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

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.

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。

人胱硫醚β-合酶（CBS）是细胞转硫化途径的关键酶，与体内重要还原型物质谷胱甘肽的合成密切相关，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/游离巯基氧化还原模块，但其功能至今不清楚。(1) 本研究通过对CBS重组蛋白进行氧化还原活性分析、质谱分析和定点突变分析表明CBS活性受到CPGC二硫键/游离巯基氧化还原模块的调控，在氧化还原应激条件下CXXC模体通过二硫键/游离巯基的相互转换调控CBS活性。同时，通过H2S探针的活细胞实时成像研究发现在细胞氧化还原应激通过介导CBS活性调控细胞内H2S的含量，该研究结果发表在J. Biol. Chem.2018, 293(7):2523-2533。另外，我们还发现体内合成H2S的另外一种酶，胱硫醚γ-裂解酶(CSE)蛋白表面也存在2个CXXC模体，其活性也受到氧化还原调控，详细机制正在研究中。(2) 建立了荧光探针法测定CBS活性的方法，通过高通量筛选6491种天然产物化合物文库，获得了6种CBS的选择性抑制剂，针对其中2种化合物金丝桃素(hypericin)和sikokianin C 分别进行了抗结肠癌细胞增殖和动物实验，结果表明2种化合物均通过抑制体内CBS活性而具有显著的抗结肠癌活性。该研究结果在Med. Chem. Commun.期刊发表论文2篇，授权发明专利2项。(3) 重组CBS和CSE可用于同型半胱氨酸的体外诊断，我们通过重组大肠杆菌发酵和亲和层析纯化获得了公斤级的高纯度CBS和CSE重组蛋白，该技术已经进行转让并进行商业化生产，重组CBS和CSE已经用于制备同型半胱氨酸测定试剂盒，试剂盒已经在医院得到应用。另外，我们通过固定化酶催化制备了S-腺苷蛋氨酸(CBS的激活剂)，该研究结果在Catalysts期刊发表论文1篇，申请发明专利1项。