Redox modification of CLOCK mediates the regulatory function of endogenous H2O2 in mouse cellular respiration

Abstract

Objective To explore the mechanisms of transcription factor circadian locomalor output cycles kaput(CLOCK) in mediating regulatory function of endogenous hydrogen peroxide(H2O2) for cell respiration. Methods Cellular oxygen consumption capacity and glycolysis in ClockC195S 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 H2O2 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 Clockwt and ClockC195S 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 ClockC195S MEFs. The treatment with Trolox decreased endogenous H2O2 level and dampened respiration capacity in Clockwt MEFs but not in ClockC195S MEFs. Conclusions CLOCK mediates the regulation of endogenous H2O2 for cellular respiration through its redox modification at Cys195.

Keywords

• cellular respiration|nicotinamide adenine denudeotide(nad) biosynthesis|redox modification of clock