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

项目名称： 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

项目编号： No.81471454

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 赵健元

作者单位： 复旦大学

项目金额： 73万元

中文摘要： 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。

中文关键词： 同型半胱氨酸化修饰；超氧化物歧化酶；活性氧；出生缺陷

英文摘要： 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.

英文关键词： Homocysteinylation;Superoxide dismutase;Reactive oxygen species;Birth defect