Journal: The Journal of Biological Chemistry

Article Title: Molecular Cloning and Characterization of First Organic Matrix Protein from Sclerites of Red Coral, Corallium rubrum *

doi: 10.1074/jbc.M112.352005

Figure Lengend Snippet: Silver staining of electrophoresis gels and Western blots on organic matrix extracted from sclerites of C. rubrum . a , one-dimensional SDS-PAGE (10 μg protein/lane; BisTris 12% polyacrylamide gel, silver stain molecular mass marker (M6539; Sigma) as protein marker). b , one-dimensional SDS-PAGE (10 μg protein/lane, Tris-Tricine 16.5% polyacrylamide gel, kaleidoscope polypeptide standards (161-0325; Bio-Rad) as protein marker); c , two-dimensional electrophoresis gel (Tris-Tricine 16.5% polyacrylamide gel, kaleidoscope polypeptide standards (161-0325) as protein marker) of the major protein band (18*) excised from one dimensional SDS-PAGE. d , Western blot (5 μg of proteins/lane; BisTris 12% polyacrylamide gel, Precision Plus Protein WesternC TM standard (161-0376; Bio-Rad) as protein marker) with antibodies against phosphorylated serine. e , Western blot with antibodies against the phosphorylated threonine (5 μg of proteins/lane; BisTris 12% polyacrylamide gel, Precision Plus Protein WesternC TM standard (161-0376) as protein marker). f , Western blot with antibodies against phosphorylated tyrosine (5 μg of proteins/lane; BisTris 12% polyacrylamide gel, Precision Plus Protein WesternC TM standard (161-0376) as protein marker). g , Western blot with anti-scleritin antibodies (Tris-Tricine 16.5% polyacrylamide gel, Precision Plus Protein WesternC TM standard (161-0376) as protein marker). MW , molecular mass; M , protein marker; and triangles show apparent molecular mass of proteins (in kDa).

Article Snippet: The protein markers used were: Silver Stain Molecular Weight Marker (M6539; Sigma) for the BisTris silver-stained electrophoresis gel, Kaleidoscope Polypeptide Standards (Bio-Rad 161-0325) for the Tris-Tricine silver-stained electrophoresis gels, and Precision Plus Protein WesternC TM standard (161-0376 Bio-Rad) both for the BisTris and Tris-Tricine gels of Western blots.

Techniques: Silver Staining, Electrophoresis, Western Blot, SDS Page, Marker

Journal: Cells

Article Title: A Phospho-SIM in the Antiviral Protein PML is Required for Its Recruitment to HSV-1 Genomes

doi: 10.3390/cells3041131

Figure Lengend Snippet: Mutation of PML phosphorylation sites does not affect colocalization of PML-I and ICP0 during HSV-1 infection. HA-shPML cells transduced with FLAG-, CFP-tagged PML-I or each PML-I mutant were infected with HSV-1 at 2 PFU/cell. At 2 hpi, the cells were fixed and stained with antibodies against ICP0 and ICP4. PML is shown as red, ICP0 as green, and ICP4 as blue in the merged image. Scale bar = 10 μm.

Article Snippet: The transduced and infected cells were probed first with antibodies against ICP0 (H11060, Santa Cruz Biotechnology) and ICP4 (EastCoastBio) diluted in 5% rabbit serum in PBS and then probed with goat-anti-mouse IgG1 Dylight 594 and goat-anti-mouse IgG2b Dylight 488 as listed above for Sp100 and Daxx staining.

Techniques: Mutagenesis, Phospho-proteomics, Infection, Transduction, Staining

Journal: Cells

Article Title: A Phospho-SIM in the Antiviral Protein PML is Required for Its Recruitment to HSV-1 Genomes

doi: 10.3390/cells3041131

Figure Lengend Snippet: ICP0 induces degradation of PML-I regardless of mutated PML phosphorylation sites. HEp-2 were transfected with 900 ng of an empty vector (pGEM-3), a vector encoding ICP0, or a vector encoding the ICP0 mutant n212 and 100 ng of a plasmid encoding FLAG-, eCFP-tagged PML-I or a PML-I mutant. Twenty-four hours later, cells were lysed, resolved by SDS-PAGE, and analyzed by western blot with an anti-FLAG or anti-β-actin antibody.

