Mitochondrial Targeting of Cytochrome P450 (CYP) 1B1 and its Role in Polycyclic Aromatic Hydrocarbon-Induced Mitochondrial Dysfunction [Membrane Biology]

February 4th, 2014 by Bansal, S., Leu, A., Gonzalez, F. J., Guengerich, F. P., Roy Chowdhury, A., Anandathheerthavarada, H. K., Avadhani, N. G.

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 37-41 of human CYP1B1. Benzo[a]pyrene (BaP) treatment induced oxidative stress, mitochondrial respiratory defects, and mtDNA damage that were attenuated by a CYP1B1-specific inhibitor, 2,3,4,5-tetramethoxystilbene (TMS). In support, the mitochondrial CYP1B1 supported by mitochondrial ferredoxin (Adx) and ferredoxin reductase (Adr) showed high aryl hydrocarbon hydroxylase activity. Administration of BaP or 2,3,7,8-tetrachlorodibenzodioxin (TCDD)-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 (NPR) or mitochondrial Adx. Our results not only show conservation of the endo-protease 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.
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