Regulation of Heme Metabolism in the Liver

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DESCRIPTION: (Adapted from the Applicant's Abstract): Heme plays a pivotal role as the prosthetic moiety of vital cellular hemoproteins that include hepatic cytochromes P450 (CYPs) and tryptophan 2,3-dioxygenase (TDO), the research focus of this application. These hepatic hemoproteins play distinct functional roles: CYPs are committed to synthesis/metabolism/detoxification of endobiotics and xenobiotics (drugs and toxins), whereas TDO is the key rate-limiting enzyme in the irreversible oxidative L-tryptophan (L-Trp) degradation that controls L-Trp levels and its flux into serotonergic pathways. Because CYPs are the major consumers of hepatic heme, CYP heme destruction results in acute heme depletion, and in genetically predisposed individuals this can trigger the clinical conditions known as the acute hepatic porphyrias. In these individuals, such heme depletion impairs both CYP and TDO function by depriving them of heme, with consequently reduced xenobiotic metabolizing potential and increased L-Trp conversion to the neurotransmitter serotonin that may be responsible for the neurologic symptoms of these heme-deficient states. Thus, normal physiologic function of these enzymes is critically dependent on the hepatic heme status for their structural integrity. However, our studies to date reveal that heme also regulates these enzymes transcriptionally, translationally and/or posttranslationally, thereby revealing a role for heme in their de novo synthesis and/or degradation. Accordingly, studies are proposed to mechanistically characterize this heme regulation by examining its precise role in (i) TDO and (ii) CYP2B transcription by determining whether it acts through specific "heme responsive elements" in their gene promoter regions and/or heme-dependent transcription factors (TFs); (iii) posttranslational regulation of suicidally inactivated CYP2C1 1 by characterizing the role of heme-sensitive phosphorylation in its enhanced proteolytic degradation; and (iv) to determine whether heme also serves as a trigger for CYP3A proteolytic turnover through irreversible posttranslational heme modification of its protein. The studies proposed entail various state-of-the-art approaches (DNAse I footprint, promoter deletion, gel-mobility, and methylation interference analyses, site-directed mutagenesis and TF cotransfection analyses) to examine the mechanics of transcriptional activation, as well as E. coli enzyme expression and purification, immunoprecipitation, heme isolation and/or structural characterization through HPLC-peptide mapping, amino acid sequencing and mass spectrometry for examination of the postranslational modification (phosphorylation and heme-modification) of CYPs. It is believed that collectively, these studies will provide insight into the versatile, albeit ill-understood multiple roles of heme in the regulation of these important liver hemoproteins