title: Circadian Oscillations of NADH Redox State Using a Heterologous Metabolic Sensor in Mammalian Cells.
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.
authors.affiliation: From the Department of Neuroscience and guocun.huang@utsouthwestern.edu.|Center for Circadian Clocks, Soochow University, Suzhou 215123, China.
authors.forename: Guocun
authors.initials: G
authors.lastname: Huang
authors.affiliation: Center for Circadian Clocks, Soochow University, Suzhou 215123, China.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: From the Department of Neuroscience and.
authors.forename: Yongli
authors.initials: Y
authors.lastname: Shan
authors.affiliation: Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390 and.
authors.forename: Shuzhang
authors.initials: S
authors.lastname: Yang
authors.affiliation: Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390 and.
authors.forename: Yogarany
authors.initials: Y
authors.lastname: Chelliah
authors.affiliation: Center for Circadian Clocks, Soochow University, Suzhou 215123, China.
authors.forename: Han
authors.initials: H
authors.lastname: Wang
authors.affiliation: From the Department of Neuroscience and joseph.takahashi@utsouthwestern.edu.|Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390 and.
authors.forename: Joseph S
authors.identifier: http://orcid.org/0000-0003-0384-8878
authors.initials: JS
authors.lastname: Takahashi
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D015374:Biosensing Techniques; D057906:Circadian Clocks; D057809:HEK293 Cells; D017405:Herpes Simplex Virus Protein Vmw65 / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D017405:Herpes Simplex Virus Protein Vmw65; D011993:Recombinant Fusion Proteins; D009243:NAD
keywords: Rex; biosensor; cell metabolism; circadian; nicotinamide adenine dinucleotide (NADH); oscillation; oxidation-reduction (redox)