The overall objective of this research is to understand the structural basis for the production of a functional cytochrome P450 (P450) molecule. Emphasis is on the cellular targeting to the endoplasmic reticulum (ER), folding of the protein and membrane topology, and amino acids involved in substrate interactions. Previous work has demonstrated that P450 is exclusively retained in the ER, that redundant ER retention signals are present in both the N-terminal signal anchor sequence and the cytoplasmic catalytic domain, that the sequence linking (""""""""linker"""""""") these two regions may be critical for folding of the protein, and that six amino acid substitutions confer a novel steroid hydroxylase activity to P450 2C2.
The specific aims are: 1) to examine the ER retention signals, 2) determine the requirements of the ER retention properties for the N-terminal signal anchor sequence, 3) examine the mechanism by which mutations in the """"""""linker"""""""" sequence reduce activity, 4) examine the kinetic and substrate binding properties of P450 2C2 mutants with novel steroid hydroxylase activity, and 5) express large amounts of soluble P45O-2C2 and variants for potential structural studies. Experiments will include the generation of mutants, the production of adequate protein in insect cells and bacteria for membrane and structural studies, and kinetic analysis of the different chimeric proteins to examine the effect of the modulations that are imposed on the leader sequences and other membrane retention signals.
|Szczesna-Skorupa, Elzbieta; Kemper, Byron (2011) Progesterone receptor membrane component 1 inhibits the activity of drug-metabolizing cytochromes P450 and binds to cytochrome P450 reductase. Mol Pharmacol 79:340-50|
|Szczesna-Skorupa, Elzbieta; Kemper, Byron (2011) The signal-anchor sequence of CYP2C1 inserts into the membrane as a hairpin structure. Biochem Biophys Res Commun 415:405-9|
|Li, Bin; Yau, Peter; Kemper, Byron (2011) Identification of cytochrome P450 2C2 protein complexes in mouse liver. Proteomics 11:3359-68|
|Hu, Gang; Johnson, Eric F; Kemper, Byron (2010) CYP2C8 exists as a dimer in natural membranes. Drug Metab Dispos 38:1976-83|
|Szczesna-Skorupa, Elzbieta; Kemper, Byron (2008) Proteasome inhibition compromises direct retention of cytochrome P450 2C2 in the endoplasmic reticulum. Exp Cell Res 314:3221-31|
|Szczesna-Skorupa, Elzbieta; Kemper, Byron (2008) Influence of protein-protein interactions on the cellular localization of cytochrome P450. Expert Opin Drug Metab Toxicol 4:123-36|
|Ozalp, Cengiz; Szczesna-Skorupa, Elzbieta; Kemper, Byron (2006) Identification of membrane-contacting loops of the catalytic domain of cytochrome P450 2C2 by tryptophan fluorescence scanning. Biochemistry 45:4629-37|
|Szczesna-Skorupa, Elzbieta; Kemper, Byron (2006) BAP31 is involved in the retention of cytochrome P450 2C2 in the endoplasmic reticulum. J Biol Chem 281:4142-8|
|Ozalp, Cengiz; Szczesna-Skorupa, Elzbieta; Kemper, Byron (2005) Bimolecular fluorescence complementation analysis of cytochrome p450 2c2, 2e1, and NADPH-cytochrome p450 reductase molecular interactions in living cells. Drug Metab Dispos 33:1382-90|
|Kemper, Byron (2004) Structural basis for the role in protein folding of conserved proline-rich regions in cytochromes P450. Toxicol Appl Pharmacol 199:305-15|
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