蛋白质巯基亚硝基化对RSC-364细胞cAMP反应元件结合蛋白活性的影响

Effect of protein S-nitrosylation on cAMP response element-binding protein activity in RSC-364 cells

目的:观察RSC-364细胞外源性亚硝基化对cAMP反应元件结合蛋白(CREB)活性的影响。方法:提取RSC-364细胞总蛋白,与100μmol/L还原型谷胱甘肽(GSH)、100μmol/L NO供体亚硝基化谷胱甘肽(GSNO)、10mmol/L亚硝基化抑制剂二硫苏糖醇(DTT)及溶剂单独或联合应用孵育15min完成外源性亚硝基化,采用生物素转化法与Western blotting技术检测亚硝基化蛋白表达水平;分别用溶剂或重组大鼠白细胞介素-1β(rIL-1β)10μg/L孵育RSC-364细胞1h,提取细胞核蛋白,经外源性亚硝基化后用电泳迁移率改变分析(elec-trophoresis mobility shift assay,EMSA)观察亚硝基化作用对CREB活性的影响。结果:RSC-364细胞总蛋白经GS-NO孵育后亚硝基化蛋白表达水平明显增高,而应用DTT可抑制亚硝基化水平;GSNO与RSC-364细胞核蛋白孵育后,明显抑制了rIL-1β诱导的CREB活性(P<0.01),GSNO的作用可被DTT所逆转(P<0.01)。结论:NO可通过亚硝基化作用抑制RSC-364细胞CREB活性。

AIM ： To investigate the effect of protein S-nitrosylation on cAMP response element-binding protein（CREB） activity in RSC- 364 cells. METHODS：①RSC -364 cells cytoplasmic extracts were incubated with 100μmol/L GSH （control chemical） , 100μmol/L GSNO （a donor of NO） in the presence or absence of 10 mmol/L DTT（inhibitor of S-nitrosylation） for 15 min at room temperature. The S-nitrosylated proteins were determined by biotin switch assay. The expression of S-nitrosylated proteins was assayed by Western blotting. ②RSC - 364 cell nuclear extracts were subjected to S -nitrosylation and electrophoretic mobility shift assay （EMSA） to analyze the CREB DNA binding activity after 1 h stimulation of rIL-1β（ 10μg/L）. RESULTS： GSNO obviously increased the expression of nitrosy-lated proteins and inhibited the CREB activity, which was reversed by DTT. CONCLUSION ： S-nitrosylation may inhibit the CREB activity in RSC-364 cells.