Tyr163和Tyr223硝基化修饰参与同型半胱氨酸诱导的胱硫醚β-合酶失活  

Nitration of tyr163 and tyr223 involved in homocysteine-mediated cystathionine β-synthase inactivation

作　　者：王环愿 徐嘉惠[1,2] 周艺[1,2] 孙琪[1,2] 董玉[1,2] 王雯[1,2] 

机构地区：[1]首都医科大学基础医学院生理学与病理生理学系,北京100069 [2]代谢紊乱相关心血管疾病北京市重点实验室

出　　处：《中南医学科学杂志》2017年第1期27-31,共5页Medical Science Journal of Central South China

基　　金：国家自然科学基金(编号81370450)

摘　　要：目的探讨酪氨酸(tyrosine,Tyr,Y)残基(Tyr163,Tyr223)硝基化修饰与同型半胱氨酸(homocysteine,Hcy)诱导的胱硫醚β-合酶(cystathionineβ-synthase,CBS)活性降低之间的关系。方法运用体外基因突变的方法,将CBS可能发生硝基化修饰的酪氨酸残基替换为苯丙氨酸(phenylalanine,F)或丙氨酸(alanine,A),构建野生型(WT)和突变型(Y163F,Y223A)质粒,转染HEK293H工具细胞,诱导重组基因表达;利用Hcy刺激后,提取细胞蛋白,分析硝基化CBS的含量及其酶活性。结果 WT,Y163A和Y223F质粒均能正常表达CBS并且具有活性;Hcy(500μmol/L,24h)诱导WT CBS发生硝基化修饰,但是Y163A和Y223F突变使CBS的硝基化程度明显下降(P<0.05,P<0.01);WT+Hcy组CBS活性明显低于WT组(P<0.01);Y163A+Hcy组和Y223F+Hcy组CBS活性高于WT+Hcy(P<0.01,P<0.05)。结论 Hcy可以导致CBS活性降低,与Hcy诱导CBS酪氨酸残基(Tyr163,Tyr223)发生硝基化修饰有关。Objective To explore the role of Tyr163 and Tyr223 nitration in homocysteine（ Hcy）-mediated CBS inactivation. Methods Construct three CBS mutant plasmids（ WT,Y163 A and Y223F）,by replacing two possible nitrated tyrosine residues to phenylalanine or alanine.HEK293 H cells were transfected with three plasmids to induce the expression of reconstructed genes.After the stimulation of Hcy（ 500 μmol / L,24h）,both the level of nitrated CBS and activity of CBS were determined in transfected cells. Results WT,Y163 A and Y223 F plasmids were all expressed successfully and the mutant CBS could have activities. Hcy induced high nitration of WT CBS,meanwhile,nitration of Y163 A and Y223 F CBS was decreased significantly（ P0.05,P0.01）.Compared with WT group,the CBS activity was decreased significantly（ P0.01） in WT ＋Hcy group.Compared with WT ＋ Hcy group,the CBS activities were both increased in Y163 A ＋ Hcy and Y223 F ＋ Hcy group（ P0.01,P0.05）. Conclusion Stimulation with homocysteine can lead to the decreased bioactivity of CBS,which is related with the CBS nitration at Tyr163,Tyr223 induced by the enhanced nitrative stress.

关 键 词：胱硫醚Β-合酶 硝基化修饰 同型半胱氨酸 

分 类 号：R363[医药卫生—病理学]