Mitochondrial targeting of mouse NQO1 and CYP1B1 proteins

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Highlights

  • Dioxin-treated wild-type, mc1A1, mtt1a1, and 1A1 KO, 1B1 KO and NQO1 KO knockout mice were compared.

  • Analysis included both mRNA and protein quantification of CYP1A1, CYP1B1 and NQO1.

  • NQO1 protein locates to cytosol, ER and mitochondria.

  • CYP1B1 protein (similar to CYP1A1 and CYP1A2 proteins) traffics to mitochondria as well as ER.

  • NQO1 and CYP1B1 targeting to MT or ER membranes is independent of CYP1A1 presence in that membrane.

Abstract

Four dioxin-inducible enzymes––NAD(P)H: quinone oxidoreductase-1 (NQO1) and three cytochromes P450 (CYP1A1, CYP1A2 & CYP1B1)––are implicated in both detoxication and metabolic activation of various endobiotics and xenobiotics. NQO1 is generally regarded as a cytosolic enzyme; whereas CYP1 proteins are located primarily in endoplasmic reticulum (ER), CYP1A1 and CYP1A2 proteins are also targeted to mitochondria. This lab has generated Cyp1a1(mc/mc) and Cyp1a1(mtt/mtt) knock-in mouse lines in which CYP1A1 protein is targeted exclusively to ER (microsomes) and mitochondria, respectively. Comparing dioxin-treated Cyp1(+/+) wild-type, Cyp1a1(mc/mc), Cyp1a1(mtt/mtt), and Cyp1a1(−/−), Cyp1b1(−/−) and Nqo1(−/−) knockout mice, in the present study we show that [a] NQO1 protein locates to cytosol, ER and mitochondria, [b] CYP1B1 protein (similar to CYP1A1 and CYP1A2 proteins) traffics to mitochondria as well as ER, and [c] NQO1 and CYP1B1 targeting to mitochondrial or ER membranes is independent of CYP1A1 presence in that membrane.

Introduction

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] and oral benzo[a]pyrene [39], [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 [6], [42]. 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.

Section snippets

Chemicals

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin; also called “dioxin”) was purchased from Accustandard, Inc. (New Haven, CT). Anti-CYP1A1/1A2 polyclonal antibody (α-1A1/1A2) was bought from BD Gentest (Woburn, MA). Anti-CYP1B1 polyclonal antibody (α-1B1) was generated in chicken against the mouse protein [25]. Anti-NQO1 polyclonal antibody (α-NQO1) was made in rabbits against the rat protein [29]. Anti-P450 oxidoreductase (α-POR) and anti-prohibitin (α-PHB) were purchased from Abcam (Cambridge, MA).

Results and discussion

Continuity of ER with the outer membrane of mitochondria [12], [32] makes it extremely difficult by differential centrifugation to separate microsomal and MT fractions without some degree of contamination of one organelle by the other. This is especially true with ER-rich liver tissue; hence, we preferred to use nonhepatic tissues for better separations of microsomal and MT fractions [10]. Another reason for not studying liver whencharacterizing the subcellular localization of CYP1B1 protein is

Acknowledgments

We thank our colleagues, especially Narayan Avadhani for fruitful discussions, and careful readings of this manuscript. We thank Bin Wang for excellent technical assistance, and Lucia F Jorge-Nebert for precise genotyping of all mouse lines. Supported, in part, by NIH Grants R01 ES08147 (D.W.N.), R01 ES014403 (D.W.N.), and Center for Environmental GeneticsP30 ES06096 (H.G.S., M.B.G., D.W.N.).

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    Current address: Department of Clinical Immunology, University Colorado Health Sciences Center, Aurora, CO 80045, United States.

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