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

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK026506-25
Application #
6847850
Study Section
Alcohol and Toxicology Subcommittee 4 (ALTX)
Program Officer
Doo, Edward
Project Start
1980-07-01
Project End
2007-06-30
Budget Start
2005-02-01
Budget End
2007-06-30
Support Year
25
Fiscal Year
2005
Total Cost
$490,541
Indirect Cost
Name
University of California San Francisco
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Lewis-Ballester, Ariel; Forouhar, Farhad; Kim, Sung-Mi et al. (2016) Molecular basis for catalysis and substrate-mediated cellular stabilization of human tryptophan 2,3-dioxygenase. Sci Rep 6:35169
Kim, Sung-Mi; Wang, YongQiang; Nabavi, Noushin et al. (2016) Hepatic cytochromes P450: structural degrons and barcodes, posttranslational modifications and cellular adapters in the ERAD-endgame. Drug Metab Rev 48:405-33
Kim, Sung-Mi; Grenert, James P; Patterson, Cam et al. (2016) CHIP(-/-)-Mouse Liver: Adiponectin-AMPK-FOXO-Activation Overrides CYP2E1-Elicited JNK1-Activation, Delaying Onset of NASH: Therapeutic Implications. Sci Rep 6:29423
Wang, YongQiang; Kim, Sung-Mi; Trnka, Michael J et al. (2015) Human liver cytochrome P450 3A4 ubiquitination: molecular recognition by UBC7-gp78 autocrine motility factor receptor and UbcH5a-CHIP-Hsc70-Hsp40 E2-E3 ubiquitin ligase complexes. J Biol Chem 290:3308-32
Correia, Maria Almira; Wang, YongQiang; Kim, Sung-Mi et al. (2014) Hepatic cytochrome P450 ubiquitination: conformational phosphodegrons for E2/E3 recognition? IUBMB Life 66:78-88
Wang, YongQiang; Guan, Shenheng; Acharya, Poulomi et al. (2012) Multisite phosphorylation of human liver cytochrome P450 3A4 enhances Its gp78- and CHIP-mediated ubiquitination: a pivotal role of its Ser-478 residue in the gp78-catalyzed reaction. Mol Cell Proteomics 11:M111.010132
Acharya, Poulomi; Liao, Mingxiang; Engel, Juan C et al. (2011) Liver cytochrome P450 3A endoplasmic reticulum-associated degradation: a major role for the p97 AAA ATPase in cytochrome P450 3A extraction into the cytosol. J Biol Chem 286:3815-28
Wang, YongQiang; Guan, Shenheng; Acharya, Poulomi et al. (2011) Ubiquitin-dependent proteasomal degradation of human liver cytochrome P450 2E1: identification of sites targeted for phosphorylation and ubiquitination. J Biol Chem 286:9443-56
Correia, Maria Almira; Sinclair, Peter R; De Matteis, Francesco (2011) Cytochrome P450 regulation: the interplay between its heme and apoprotein moieties in synthesis, assembly, repair, and disposal. Drug Metab Rev 43:1-26
Kim, Sung-Mi; Acharya, Poulomi; Engel, Juan C et al. (2010) Liver cytochrome P450 3A ubiquitination in vivo by gp78/autocrine motility factor receptor and C terminus of Hsp70-interacting protein (CHIP) E3 ubiquitin ligases: physiological and pharmacological relevance. J Biol Chem 285:35866-77

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