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.
出处
《中南医学科学杂志》
CAS
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)
