Mitochondrial Targeting of Cytochrome P450 (CYP) 1B1 and Its Role in Polycyclic Aromatic Hydrocarbon-induced Mitochondrial Dysfunction

• Published in: The Journal of biological chemistry 2014-04, Vol.289 (14), p.9936-9951
• Main Authors: Bansal, Seema, Leu, Adrian N., Gonzalez, Frank J., Guengerich, F. Peter, Chowdhury, Anindya Roy, Anandatheerthavarada, Hindupur K., Avadhani, Narayan G.
• Format: Article
• Language: English
• Subjects: Adrenodoxin - genetics; Adrenodoxin - metabolism; Animals; Aryl Hydrocarbon Hydroxylases - genetics; Aryl Hydrocarbon Hydroxylases - metabolism; Benzo(a)pyrene - adverse effects; Benzo(a)pyrene - pharmacology; Carcinogenesis; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Cytochrome P-450 CYP1B1; Cytochrome P450; DNA Damage; Female; Humans; Male; Membrane Proteins; Metabolism; Mice; Mice, Knockout; Mitochondria; Mitochondria - enzymology; Mitochondria - genetics; Mitochondria - pathology; Mitochondrial DNA; Mutagenesis; Oxidation-Reduction - drug effects; Oxygen Consumption - drug effects; Polychlorinated Dibenzodioxins - adverse effects; Polychlorinated Dibenzodioxins - pharmacology; Protein Sorting Signals; Protein Transport - drug effects; Teratogens
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.525659

We report that polycyclic aromatic hydrocarbon (PAH)-inducible CYP1B1 is targeted to mitochondria by sequence-specific cleavage at the N terminus by a cytosolic Ser protease (polyserase 1) to activate the cryptic internal signal. Site-directed mutagenesis, COS-7 cell transfection, and in vitro import studies in isolated mitochondria showed that a positively charged domain at residues 41–48 of human CYP1B1 is part of the mitochondrial (mt) import signal. Ala scanning mutations showed that the Ser protease cleavage site resides between residues 37 and 41 of human CYP1B1. Benzo[a]pyrene (BaP) treatment induced oxidative stress, mitochondrial respiratory defects, and mtDNA damage that was attenuated by a CYP1B1-specific inhibitor, 2,3,4,5-tetramethoxystilbene. In support, the mitochondrial CYP1B1 supported by mitochondrial ferredoxin (adrenodoxin) and ferredoxin reductase showed high aryl hydrocarbon hydroxylase activity. Administration of benzo[a]pyrene or 2,3,7,8-tetrachlorodibenzodioxin induced similar mitochondrial functional abnormalities and oxidative stress in the lungs of wild-type mice and Cyp1a1/1a2-null mice, but the effects were markedly blunted in Cyp1b1-null mice. These results confirm a role for CYP1B1 in inducing PAH-mediated mitochondrial dysfunction. The role of mitochondrial CYP1B1 was assessed using A549 lung epithelial cells stably expressing shRNA against NADPH-cytochrome P450 oxidoreductase or mitochondrial adrenodoxin. Our results not only show conservation of the endoprotease cleavage mechanism for mitochondrial import of family 1 CYPs but also reveal a direct role for mitochondrial CYP1B1 in PAH-mediated oxidative and chemical damage to mitochondria. Background: Cytochrome P450 (CYP) 1B1 activates diverse polycyclic aromatic hydrocarbons (PAH) to reactive species. Results: Processing by a cytosolic Ser protease activates a mitochondrial (mt) targeting signal of CYP1B1. Conclusion: Mitochondrial CYP1B1 plays a role in PAH-induced mtDNA damage and mitochondrial dysfunction. Significance: PAH-induced mitochondrial dysfunction may be important in tissue injury and inflammation.