Mitochondrial targeting of cytochrome P450 (CYP) 1B1 and its role in polycyclic aromatic hydrocarbon-induced mitochondrial dysfunction.
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.
From the Department of Animal Biology and the Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
Seema
S
Bansal
Adrian N
AN
Leu
Frank J
FJ
Gonzalez
F Peter
FP
Guengerich
Anindya Roy
AR
Chowdhury
Hindupur K
HK
Anandatheerthavarada
Narayan G
NG
Avadhani
D000325:Adrenodoxin / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D001564:Benzo(a)pyrene / Q000009:adverse effects* / Q000494:pharmacology; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D065633:Cytochrome P-450 CYP1B1; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D016296:Mutagenesis; D010084:Oxidation-Reduction / Q000187:drug effects; D010101:Oxygen Consumption / Q000187:drug effects*; D000072317:Polychlorinated Dibenzodioxins / Q000009:adverse effects* / Q000494:pharmacology; D021382:Protein Sorting Signals; D021381:Protein Transport / Q000187:drug effects; D013723:Teratogens
D000072317:Polychlorinated Dibenzodioxins; D021382:Protein Sorting Signals; D013723:Teratogens; D000325:Adrenodoxin; D001564:Benzo(a)pyrene; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; C585498:Cyp1b1 protein, mouse; D065633:Cytochrome P-450 CYP1B1
Carcinogenesis; Cytochrome P450; DNA Damage; Membrane Proteins; Mitochondria; Mitochondrial DNA