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

Seema Bansal,Adrian N Leu,Frank J Gonzalez,F Peter Guengerich,Anindya Roy Chowdhury,Hindupur K Anandatheerthavarada,Narayan G Avadhani

doi:10.1074/jbc.m113.525659

Abstract

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.

Highlights

Cytochrome P450 (CYP) 1B1 activates diverse polycyclic aromatic hydrocarbons (PAH) to reactive species
A marked increase in CYP1B1 and CYP1A1 proteins in both the mitochondrial and microsomal fractions was seen in cells treated with BaP but not in cells treated with the vehicle DMSO
In this study using MCF-7 breast carcinoma and A549 lung carcinoma cells, both of which express Cyp1 genes in response to PAHs, and lungs from WT, Cyp1a1/1a2-null, and Cyp1b1-null mice, we showed that human CYP1B1 is targeted to mitochondria through a pathway similar to that shown for rat and mouse CYP1A1 [32, 35, 37]

Summary

Background

Cytochrome P450 (CYP) 1B1 activates diverse polycyclic aromatic hydrocarbons (PAH) to reactive species. We described two distinct mechanisms for the activation of cryptic signals as follows: 1) PKA- and/or PKC-mediated phosphorylation at the Nterminal phosphorylation sites of CYP2B1, CYP2E1, and CYP2D6 and C-terminal sites of glutathione transferases, increasing the affinity of nascent chains for binding to the mitochondrial chaperones HSP70 and HSP90 (33, 39 – 43); 2) sequence-specific endoprotease processing past the Nterminal transmembrane domain by a xenobiotic-inducible Ser protease in the case of CYP1A1, as a mechanism of activation of cryptic signal [32, 35, 37, 44]. Use of A549 lung epithelial cells expressing shRNA against NPR and Adx show that mitochondrial CYP1B1 is likely to be responsible for the BaP- and TCDD-induced mitochondrial dysfunction

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION