We propose to determine how the heme responsive motif (HRM) promotes regulation of the structure, function and proteasomal degradation of two proteins with key metabolic roles: Rev-erb? and heme oxygenase-2 (HO-2). Our working hypothesis is that the HRMs confer the ability to be regulated by heme, redox and gaseous signaling molecules (CO, NO, H2S) and by proteasomal degradation. Rev-erb? is a heme-responsive nuclear hormone receptor, which links the circadian clock to metabolism and inflammation by acting as a transcriptional repressor of diverse genes involved in lipid, glucose and bile acid metabolism as well as regulating the circadian cycle, cellular differentiation and the proinflammatory response. Our guiding hypothesis is that the HRMs in Rev-erb? constitute thiol-disulfide redox switches that link oxidative stress to the circadian cycle and to metabolic regulation. Our two aims related to Rev-erb? are to: (1) determine how the C384-C374 thiol-disulfide redox switch affects Rev-erb? repressor activity, proteosomal degradation and interaction with partner proteins and (2) quantify how the state of the HRM- bound heme affects the properties of Rev-erb?. HO1 and HO2 catalyze the rate-limiting step in O2-dependent degradation of heme to biliverdin, CO and Fe with electrons delivered from NADPH via cytochrome P450 reductase (CPR). With respect to characterizing the properties of the HRMs in human HO2, which sits at the nexus of major cellular redox and metal regulatory systems, we plan to: (3) perform kinetic, spectroscopic and hydrogen-deuterium exchange mass spectrometric (HDX-MS) studies to investigate heme transfer from the HRMs in the tail to the catalytic core of HO2 and interactions between the tail and core; (4) determine the role of the N-terminal extension of HO2; and (5) determine the effect of the HRM on stability of HO2. Our studies will reveal how HRMs regulate Rev-erb?, which, helping explain why abnormal circadian rhythms are associated with cancer, disturbances in the cell cycle and metabolic disorders. Our research will also help understand how HO2 is involved in cellular protection against renal, cardiovascular and central nervous system pathologies induced by oxidative stress by controlling the cellular levels of heme, biliverdin, iron and carbon monoxide.
Successful completion of the goals of this project will reveal heme responsive motifs (HRMs) are involved in the multi-tier regulation of Rev-Erb? and heme oxygenase-2 (HO2). Studies on Rev-Erb, which links the circadian clock to metabolism and inflammation, will help explain why abnormal circadian rhythms are associated with cancer, disturbances in the cell cycle and metabolic disorders. Research on HO2 will help understand the regulation of HO2, which itself regulates heme and carbon monoxide levels and protects cells against renal, cardiovascular and central nervous system pathologies induced by oxidative stress.
|Fleischhacker, Angela S; Carter, Eric L; Ragsdale, Stephen W (2018) Redox Regulation of Heme Oxygenase-2 and the Transcription Factor, Rev-Erb, Through Heme Regulatory Motifs. Antioxid Redox Signal 29:1841-1857|
|Fleischhacker, Angela S; Ragsdale, Stephen W (2018) An unlikely heme chaperone confirmed at last. J Biol Chem 293:14569-14570|
|Carter, Eric L; Ramirez, Yanil; Ragsdale, Stephen W (2017) The heme-regulatory motif of nuclear receptor Rev-erb? is a key mediator of heme and redox signaling in circadian rhythm maintenance and metabolism. J Biol Chem 292:11280-11299|