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Abstract
It is known that there are mechanistic links between circadian clocks and metabolic cycles. Reduced nicotinamide adenine dinucleotide (NADH) is a key metabolic cofactor in all living cells; however, it is not known whether levels of NADH oscillate or not. Here we employed REX, a bacterial NADH-binding protein, fused to the VP16 activator to convert intracellular endogenous redox balance into transcriptional readouts by a reporter gene in mammalian cells. EMSA results show that the DNA binding activity of both T- and S-REX::VP16 fusions is decreased with a reduced-to-oxidized cofactor ratio increase. Transient and stabilized cell lines bearing the REX::VP16 and the REX binding operator (ROP) exhibit two circadian luminescence cycles. Consistent with these results, NADH oscillations are observed in host cells, indicating REX can act as a NADH sensor to report intracellular dynamic redox homeostasis in mammalian cells in real time. NADH oscillations provide another metabolic signal for coupling the circadian clock and cellular metabolic states.
Highlights
In the mammalian circadian feedback loop, the CLOCK: BMAL13 complex is the positive element that activates expression of its own transcriptional repressors, Period (Per1 and Per2) and Cryptochrome (Cry1 and Cry2) [1]
When bacteria are growing under an aerobic condition, Rex binds to its operator (ROP) sites upstream of several
Generation of the Rex Transactivator and Its DNA-binding ROPs—To test if Rex could sense NADH/NADϩ in mammalian cells, we adopted the strategy based on the tetracycline-controlled transactivator system which was developed as a genetic switch in eukaryotes [26]
Summary
In the mammalian circadian feedback loop, the CLOCK: BMAL13 (or NPAS2:BMAL1) complex is the positive element that activates expression of its own transcriptional repressors (the negative elements), Period (Per1 and Per2) and Cryptochrome (Cry1 and Cry2) [1]. We employed REX, a bacterial NADH-binding protein, fused to the VP16 activator to convert intracellular endogenous redox balance into transcriptional readouts by a reporter gene in mammalian cells. Among Gram-positive bacteria, the Rex protein is known to act as a redox sensor in response to the cellular NADH/NADϩ ratio changes.
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