氧化还原电位介导人胱硫醚β合酶活性调控的分子机制及其对细胞硫化氢代谢的影响

项目名称： 氧化还原电位介导人胱硫醚β合酶活性调控的分子机制及其对细胞硫化氢代谢的影响

项目编号： No.31471086

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 生理学与整合生物学

项目作者： 牛卫宁

作者单位： 西北工业大学

项目金额： 86万元

中文摘要： 人胱硫醚β合酶（CBS）是细胞转硫化途径的关键酶，与谷胱甘肽合成密切相关，同时催化合成信号分子H2S，其突变导致多种疾病。CBS序列中包含CPGC二硫键/巯基氧化还原模块，但其功能至今不清楚。前期研究我们建立了以不含游离巯基的甲基半胱氨酸为底物的CBS活性测定方法，据此发现在体外CBS活性受到氧化还原电位调控，定点突变结果表明CPGC模块可能是CBS感受氧化还原电位变化的分子开关，但其分子机制和生理意义并不清楚。本项目研究内容：1.研究CBS感受氧化还原电位的分子开关以及氧化还原电位调控CBS活性的分子机制和结构基础。2.借助活细胞氧化还原电位实时测定技术，研究细胞中CBS的CPGC模块氧化还原状态的变化及其对酶活性的调控机制3.借助H2S测定荧光探针，在细胞和组织中研究氧化还原电位介导CBS的活性调控对H2S代谢的影响。最终阐明氧化还原电位调控CBS活性的分子机制及对H2S代谢的影响。

中文关键词： 氧化还原电位；人胱硫醚β合酶；自由基；硫化氢；活性调控

英文摘要： Human cystathionine β-synthase(CBS) is the key enzyme in the first step of the transsulfuration pathway,and is closely related the biosynthesis of glutathione(GSH). Alternatively,CBS can generate hydrogen sulfide(H2S), a gaseous signal molecule,by condensation of cysteine and homocysteine. CBS deficiency or mutation causes the enzymatic inactivation or the reduced activity,resulting in a variety of diseases.Human CBS includes the CPGC redox motif,which has been observed in the oxidized disulfide and reduced dithiol states, but to date the function of CPGC motif remains unclearly.In our previous study,we established the novel method for measuring the CBS activity by using methylcysteine(without free thiol groups)as substrate.Based on this method we found that in vitro CBS activity was modulated by redox potential.The result of site-directed mutagensis indicated that CPGC motif could be a molecular switch of CBS that senses the change of redox potential, but the detailed molecular mechanism and physiological relevance remain unclear. The main research contents of this project are as follows: (1) confirm the molecular switch of CBS in sensing the change of redox potential,and elucidate the molecular mechanism and structral basis on redox potential modulating CBS activity. (2)In cells,confirm the change of redox state of CPGC motif in CBS,and elucidate the molecular mechanism of CBS redox state modulating the enzymatic activity by means of the real-time imaging of the intracellular redox potential. (3)In cells and tissue,research the effect of CBS activity mediated by redox potential on the metablism of H2S by using the fluorescent probe for detecting H2S.The results will elucidate the molecular mechanism of redox potential modulating CBS activity and its effect on the metabolism of H2S.

英文关键词： redox potential;human cystathionine β synthase;free radicals;hydrogen sulfide;activity regulation