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Characterization of Interactions Among CYP1A2, CYP2B4, and NADPH-cytochrome P450 Reductase: Identification of Specific Protein Complexes. 2018

J Patrick Connick, and James R Reed, and Wayne L Backes
Department of Pharmacology and Experimental Therapeutics and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana.

Cytochromes P450s (P450s) catalyze oxygenation reactions via interactions with their redox partners. However, other proteins, particularly other P450s, also have been shown to form complexes that modulate P450 function. Previous studies showed that CYP1A2 and CYP2B4 form a complex when reconstituted into phospholipid vesicles; however, details of the interactions among the P450s and NADPH-cytochrome P450 reductase (POR) have not been fully characterized. The goal of this study was to examine P450 complex formation in living cells, using bioluminescence resonance energy transfer (BRET). Various pairs of P450 and POR constructs were tagged with either green fluorescent protein or Renilla luciferase, and transfected into human embryonic kidney 293T cells. Complexes were demonstrated by measuring energy transfer between the tags, and disruption of the complex was verified by cotransfection with unlabeled P450-system proteins. CYP1A2 and CYP2B4 formed a stable complex that could not be disrupted by cotransfection of untagged POR. Interactions of both P450s with POR were detected, with untagged CYP1A2 disrupting the POR-CYP2B4 interaction. In contrast, untagged CYP2B4 did not affect the POR-CYP1A2 interaction. These data are consistent with POR preferentially binding to the CYP1A2 moiety of CYP1A2-CYP2B4. BRET-detectable homomeric CYP1A2-CYP1A2 also was detected, and was disrupted by cotransfection of either POR or CYP2B4. Both CYP1A2 and CYP2B4 activities were affected by their coexpression in a manner consistent with formation of the high-affinity POR-CYP1A2-CYP2B4 complex. These findings demonstrate that CYP1A2 and CYP2B4 form a heteromeric POR-CYP1A2-CYP2B4 complex in living cells that has altered catalytic activities relative to the homomeric enzymes.

UI MeSH Term Description Entries
D009251 NADPH-Ferrihemoprotein Reductase A flavoprotein that catalyzes the reduction of heme-thiolate-dependent monooxygenases and is part of the microsomal hydroxylating system. EC 1.6.2.4. Cytochrome P-450 Reductase,Ferrihemoprotein P-450 Reductase,NADPH Cytochrome P-450 Oxidoreductase,NADPH Cytochrome P-450 Reductase,NADPH Cytochrome c Reductase,Cytochrome P-450 Oxidase,Cytochrome P450 Reductase,Ferrihemoprotein P450 Reductase,NADPH Cytochrome P450 Oxidoreductase,NADPH Cytochrome P450 Reductase,NADPH-Cytochrome P450 Reductase,NADPH-P450 Reductase,Cytochrome P 450 Oxidase,Cytochrome P 450 Reductase,Ferrihemoprotein P 450 Reductase,NADPH Cytochrome P 450 Oxidoreductase,NADPH Cytochrome P 450 Reductase,NADPH Ferrihemoprotein Reductase,NADPH P450 Reductase,Oxidase, Cytochrome P-450,P-450 Oxidase, Cytochrome,P450 Reductase, Cytochrome,P450 Reductase, NADPH-Cytochrome,Reductase, Cytochrome P-450,Reductase, Cytochrome P450,Reductase, Ferrihemoprotein P-450,Reductase, Ferrihemoprotein P450,Reductase, NADPH-Cytochrome P450,Reductase, NADPH-Ferrihemoprotein,Reductase, NADPH-P450
D010084 Oxidation-Reduction A chemical reaction in which an electron is transferred from one molecule to another. The electron-donating molecule is the reducing agent or reductant; the electron-accepting molecule is the oxidizing agent or oxidant. Reducing and oxidizing agents function as conjugate reductant-oxidant pairs or redox pairs (Lehninger, Principles of Biochemistry, 1982, p471). Redox,Oxidation Reduction
D010743 Phospholipids Lipids containing one or more phosphate groups, particularly those derived from either glycerol (phosphoglycerides see GLYCEROPHOSPHOLIPIDS) or sphingosine (SPHINGOLIPIDS). They are polar lipids that are of great importance for the structure and function of cell membranes and are the most abundant of membrane lipids, although not stored in large amounts in the system. Phosphatides,Phospholipid
D011485 Protein Binding The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments. Plasma Protein Binding Capacity,Binding, Protein
D002384 Catalysis The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction. Catalyses
D002460 Cell Line Established cell cultures that have the potential to propagate indefinitely. Cell Lines,Line, Cell,Lines, Cell
D003577 Cytochrome P-450 Enzyme System A superfamily of hundreds of closely related HEMEPROTEINS found throughout the phylogenetic spectrum, from animals, plants, fungi, to bacteria. They include numerous complex monooxygenases (MIXED FUNCTION OXYGENASES). In animals, these P-450 enzymes serve two major functions: (1) biosynthesis of steroids, fatty acids, and bile acids; (2) metabolism of endogenous and a wide variety of exogenous substrates, such as toxins and drugs (BIOTRANSFORMATION). They are classified, according to their sequence similarities rather than functions, into CYP gene families (>40% homology) and subfamilies (>59% homology). For example, enzymes from the CYP1, CYP2, and CYP3 gene families are responsible for most drug metabolism. Cytochrome P-450,Cytochrome P-450 Enzyme,Cytochrome P-450-Dependent Monooxygenase,P-450 Enzyme,P450 Enzyme,CYP450 Family,CYP450 Superfamily,Cytochrome P-450 Enzymes,Cytochrome P-450 Families,Cytochrome P-450 Monooxygenase,Cytochrome P-450 Oxygenase,Cytochrome P-450 Superfamily,Cytochrome P450,Cytochrome P450 Superfamily,Cytochrome p450 Families,P-450 Enzymes,P450 Enzymes,Cytochrome P 450,Cytochrome P 450 Dependent Monooxygenase,Cytochrome P 450 Enzyme,Cytochrome P 450 Enzyme System,Cytochrome P 450 Enzymes,Cytochrome P 450 Families,Cytochrome P 450 Monooxygenase,Cytochrome P 450 Oxygenase,Cytochrome P 450 Superfamily,Enzyme, Cytochrome P-450,Enzyme, P-450,Enzyme, P450,Enzymes, Cytochrome P-450,Enzymes, P-450,Enzymes, P450,Monooxygenase, Cytochrome P-450,Monooxygenase, Cytochrome P-450-Dependent,P 450 Enzyme,P 450 Enzymes,P-450 Enzyme, Cytochrome,P-450 Enzymes, Cytochrome,Superfamily, CYP450,Superfamily, Cytochrome P-450,Superfamily, Cytochrome P450
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000072467 Cytochrome P450 Family 2 A cytochrome P450 enzyme family that includes members which function in the metabolism of STEROIDS; COUMARINS; and NICOTINE. CYP2 Enzymes,CYP2 Family
D001189 Aryl Hydrocarbon Hydroxylases A large group of cytochrome P-450 (heme-thiolate) monooxygenases that complex with NAD(P)H-FLAVIN OXIDOREDUCTASE in numerous mixed-function oxidations of aromatic compounds. They catalyze hydroxylation of a broad spectrum of substrates and are important in the metabolism of steroids, drugs, and toxins such as PHENOBARBITAL, carcinogens, and insecticides. Microsomal Monooxygenases,Xenobiotic Monooxygenases,Hydroxylases, Aryl Hydrocarbon,Monooxygenases, Microsomal,Monooxygenases, Xenobiotic

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