氧化还原应激介导CXXC模体调控人胱硫醚γ-裂解酶/硫化氢体系的机制

Human cystathionine γ-lyase (CSE) is the one of three key enzymes which catalyzes the synthesis of hydrogen sulfide (H2S) in vivo, also involved in the biosynthesis of antioxidants, cysteine and glutathione (GSH). The change of CSE activity is closely correlated to many diseases. Human cystathionine γ-lyase contains two CXXC motifs, but the function remains elusive. In our previous study, a method for determining the CSE activity had been developed by using S-methylmethionine as substrate. Based on this method we found that CSE activity was redox-regulated in vitro. The results of mass spectrometry analysis and site-directed mutagenesis of CXXC motifs revealed that one of the CXXC motifs (CYLC motif) could be the redox sensor of CSE, but the detailed regulatory mechanism and biological functions remain unclear. The research contents of this proposal are as follows: (1) Clarify the molecular mechanism and the structural basis of CYLC motif as redox sensor modulating the CSE activity. (2) In human aortic smooth muscle cells, the cytosolic glutathione redox potential (EGSH) is determined by using the fluorescent probe. The effect of redox stress on the thiol-disulfide state of CYLC motif in CSE is elucidated. (3) In human aortic smooth muscle cells, the effect of redox stress on the CSE/H2S system is studied by using the H2S fluorescent probe. These results will elucidate the mechanism of redox stress modulating the redox state change of CXXC motif in CSE, and its effect on the CSE/H2S system. This research will provide a new perspective for the studies of diseases relating to the CSE/H2S system.

胱硫醚γ-裂解酶(CSE)是人体内催化合成硫化氢的三种关键酶之一，同时参与抗氧化物质谷胱甘肽的合成，其活性改变与多种疾病密切相关。CSE序列中包含两个CXXC模体，但其生物学功能仍未阐明。我们前期研究发现CSE活性受到氧化还原调控，质谱分析和定点突变结果表明其中一个CYLC模体可能是CSE的氧化还原感受器，但其详细的调控机制及生物学功能并不清楚。本项目研究内容：(1)研究CYLC模体作为氧化还原感受器调控CSE活性的分子机制和结构基础。(2)通过荧光探针实时测定活细胞氧化还原状态，研究氧化还原应激对细胞内CSE的CYLC模体游离巯基/二硫键两种状态转换的调控。(3)通过荧光探针实时测定活细胞内硫化氢含量变化，研究氧化还原应激对细胞CSE/H2S体系的影响。最终阐明氧化还原应激介导CXXC模体的氧化还原状态改变，继而调控CSE/H2S体系的机制，为CSE/H2S体系相关疾病的研究提供新视角。

胱硫醚γ-裂解酶（CSE）是细胞转硫化途径的关键酶，与体内重要的抗氧化物质谷胱甘肽的合成密切相关。同时CSE也是人体催化合成信号分子硫化氢（H2S）的三种关键酶之一， 尤其在心血管系统中CSE 是合成H2S 的主要酶，CSE/H2S 体系与多种心血管疾病密切相关。CSE蛋白序列中包含两个CXXC模体，但其功能并不清楚。本项目主要研究结果如下：（1）在氧化还原应激条件下CSE蛋白中C252YLC255模体通过二硫键/游离巯基的相互转换调控酶活性，氧化型CSE催化合成H2S的活性是还原型CSE的2.5倍。借助H2S荧光探针细胞成像研究表明血管平滑肌细胞氧化应激能增强CSE活性，继而显著提高细胞内H2S含量；（2）硫巯基化修饰（-SSH）能反馈抑制CSE/H2S活性。在氧化应激条件下增加的CSE活性能产生过量的H2S，提高浓度的H2S对CSE蛋白的137位Cys残基进行硫巯基化修饰，从而反馈抑制CSE活性。CSE通过CYLC模体氧化还原调控以及137位Cys残基的硫巯基化修饰联合调控细胞CSE/H2S活性。（3）发现了一种新的大肠杆菌H2S合成酶—半胱氨酸脱硫酶（IscS）。在厌氧条件下大肠杆菌细胞合成H2S的主要酶是IscS，而不是报道的在好氧条件下合成H2S的3-巯基丙酮酸硫转移酶（3-MST）。进一步研究表明外源H2S能恢复IscS缺陷大肠杆菌的增殖和细胞能量代谢。（4）外源性H2S能促进半胱氨酸脱硫酶（IscS）缺陷的大肠杆菌和哺乳动物细胞铁硫簇的合成。通过代谢组学、蛋白质组学、基因敲除和生化酶学研究表明外源性H2S能为IscS缺陷细胞铁硫簇合成提供硫源，从而恢复细胞线粒体功能。本研究为铁硫簇合成障碍相关疾病的治疗提供新视角。