| 王环愿,徐嘉惠,周艺,孙琪,董玉,王雯.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.[J].中南医学科学杂志.,2017,(1):27-31. |
| 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-mediatedcystathionine β-synthase inactivation |
| 投稿时间：2017-10-09 |
| DOI：10.15972/j.cnki.43-1509/r.2017.01.007 |
| 中文关键词: 胱硫醚β-合酶 硝基化修饰 同型半胱氨酸 |
| 英文关键词:cystathionine β-synthase nitration homocysteine |
| 基金项目:国家自然科学基金(编号81370450). |
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| 中文摘要: |
| 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(cystathionine β-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。 |
| 英文摘要: |
| Objective To explore the role of Tyr163 and Tyr223 nitration in homocysteine (Hcy) -mediated CBS inactivation.MethodsConstruct three CBS mutant plasmids (WT,Y163A and Y223F),by replacing two possible nitrated tyrosine residues to phenylalanine or alanine.HEK293H 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.ResultsWT,Y163A and Y223F plasmids were all expressed successfully and the mutant CBS could have activities.Hcy induced high nitration of WT CBS,meanwhile,nitration of Y163A and Y223F CBS was decreased significantly (P<0.05,P<0.01).Compared with WT group,the CBS activity was decreased significantly (P<0.01) in WT +Hcy group.Compared with WT +Hcy group,the CBS activities were both increased in Y163A+Hcy and Y223F+Hcy group (P<0.01,P<0.05).ConclusionStimulation 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. |
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