Mitochondrial targeting of mouse NQO1 and CYP1B1 proteins

Membrane-bound cytochrome P450 (CYP) monooxygenases catalyzing the oxygenation of innumerable endogenous compounds and foreign chemicals; there are 103 functional protein-coding Cyp genes in the mouse genome, and 57 CYP genes in the human genome [23;24]. Members of the CYP1, CYP2, CYP3 and CYP4 families are largely involved in metabolism of drugs and environmental pollutants [20;21;28], although lipid mediators including eicosanoids [22] and many other endogenous compounds [23] are also substrates. The mammalian CYP1 family has three members—CYP1A1, CYP1A2 and CYP1B1—which are highly conserved between mouse and human. CYP1A1 and CYP1B1 are best known for polycyclic aryl hydrocarbon (PAH) metabolism, whereas CYP1A2 preferentially metabolizes arylamines [21]. All three monooxygenases are known to transform numerous environmental chemicals into reactive intermediates that can cause genotoxicity, mutagenesis, and oxidative stress—associated in laboratory animals with increased risk of toxicity, birth defects, mutagenesis and cancer. Cyp1 knockout mouse studies have confirmed the relevance of CYP1-mediated metabolic activation of PAHs such as benzo[a]pyrene and 7,12-dimethylbenzo[a]anthracene and arylamines such as 4-aminobiphenyl and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, a food mutagen) in causing toxicity or tumorigenesis. Toxic or carcinogenic effects depend on the CYP1 enzyme present, route-of-administration, dose and rate of exposure, and the target organ being studied [7;13;17;18;35;36;38;40]. Similarly, these knockout mouse lines have helped us understand the importance of CYP1-mediated detoxication of topical 4-aminobiphenyl [38–40] and oral benzo[a]pyrene [38–40]. NAD(P)H:quinone oxidoreductase-1 (NQO1) is historically regarded as a cytosolic flavoenzyme. NQO1 catalyzes the obligatory 2-electron reduction of toxic quinones to hydroquinones, thereby detoxifying reactive intermediates which can be generated via 1-electron reduction “recycling” pathways {19, 8}. Quinones can be toxic as electrophiles, and may also undergo 1-electron reduction via redox cycling to generate semiquinones that cause oxidative stress due to formation of reactive oxygen species (ROS) [16]. CYP1 proteins were historically regarded as located only in the endoplasmic reticulum (ER). Over the past two decades, however, studies by the Avadhani lab have convincingly demonstrated that CYP1A1 protein is partially targeted to mitochondrial (MT) inner membrane; MT- vs ER-targeting is determined by the NH2-terminal protein sequence [2;5]. Using Cyp1a1(−/−) and Cyp1a2(−/−) knockout mouse lines, we confirmed the unequivocal presence of not only CYP1A1 but also CYP1A2 protein in MT [33]. Recently, we generated Cyp1a1 knock-in mouse lines in which the CYP1A1 protein is exclusively targeted to either ER or MT [10]. Examining NH2-terminal sequences of CYP1B1 and NQO1, we posited that these two redox enzymes might also be trafficked to MT. Comparing Cyp1b1(−/−) and Nqo1(−/−) knockout mice with our previously generated ER-specific- and MT-specific CYP1A1 lines, plus Cyp1a1(−/−) knockout and Cyp1(+/+) wild-type (WT) mice as controls, we set out to prove unequivocally the existence of mitochondrial CYP1B1 and NQO1 protein.