title: High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear Receptor Rev-erbβ Leads to Its Degradation and Indirectly Regulates Its Interaction with Nuclear Receptor Corepressor.
abstract: Rev-erbα and Rev-erbβ are heme-binding nuclear receptors (NR) that repress the transcription of genes involved in regulating metabolism, inflammation, and the circadian clock. Previous gene expression and co-immunoprecipitation studies led to a model in which heme binding to Rev-erbα recruits nuclear receptor corepressor 1 (NCoR1) into an active repressor complex. However, in contradiction, biochemical and crystallographic studies have shown that heme decreases the affinity of the ligand-binding domain of Rev-erb NRs for NCoR1 peptides. One explanation for this discrepancy is that the ligand-binding domain and NCoR1 peptides used for in vitro studies cannot replicate the key features of the full-length proteins used in cellular studies. However, the combined in vitro and cellular results described here demonstrate that heme does not directly promote interactions between full-length Rev-erbβ (FLRev-erbβ) and an NCoR1 construct encompassing all three NR interaction domains. NCoR1 tightly binds both apo- and heme-replete FLRev-erbβ·DNA complexes; furthermore, heme, at high concentrations, destabilizes the FLRev-erbβ·NCoR1 complex. The interaction between FLRev-erbβ and NCoR1 as well as Rev-erbβ repression at the Bmal1 promoter appear to be modulated by another cellular factor(s), at least one of which is related to the ubiquitin-proteasome pathway. Our studies suggest that heme is involved in regulating the degradation of Rev-erbβ in a manner consistent with its role in circadian rhythm maintenance. Finally, the very slow rate constant (10(-6) s(-1)) of heme dissociation from Rev-erbβ rules out a prior proposal that Rev-erbβ acts as an intracellular heme sensor.
authors.affiliation: From the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109.
authors.forename: Eric L
authors.initials: EL
authors.lastname: Carter
authors.affiliation: From the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109.
authors.forename: Nirupama
authors.initials: N
authors.lastname: Gupta
authors.affiliation: From the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109 sragsdal@umich.edu.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Ragsdale
mesh_terms: D020816:Amino Acid Motifs; D001059:Apoproteins / Q000737:chemistry; D002940:Circadian Rhythm; D003513:Cycloheximide / Q000737:chemistry; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006418:Heme / Q000737:chemistry*; D006801:Humans; D007249:Inflammation; D007477:Ions; D008024:Ligands; D013058:Mass Spectrometry; D008670:Metals / Q000737:chemistry; D009211:Myoglobin / Q000737:chemistry; D056971:Nuclear Receptor Co-Repressor 1 / Q000737:chemistry*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry*; D012097:Repressor Proteins / Q000737:chemistry*; D014162:Transfection; D025801:Ubiquitin / Q000737:chemistry
chemical_list: D001059:Apoproteins; D007477:Ions; D008024:Ligands; D008670:Metals; D009211:Myoglobin; C539127:NCOR1 protein, human; C090977:NR1D2 protein, human; D056971:Nuclear Receptor Co-Repressor 1; D018160:Receptors, Cytoplasmic and Nuclear; D012097:Repressor Proteins; D025801:Ubiquitin; C010857:apomyoglobin; D006418:Heme; D003513:Cycloheximide
keywords: DNA-protein interaction; heme; ligand-binding protein; metal ion-protein interaction; nuclear receptor; protein-protein interaction; proteolysis; transcription corepressor; transcription repressor