Mitochondrial targeting of cytochrome P450 (CYP) 1B1 and its role in polycyclic aromatic hydrocarbon-induced mitochondrial dysfunction

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.

Keywords: Carcinogenesis; Cytochrome P450; DNA Damage; Membrane Proteins; Mitochondria; Mitochondrial DNA.

FIGURE 1.

Induction of CYP1B1 and mitochondrial dysfunctions in MCF-7 cells exposed to BaP. A, induction of CYP1B1 in the mitochondria (Mito) and microsomes (Micro) isolated from MCF-7 cells treated with BaP (20 μm) for 48 or 72 h or with DMSO alone. Mitochondrial and microsomal proteins (50 μg) each were resolved by SDS-PAGE and subjected to immunoblot analysis with CYP1B1 antibody (1:1500, v/v) and CYP1A1 antibody (1:4000, v/v). The blot was also codeveloped with NPR (1:2500, v/v) and Tom20 (1:2000, v/v) antibodies to assess levels of cross-contamination. B and C, rate of O₂ consumption (OCR) was measured using a Seahorse Bioscience XF24 Extracellular Flux Analyzer. Cells (50,000) from control, BaP treatment (20 μm), and TMS + BaP were cultured in DMEM/F-12 medium for 72 h followed by a change with XF medium (low buffered and bicarbonate-free, pH 7.4) for 1 h before the assay. TMS was used at a concentration of 0.8 μg/ml. The plate was incubated without CO₂ for 1 h before recording the respiration rates. Oligomycin (2 μg/ml), 2,4-dinitrophenol (75 μm), and rotenone (1 μm) were injected through ports A, B, and C, respectively. Basal OCR (pmol of O₂/min) and maximum reserve capacity (%OCR) were analyzed using XF software. D, BaP hydroxylation activity (AHH) was assayed using MCF-7 cells treated with 20 μm BaP for 48, 72, and 96 h. Digitonin-treated mitochondria and microsomes (200 μg of protein each) were incubated and used for assaying AHH activity as described under “Experimental Procedures.” The values in parentheses show specific activity (pmol of product formed/nmol of CYP/min). E, bacterially expressed and purified human CYP1B1 (0.2 nmol of CYP) was assayed for AHH activity by reconstituting with 0.5 nmol of NPR (along with phospholipid vesicles) and Adx + AdxR as described under “Experimental Procedures.” TMS was added at a concentration of 10 μg/ml to study the reaction specificity. The means ± S.D. were calculated from three independent measurements.

FIGURE 2.

Localization of mitochondria targeting signal of CYP1B1. A, cDNA constructs used for in vitro import studies. Full-length CYP1B1 and other mutants (+34, +54, +141) were cloned in PCDNA3.1 and PCMV4 for in vitro transcription-linked translation and cell transfection, respectively. B, ³⁵S-labeled translation products of WT CYP1B1, +34, +54, +141 1B1 cloned in PCDNA3.1. C, import of ³⁵S-labeled translation products in isolated rat liver mitochondria. D, DHFR and Su-9 DHFR, in which the pre-sequence of subunit 9 of Neurospora crassa F₀F₁-ATPase was fused to DHFR used as negative and positive controls, respectively. In all experiments, trypsin digestion (150 μg/ml) of mitochondria was performed for 20 min on ice. Proteins (50 μg each) were subjected to SDS-PAGE and fluorography. C on the top of lanes represents control experiments in which total protein bound and imported into mitochondria is present; T represents trypsin-treated mitochondria in which only the protein imported into mitochondria is present. In the lanes marked In, 10% of the counts used as input for the import reactions were loaded. E, patterns of mitochondrial and microsomal CYP1B1 in COS-7 cells transfected with WT CYP1B1 cDNA for 48 h. Fifty μg of protein each was loaded onto a 12% Tricine gel (w/v), and proteins were probed with CYP1B1 antibody (1:1500, v/v).

FIGURE 3.

Putative mitochondrial targeting signal of CYP1B1 and endoprotease-processing site. A, sequence of putative mitochondrial signal region (WT) and two mutant versions (RMut1 and RMut2) are shown. B, in vivo targeting patterns of WT and Mut proteins in cells transfected with respective cDNA constructs. Proteins (50 μg each) were resolved on SDS-PAGE and subjected to immunoblot analysis with CYP1B1 antibody (1:1500, v/v). The blot was also codeveloped with NPR (1:2500, v/v) and TOM20 (1: 2000, v/v) antibodies to detect any cross-contamination. C, conservation of putative Ser protease cleavage site in the N-terminal signal region of CYP1B1. The Ser protease cleavage site of CYP1A1 was used for comparison. D, COS-7 cells were cotransfected with varying amounts of full-length CYP1B1 and AK131261 cDNA. Mitochondria (Mt) and microsomes (Mc) were isolated as described under the “Experimental Procedures” and subjected to trypsin digestion (100 μg of trypsin/mg of protein for 20 min on ice). An equal amount of protein (50 μg each) was loaded onto SDS-PAGE and subjected to immunoblot analysis with CYP1B1 antibody. The blots were also probed for NPR, an ER-specific marker: Tom20, an outer mitochondrial membrane marker, and SDH, an inner mitochondrial membrane-specific marker protein.

