蛋白质赖氨酸同型半胱氨酸修饰的底物鉴定及其生理病理效应

Elevated homocysteine levels are known to be a risk factor for birth defects and cardiovascular diseases. The unknown mechanism underlying this effect causes that the hyperhomocysteinemia associated diseases could not be prevented completely through the supplementation of folic acid. In our previous genetics studies, we found that several genetic variants in folate metabolism pathway genes were correlated with elevated plasma homocysteine concentrations and the risk of birth defect simultaneously, indicating the genetic component for hyperhomocysteinemia and the pathological role of homocysteine in the birth defects. Elevated homocysteine concentration increases the level of protein lysine homocysteinylation. However, the effect of protein homocysteinylation on the disease development is not clear. In the current study, we generate polyclonal antibody of homocysteinylated lysine and identify the cell-wide homocysteinylated proteins. Hyperhomocysteinemia is known to be associated with increased cellular reactive oxygen species (ROS) levels. We therefore study the influence of homocysteinylation on the superoxide dismutases (SOD) activities. The contribution of SOD homocysteinylation on ROS modulation is also investigated. To further confirm the notion that homocysteinylation modulated ROS variantions regulates central pathway in development and prolifieration, we survey the Wnt/β-catenin pathway activation caused by homocysteinylation. This study will reveal novel pathological mechanism of protein lysine homocysteinylation and shed lights on novel intervening strategies of birth defects and cardiovascular diseases.

同型半胱氨酸（Hcy）是出生缺陷和心血管疾病的独立风险因子，致病机理不清导致叶酸补服并不能完全预防相关疾病。申请人前期系列遗传学研究发现与出生缺陷相关的多个叶酸代谢酶基因突变导致Hcy水平上升。Hcy浓度的升高会引起细胞内蛋白质赖氨酸同型半胱氨酸化（L-Hcy）修饰水平的上升，然而，L-Hcy在相关疾病发生中的分子细胞机理有待阐明。本研究将利用实验室已获得的L-Hcy高亲和特异抗体，借助我们成熟的修饰谱检测技术平台，在全细胞水平富集并检测L-Hcy修饰底物及位点。在此基础上，选择与出生缺陷相关的蛋白，如超氧化物歧化酶SOD为研究对象，在细胞系及动物模型中探明Hcy通过L-Hcy修饰对SOD活性的影响，以及对活性氧（ROS）水平和发育相关Wnt/β-catenin通路的调控作用。项目的实施将揭示L-Hcy的分子病理基础，为通过降低L-Hcy实现对出生缺陷和心血管疾病的干预新手段提供全新思路。

同型半胱氨酸（Hcy）是出生缺陷、心血管疾病和肿瘤的独立风险因子，致病机理不清导致叶酸补服并不能完全预防相关疾病。申请人前期系列遗传学研究发现与出生缺陷相关的多个叶酸代谢酶基因突变导致 Hcy 水平上升。Hcy 浓度的升高会引起细胞内蛋白质赖氨酸同型半胱氨酸化（K-Hcy）修饰水平的上升，然而，K-Hcy的修饰底物蛋白、修饰位点及其在相关疾病发生中的致病分子机理有待阐明。本项目应用修饰蛋白组学、生物化学、细胞分子生物学、遗传学等方法，在分子细胞、动物模型和临床样本中完成下述工作：1）建立了蛋白质K-Hcy修饰的富集检测方法，构建K-Hcy修饰的功能蛋白资源库，为探索K-Hcy修饰对发育、肿瘤等重大疾病的影响提供研究平台。2）开发了能够用于现场及临床研究的分子诊断技术，为在人群中筛查风险性K-Hcy修饰、预防高K-Hcy修饰相关疾病以及病例研究奠定方法学基础。3）设计合成了干预K-Hcy修饰的系列小分子，为叶酸不应答群体提供了新型疾病预防与治疗策略。4）阐明了K-Hcy通过修饰SOD、ATR等蛋白改变发育相关信号通路并诱发疾病的分子机制。5）发现了FIGN介导的叶酸吸收调控新机制，阐明了叶酸利用效率是先天性心脏病罹患风险的决定因素之一，有可能为困扰临床多年的血液叶酸水平与出生缺陷发病率关联性欠佳这一问题提供答案。