Nuclear hormone receptors (NRs) are eukaryotic transcription factors that recognize small signaling molecules, which in turn modulate the regulatory properties of NRs. Rev-erb? is a NR that binds heme leading to the recruitment of the NCoR-HDAC1 corepressor complex with concomitant repression of genes involved in circadian rhythm maintenance and metabolism. Rev-erb?-heme binds CO and NO, gaseous signaling molecules involved in diurnal cycling; additionally, Rev-erb? contains a thiol-disulfide redox switch that modulates heme-binding in accordance with redox poise. The first specific aim of this proposal is to describe the interactions of pure full-length Rev-erb? (FLRev-erb?) with DNA and corepressors. Rev-erb? exerts its transcriptional control by binding to a promoter sequence of its target genes called ROR-RE. First, the mid- point redox potential of the thiol-disulfide switch will be calculated by plotting the ratio of dithiol:disulfide populations contributing to th position of the FLRev-erb?:heme Soret band (determined by deconvolution of the UV-visible spectrum) as a function of the ambient redox potential that will be controlled using the reduced/oxidized glutathione couple. Next, fluorescence anisotropy (FA) will be utilized to determine how variations in redox poise and heme concentrations affect the interaction between fluorescein (FSN) labeled ROR-RE oligonucleotides and FLRev-erb? (and site-directed variants containing substitutions of the redox switch and heme-ligands). Using FA, I will also determine the influences of redox and heme on the affinity of FLRev-erb? for FSN-NCoR ID1 and ID2 peptides, which mimic the binding of FLRev-erb? to NCoR. Additional efforts will focus on the production and purification of recombinant full-length or truncated forms of NCoR or HDAC1 and their interactions with FLRev-erb?. The structure-function relationship of gasotransmitters binding to FLRev-erb?-heme will be explored with UV-visible spectroscopy and electron paramagnetic resonance to determine a Kd for the interaction between CO, NO or H2S and FLRev-erb?, and to characterize the coordination environment of heme-gas complexes. Lastly, I will determine the effect of NO, CO and H2S on the interaction between FLRev-erb? and ROR-RE/corepressors. The second specific aim will focus on the characterization of novel and pre-existing synthetic ligands that modulate Rev-erb? function. Virtual screening techniques will be used to screen chemical libraries for candidates that favorably interact with the Rev-erb? heme-binding pocket. The thermodynamics of candidate compounds and previously described tertiary amine- based agonists/antagonists binding to FLRev-erb? will be measured with ITC and the influence of the ligands on heme-binding will be tested with UV-visible spectroscopy and EPR. Lastly, I will determine the effect of synthetic ligands on the interaction between FLRev-erb? and ROR-RE/corepressors. Results obtained during pursuance of these specific aims will lead to a coherent biological model explaining how cellular redox poise and heme control the regulatory output of Rev-erb?.
The studies described in this proposal will lend insight into those cellular factors controlling the regulatory mechanisms of Rev-erb?, a protein essential for the maintenance of human circadian rhythms and metabolic homeostasis. Furthermore, efforts to characterize novel and pre-existing synthetic Rev-erb ligands may lead to the development of pharmaceutical agents to treat sleep and metabolic disorders by means of controlling Rev-erb function in vivo.
| 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 |
| Carter, Eric L; Gupta, Nirupama; Ragsdale, Stephen W (2016) High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear Receptor Rev-erb? Leads to Its Degradation and Indirectly Regulates Its Interaction with Nuclear Receptor Corepressor. J Biol Chem 291:2196-222 |
