摘要
目的探究生物钟核心转录因子昼夜节律运动输出周期蛋白(CLOCK)介导内源性过氧化氢(H_(2)O_(2))对细胞呼吸的调节作用和机制。方法利用Seahorse细胞能量代谢分析仪检测Clock^(C195S)小鼠胚胎成纤维细胞(MEFs)和成体成纤维细胞(MAFs)细胞氧耗能力和糖酵解能力;通过q-PCR检测细胞呼吸关键代谢酶基因表达水平;MEFs进行抗氧化剂Trolox处理后,用Amplex■ Red试剂盒检测内源性H_(2)O_(2)的含量,并通过生物素碘乙酰胺(BIAM)探针标记检测CLOCK蛋白氧化还原修饰的改变,进一步检测处理后的细胞氧耗能力和细胞呼吸关键代谢酶基因表达水平。结果Clock^(C195S)细胞氧耗能力和糖酵解能力下降,烟酰胺腺嘌呤二核苷酸(NAD)合成关键酶烟酰胺单核苷酸腺苷转移酶(NMNAT2)表达降低(P<0.05),NAD含量下降;Trolox处理导致细胞内源性H_(2)O_(2)含量降低,Clock wt MEFs氧耗能力降低而Clock^(C195S) MEFs变化无统计学意义。结论CLOCK蛋白可以通过195位点半胱氨酸氧化还原修饰介导内源性H_(2)O_(2)对细胞呼吸的调节作用。
Objective To explore the mechanisms of transcription factor circadian locomalor output cycles kaput(CLOCK)in mediating regulatory function of endogenous hydrogen peroxide(H_(2)O_(2))for cell respiration.Methods Cellular oxygen consumption capacity and glycolysis in Clock^(C195S) mouse embryonic fibroblasts(MEFs)and adult fibroblasts(MAFs)were tested by Agilent Seahorse XF Cell Mito Stress Test Kit and Glycolysis Stress Test Kit.The expression of key genes participating in cell respiration was detected by q-PCR.The endogenous H_(2)O_(2) levels and the redox modification of CLOCK were detected by Amplex■ Red Hydrogen Peroxide/Peroxidase Assay Kit and biotin-conjugated iodoacetamide(BIAM)were used respectively after the treatment of antioxidant Trolox.Changes in the oxygen consumption capacity and in key respiratory gene expression after the treatment of antioxidant Trolox were also tested in Clock wt and Clock^(C195S) MEFs.Results Cellular oxygen consumption capacity,glycolysis and the expression of NMNAT2,a key enzyme in nicotinamide adenine denudeotide(NAD)biosynthesis,as well as NAD content all decreased in Clock^(C195S) MEFs.The treatment with Trolox decreased endogenous H_(2)O_(2) level and dampened respiration capacity in Clock wt MEFs but not in Clock^(C195S) MEFs.Conclusions CLOCK mediates the regulation of endogenous H_(2)O_(2) for cellular respiration through its redox modification at Cys195.
作者
常薇薇
李勋凯
张祝琴
陈厚早
刘德培
CHANG Weiwei;LI Xunkai;ZHANG Zhuqin;CHEN Houzao;LIU Depei(State Key Laboratory of Medical Molecular Biology,Department of Biochemistry and Molecular Biology,Institution of Basic Medical Sciences CAMS,School of Basic Medicine PUMC,Beijing 100005,China)
出处
《基础医学与临床》
2023年第5期745-750,共6页
Basic and Clinical Medicine
基金
国家重点研究与发展计划(2021YFA0804903)
中国医学科学院医学科学创新基金(CIFMS,2021-I2M-1-008)。
