型半胱氨酸关键性的调节因子，抑制MARS或将为治疗CHD等高同半胱氨酸相关的出生缺陷提供了新策略。本项目还阐明了血叶酸水平与CHD发病率关联性欠佳的原因，证实了神经管畸形多因素阈值假说，为解决基于小鼠这一模式动物鉴定致病基因耗时耗力的困难，合作建立了基于CRISPR /Cas9系统和孤雄单倍体胚胎干细胞技术平台，并初步在神经管畸形的致病基因鉴定中显示出快捷高效的优势。综上，本项目完成预期研究计划，并取得多项原创成果，发表论文7篇，推动了CHD的遗传学机制研究的发展。