FIGURE 4.

Intramitochondrial localization of CYP1B1 by immunofluorescence and immunogold electron microscopy. A, COS-7 cells grown on coverslips were transfected with vector alone, WT, + 34 CYP1B1 cDNAs, and in one case cotransfected with AK131261 cDNA for 48 h. The cells were fixed with chilled methanol, and cells were double-immunostained with antibodies specific for CYP1B1 and CcOI. The stained patterns were overlaid, and the Pearson's coefficient for each overlay was calculated based on the scatterplot using Volocity software. B, COS-7 cells were transfected with vector and WT CYP1B1, and the trypsin-digested mitochondrial pellets were used for immunogold electron microscopy. The sections were stained with antibody to CYP1B1 and costained with gold-conjugated (10 nm gold particles) secondary antibody. C, solubility of mitochondrial and microsomal CYP1B1 in alkaline Na₂CO₃. Mitochondria and microsomes from +34CYP1B1 or WT CYP1B1 cDNA-transfected COS-7 cells were extracted with 0.10 m Na₂CO₃, pH 11.0. The total soluble protein (S) and proteins from the insoluble membrane fraction (P) were isolated and resolved by SDS-PAGE and subjected to immunoblot analysis using CYP1B1 antibody.

FIGURE 5.

Endoproteolytic processing at the N-terminal signal region of human CYP1B1 protein. A, fusion construct of DHFR fused to the N terminus of CYP1B1 was generated as described under the “Experimental Procedures.” The position of DHFR, various signal domains of CYP1B1, and the putative processing sites are shown. B, COS-7 cells were transfected with mock, WT CYP1B1, or DHFR-1B1 cDNAs with or without AK131261 cDNA as indicated. Total cell extracts (50 μg of protein each) were loaded on SDS-PAGE, and the immunoblot was developed with CYP1B1 antibody. The blot was also developed with actin antibody to assess loading levels. C, subcellular distribution of fusion protein and processed product. COS-7 cells were transfected with mock, DHFR-1B1, and with varying amounts of AK131261 cDNA as described in B. Mitochondria (Mito), cytosol (Cyto), and microsomes (Micro) were isolated from transfected cells, and 50 μg of protein each was subjected to immunoblot analysis using CYP1B1 antibody (1:1500, v/v). The same blot was also developed with NPR, SDH, and β-actin antibodies. Std., standard.

FIGURE 6.

Sequence specificity of processing by the endoprotease. A, predicted processing sites and sequence of WT CYP1B1 and DHFR-1B1 and alanine scan mutations are shown. Constructs where amino acids at positions 37/38, 38/39, 39/40, and 40/41 were mutated to alanines were designated as M1, M2, M3 and M4, respectively. B, processing of WT and mutant (M1–M4) CYP1B1 protein cotransfected with 4 μg of AK131261 cDNA in COS-7 cells. Mitochondrial and microsomal samples (50 μg each) were subjected to immunoblot analysis. The numbers below the blot in parentheses indicate relative band intensities of mitochondrial CYP1B1 protein. C, experiment was performed similar to that in B except that mitochondria and microsomes were treated with trypsin (100 μg/mg at 4 °C for 20 min). D, DHFR-1B1 fusion constructs and Ala scan mutant constructs were cotransfected with 4 μg of AK131261 cDNA in COS cells. Mitochondrial (Mito), microsomal (Micro), and cytosolic (Cyto) proteins (50 μg per lane) were subjected to immunoblot analysis with CYP1B1 antibody to detect the levels of precursor and processed protein. The blot was also codeveloped with antibody to β-actin and CcO IVi1 to detect the level of cytosolic contamination of mitochondrial isolates. Std, standard.

FIGURE 7.

In vivo induction of Cyp1b1 mRNA and protein in WT, Cyp1a1/1a2-null, and Cyp1b1-null mice. A–D, total RNA from control and treated lungs copied into cDNA was used for quantifying various Cyp1 mRNAS as described under “Experimental Procedures.” Means ± S.D. were calculated from three independent assays. A, Cyp1B1 mRNA quantification in control and Cyp1b1-null mice. B, Cyp1a1 mRNA quantified from WT, Cyp1a1/1a2-null, and Cyp1b1-null mice. C, Cyp1a2 mRNA quantified from WT, Cyp1a1/1a2-null, and Cyp1b1-null mice. D, Cyp1b1 mRNA quantification from WT, Cyp1a1/1a2-null, and Cyp1b1-null mice. E, mitochondria and microsomes isolated from lungs of control, BaP-, and TCDD-treated mice (50 μg of protein each) were subjected to immunoblot analysis using antibodies to CYP1B1 and CYP1A1. The membrane portions cut from the same blot were also developed with antibodies to NPR and porin to detect any cross-contamination. *, p < 0.05. C, control (untreated); T, treated; NS, nonspecific.

