赖氨酸同型半胱氨酸修饰促新发突变致先天性心脏病的分子机理

Elevated homocysteine levels are established risk factors for congenital heart disease (CHD) with limited underlying mechanism elucidated. In previous studies, we found that genetic variants of the genes in the folate metabolism pathway were associated with elevated homocysteine concentrations and increased risk of CHD simultaneously, indicating the genetic component for hyperhomocysteinemia and the pathological role of homocysteine in the birth defects (Circulation, 2012 and 2017; Cell Research, 2013; European Heart Journal, 2014). We next observed either elevated homocysteine level or activated methionyl-tRNA synthetase (MARS) resulted in the increased protein lysine homocysteinylation (K-Hcy). In a cell-wide proteomics survey to look for K-Hcy substrates, we identified more than 1,000 different proteins from HEK293T cells were modified by K-Hcy, including the ATR (Ataxia telangiectasia and Rad3 related), the key regulator in DNA-damage response. Since it is well known that de novo mutation induces the onset of CHD, we hypothesize that increased homocysteine lead to the occurrence of CHD through modifying ATR by homocysteinylation, inhibiting DNA damage repairing pathway and promoting the formation of de novo mutations. In the current project, we will confirm this hypothesis by performing experiments in vitro, in culture cells and human samples. To reveal the relation between the dosage of MARS and the level of K-Hcy, we will analyze the copy numbers of K-Hcy-promoting MARS and the plasma K-Hcy levels in the blood samples of CHD patients and healthy subjects. We next investigate the molecular mechanism of how MARS and Hcy coordinate K-Hcy modification, inactivate ATR and its downstream DNA damage repair pathway, accumulate DNA damage, promote the formation of de novo mutations, and finally lead to the onset of CHD. This study will reveal a novel pathological mechanism of homocysteine induced CHD and shed lights on novel intervening strategies of birth defects.

同型半胱氨酸（Hcy）作为先天性心脏病的独立风险因子，其致病机理至今未完全阐明。申请人前期研究发现，叶酸代谢中系列代谢酶的基因变异均通过升高Hcy导致先心病。后续机制研究发现Hcy及甲硫氨酰-tRNA合成酶（MARS）均会引起细胞内蛋白质赖氨酸同型半胱氨酸化（K-Hcy）修饰上升；修饰蛋白组学分析发现DNA修复关键蛋白ATR等可被K-Hcy修饰。新发突变是先心病发生的重要原因。据此我们推测：高Hcy可能通过升高K-Hcy，抑制DNA损伤修复，促进新发突变而诱发先心病。本项目拟利用遗传学、生物化学和分子细胞生物学手段，在体外、细胞系及临床样本层面探明1）MARS和K-Hcy均是先心病的风险因子；2）Hcy通过MARS上调K-Hcy；3）高K-Hcy抑制DNA损伤修复；4）高K-Hcy诱发新发突变。项目成功实施将揭示Hcy致先心病的新机制，为发展先心病及其它出生缺陷的诊断及干预新手段提供依据。

遗传和环境因素导致包括先天性心脏病（先心）等重大出生缺陷的机制长期未明。申请人前期系统揭示了遗传变异通过引起同型半胱氨酸(Hcy)代谢异常致先心（Circulation 2012, 2017；Cell Res 2013；Eur Heart J 2014），并发现代谢物信号失调致多种疾病的分子机制（J Clin Invest 2014；Signal Transd & Tar Ther 2017），提示代谢物信号可能在心脏发育中具有重要功能。后续机制研究发现 Hcy及甲硫氨酰-tRNA合成酶（MARS）均会引起细胞内蛋白质赖氨酸同型半胱氨酸化（K-Hcy）修饰上升；修饰蛋白组学分析发现DNA修复关键蛋白ATR等可被K-Hcy修饰，提示Hcy修饰可能会通过积累新发突变来增大先心风险。基于代谢物具有细胞发育信号调控功能这一原创思想，申请人在本项目支持下，系统阐释了Hcy增大先心的分子机制，并在代谢物失调致病机制相关前沿理论问题方向做出系列具有行业影响的重要进展：（1）发现Hcy可以通过对蛋白质的翻译后修饰改变蛋白功能，进而产生同型半胱氨酸细胞信号；在此基础上系统阐明同型半胱氨酸信号失调导致出生缺陷等疾病的分子机理，加深了代谢物信号失调病理价值的认识（Cell Rep 2018；EMBO Mol Med 2020）。除了翻译后修饰蛋白赖氨酸残基，我们发现Hcy还可以在内质网中翻译后修饰蛋白半胱氨酸修饰，通过对胰岛素受体的修饰诱发胰岛素抵抗（Cell Rep 2021）。（2）通过对磷酸戊糖途径关键酶TKTL1、脂肪酸氧化关键酶ECHS1蛋白功能和调控机制的全新解析，揭示细胞信号调控核糖-5-磷酸、脂肪酸氧化的分子机制，阐明了葡萄糖/脂肪酸等营养物质在细胞物质和能量合成中的分配机制（Nat Commun 2019；Cancer Res 2020）。（3）原创揭示琥珀酸、β羟基丁酸和胆酸等系列代谢物信号失调在滋养细胞和肝发育相关疾病的病理作用，扩展了发育与出生缺陷的代谢物信号研究领域（Signal Transd & Tar Ther 2021；Nat Commun 2021；J Hepatol 2021）。系列工作推进了“代谢物失调致病机制”这一领域的发展，提升了我国在相关领域的国际影响。