THIOL-BASED REDOX MODULATION OF TRANSCRIPTIONAL REGULATORS; CprK AND Rev-erbB.

[摘要] Thiol-based redox regulation of both prokaryotic and eukaryotic systems plays an important role in modulating cellular functions such as gene expression. Transcriptional factors play an important role in these modulations and exert redox-dependent activity through highly conserved cysteine residues in them. This thesis illustrates thiol-disulfide redox regulation of two such transcriptional regulators: CprK and Rev-erbβ.CprK is a positive transcriptional regulator from a prokaryotic organism, Desulfitobacterium dehalogenans. CprK has been shown to contain a thiol/disulfide redox switch that undergoes reversible inactivation upon oxidation. We have demonstrated that a disulfide bond, formed between Cys11 and Cys200 in vivo, is responsible for oxidative inactivation of CprK. Moreover, we have shown that Cys11 is also required for binding DNA and that Cys11 mutants are unable to bind DNA due to a change in their tertiary structures. Therefore, Cys11 plays dual roles in maintaining the normal activity of CprK and its redox inactivation.Rev-erbβ is a human nuclear receptor and downregulates the expression of target genes in the presence of heme. Our studies with the ligand binding domain of Rev-erbβ (Rev-erbβ LBD) have revealed that its affinity for heme is controlled by a thiol-disulfide redox-switch, where the oxidized LBD exhibits 5-fold lower affinity for heme than the reduced LBD.Oxidation of the protein also triggers a ligand switch in which the reduced Rev-erbβ LBD binds heme via Cys 384/His568 while the oxidized protein coordinates heme primarily via His/neutral residue ligand pair.Gas binding analyses with the heme-Rev-erbβ LBD complexes show a dependence of binding on the redox status of the heme and the protein. The binding constants for CO and H2S are low and below their physiological concentrations.In contrast, NO shows very low binding affinities for heme-Rev-erbβ LBD complexes. Currently, we are characterizing full-length hRev-erbβ and our preliminary results corroborate our earlier findings on the redox-regulation of heme binding and heme ligand switching in the isolated LBD.In addition, full-length hRev-erbβ shows redox regulation of DNA binding. Overall, these findings indicate a potential role for the thiol-disulfide redox-switch in Rev-erbβ in responding to changes in redox poise in the cell under oxidative stress.

[发布机构] University of Michigan

[效力级别] Biological Chemistry

[关键词] Redox Regulation;Biological Chemistry;Science;Biological Chemistry