FIGURE 8.

Aryl hydrocarbon hydroxylase activity in the mitochondria and microsomes isolated from lungs of WT, Cyp1a1/1a2-null, and Cyp1b1-null mice. A and B, mitochondrial and microsomal lung proteins (200 μg of protein each) from control and BaP-treated mice (n = 6) were used to determine the rates of benzo[a]pyrene hydroxylation as described under the “Experimental Procedures” and in Fig. 1. C and D, mitochondrial and microsomal protein (200 μg of protein each) from control and TCDD-treated mouse lungs was used to determine the rates of benzo[a]pyrene hydroxylation. Mitochondrial samples were reconstituted with Adx and Adr and the assays were carried out as detailed under the “Experimental Procedures.” TMS (33 μm) was added at the start of the reaction. The activity was plotted as nanomoles of hydroxybenzopyrene formed per min/mg of protein for all groups. The numbers in parentheses over the bars represent CYP contents of mitochondria and microsomes from treated and untreated mouse lungs. Means ± S.E. were calculated from 4 to 6 independent assays. ** indicates p < 0.001.

FIGURE 9.

ROS measurements in mitochondrial and microsomal isolates using the DCFH-DA method. A, ROS levels in treated and untreated WT, Cyp1a1/1a2-null, and Cyp1b1-null mouse lung mitochondria (Mito). B, microsomes from treated and untreated WT, Cyp1a1/1a2-null, and Cyp1b1-null mouse lung were measured using DCFH-DA (5 μm). Details were as described under “Experimental Procedures.” C, control experiment showing time-dependent increase of fluorescence using BaP-treated lung mitochondria from WT mice. Membrane-permeable catalase (10 units/ml) and SOD (30 units/ml) were added before the start of the reaction. The assays were run as described under the “Experimental Procedures” using 10 μg of brain cytosolic preparation per 200-μl reaction volume. The fluorescence was recorded at an excitation at 488 nm and emission at 525 nm for 15 min. **, p < 0.001.

FIGURE 10.

Mitochondrial respiratory complex activities and mtDNA content in response to BaP and TCDD treatment in mouse lungs. A, rotenone-sensitive complex I activity was measured in the lung mitochondria from control and Cyp1b1-null mice, treated with BaP or TCDD as NADH oxidation (decrease in absorbance at 340 nm). B, CcO activity was measured spectrophotometrically by following the oxidation of ferrocytochrome c at 550 nm. The details are described under the “Experimental Procedures.” Data are presented as means ± S.D. from six different mice, and groups were compared using an unpaired, two-tailed Student's t test. *, p < 0.05. C and D, total DNA was isolated from lung slices from control, BaP-, and TCDD-treated WT, Cyp1a1/1a2-null, and Cyp1b1-null mice. Quantitative PCR was performed with 50 ng of the template DNA using van Houten's methodology as described under the “Experimental Procedures” and resolved on 0.6% (w/v) agarose gels. E, bar graph showing the relative mtDNA content in the treated and untreated groups from WT and Cyp1b1-null mouse lungs. Primers used are listed under the “Experimental Procedures.” Data are presented as mean ± S.D. from six different mice, and groups were compared using an unpaired two-tailed Student's t test. *, p < 0.05; **, p < 0.001.

FIGURE 11.

Relative contributions of microsomal and mitochondrial CYP1B1 in inducing mitochondrial dysfunction. A, shRNA constructs targeted for NPR and Adx mRNAs were transfected in A549 cells. The crude lysates were analyzed 48 h post-transfection by immunoblotting with NPR antibody (1:2500, v/v) and Adx antibody (1:1000, v/v). B, shRNA constructs showing the maximum knockdown in A were used to generate stable expression lines in A549 cells as described under the “Experimental Procedures.” The cells were treated with DMSO alone or 20 μm BaP in DMSO for 72 h. Total cell lysate (50 μg of protein each) was subjected to immunoblot analysis with the indicated antibodies. The numbers below each lane represent relative band intensity for individual proteins. The values are average of two experiments. C–E, A549 cells stably expressing scrambled shRNA or shRNA for Adx and NPR were grown for 96 h either in presence of vehicle, DMSO alone, or with 20 μm BaP. mtDNA was quantified as in Fig. 10. C, ethidium bromide-stained pattern of 16.2-kb amplicon of mtDNA. D, PCR amplification of short mitochondrial fragment (100 bp) from different cell types treated with or without BaP as a template control. E, bar graph showing relative levels of mtDNA in different cell types treated with or without BaP. Details were as in Fig. 1 and under “Experimental Procedures.”