Mitochondrial targeting of mouse NQO1 and CYP1B1 proteins
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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Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation
2021, Redox BiologyCitation Excerpt :To our knowledge, this is the first time that endogenous CoQ has been shown to regulate NOX4 activity, which is in agreement with previous findings for synthetic quinone compounds and NQO1 overexpression [18]. Importantly, although NQO1 is known to regulate mitochondrial function, its localization on the mitochondrial membrane remains controversial [49–53]. While NQO1 can be detected in mitochondria isolated from multiple mouse tissues, it was not found in mitochondria in a human cell line [52].
Camphorquinone alters the expression of extracellular proteases in a 3D co-culture model of the oral mucosa
2021, Dental MaterialsCitation Excerpt :However, in HGFs only the transcription of NQO1 was significantly induced 24 h after the initial treatment, indicating a less pronounced and more specific activation of the machinery against oxidative stress in underlying HGFs. However, NQO1 also plays a role in the detoxification of toxic quinones and might therefore be induced to metabolize CQ or secondary CQ-dependent quinones directly [44,45]. The latter possibility would suggest that only minor oxidative stress was induced in HGFs by CQ that did not result in a detectable induction of the other antioxidant enzymes analyzed (HO-1, SOD1 and CAT).
Acute sources of mitochondrial NAD<sup>+</sup> during respiratory chain dysfunction
2020, Experimental NeurologyCitation Excerpt :A striking difference among them is that NQO2 uses dihydronicotinamide riboside (NRH), while NQO1 uses NAD(P)H as an electron donor (Wu et al., 1997), (Zhao et al., 1997). NQO1 is distributed in the cytosol and mitochondria (Ernster et al., 1962), (Dong et al., 2013), (Bianchet et al., 2004), (Eliasson et al., 1999), (Edlund et al., 1982), (Conover and Ernster, 1962), (Conover and Ernster, 1960), (Wosilait, 1960), (Colpa-Boonstra and Slater, 1958), (Lind and Hojeberg, 1981), but see (Winski et al., 2002). Total mitochondrial diaphorase activity corresponds to 3–15% of total cellular activity (Ernster et al., 1962), (Edlund et al., 1982), (Wosilait, 1960), (Colpa-Boonstra and Slater, 1958), (Lind and Hojeberg, 1981) and is localized in the matrix, since it reacts only with intramitochondrial reduced pyridine nucleotides, but is inaccessible to those added from the outside (Conover and Ernster, 1960), (Conover and Ernster, 1963).
Loss of NQO1 generates genotoxic estrogen-DNA adducts in Fuchs Endothelial Corneal Dystrophy
2020, Free Radical Biology and MedicineCitation Excerpt :There is very limited literature on the direct role of NQO1 in mitochondrial function. Although NQO1 is primarily considered a cytosolic protein [66], a recent study detected dioxin-induced NQO1 expression in the microsomal, mitochondrial and cytosolic fractions of various mouse organs [67]. In another study, increased NQO1 was shown to improve the activity of mitochondrial electron chain complexes and offer protection against mitochondrial toxins [68].
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Current address: Department of Clinical Immunology, University Colorado Health Sciences Center, Aurora, CO 80045, United States.