Article Snippet: The transduced and infected cells were probed first with antibodies against ICP0 (H11060, Santa Cruz Biotechnology) and ICP4 (EastCoastBio) diluted in 5% rabbit serum in PBS and then probed with goat-anti-mouse IgG1 Dylight 594 and goat-anti-mouse IgG2b Dylight 488 as listed above for Sp100 and Daxx staining.

Techniques: Phospho-proteomics, Transfection, Plasmid Preparation, Mutagenesis, SDS Page, Western Blot

Journal: Cells

Article Title: A Phospho-SIM in the Antiviral Protein PML is Required for Its Recruitment to HSV-1 Genomes

doi: 10.3390/cells3041131

Figure Lengend Snippet: Mutation of phosphorylation sites near the SIM compromises recruitment of PML-I to incoming HSV-1 genomes. HA-shPML cells transduced with FLAG-, eCFP-tagged PML-I or a PML-I mutant were infected with an ICP0-null virus at 0.1 PFU/cell. At 24 hpi, the cells were fixed and stained with antibody against ICP4 as a marker of viral DNA. Scale bar = 10 μm.

Article Snippet: The transduced and infected cells were probed first with antibodies against ICP0 (H11060, Santa Cruz Biotechnology) and ICP4 (EastCoastBio) diluted in 5% rabbit serum in PBS and then probed with goat-anti-mouse IgG1 Dylight 594 and goat-anti-mouse IgG2b Dylight 488 as listed above for Sp100 and Daxx staining.

Techniques: Mutagenesis, Phospho-proteomics, Transduction, Infection, Virus, Staining, Marker

Journal: Cells

Article Title: A Phospho-SIM in the Antiviral Protein PML is Required for Its Recruitment to HSV-1 Genomes

doi: 10.3390/cells3041131

Figure Lengend Snippet: Properties of PML-I and PML-I mutants.

Article Snippet: The transduced and infected cells were probed first with antibodies against ICP0 (H11060, Santa Cruz Biotechnology) and ICP4 (EastCoastBio) diluted in 5% rabbit serum in PBS and then probed with goat-anti-mouse IgG1 Dylight 594 and goat-anti-mouse IgG2b Dylight 488 as listed above for Sp100 and Daxx staining.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Different molecular forms of CYP2C8 in human liver samples. A, panels i–iii, representative immunoblots of mitochondrial and microsomal proteins (50 μg each) from human liver samples (Mt, mitochondrial fraction; Mc, microsomal fraction; HL, human liver used in labeling of some of the samples). Blots were developed with polyclonal antibodies to CYP2C8 (1:500 dilution, v/v) and TOM20 (1:2,000 dilution, v/v) and monoclonal antibody to cytochrome P450 reductase (CPR) (1:1,500 dilution, v/v). In addition to the full-length CYP2C8, a smaller form of 44 kDa (Var_3 (V3)) was seen predominantly in the mitochondrial fraction. The numbers in parentheses below the CYP2C8 immunoblot represent the ratios of Var_3 and full-length (Var_1) proteins in terms of band intensities. B, relative distribution of full-length CYP2C8 in mitochondria and microsomes of the liver samples analyzed in A. The percent distribution was calculated based on the densitometry of band intensities in A. Results represent averages from two blots.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Western Blot, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Identification of the 44-kDa species as a splice variant. A, protein sequence alignment of full-length (WT) CYP2C8 (marked 1) and its two splice variants (marked 2 and 3, respectively). Conserved residues in all three sequences are marked with asterisks. The reported Var_2 and Var_3 lack the N-terminal stretch of amino acids from the full-length protein. B, schematic representation of differential splicing of pre-mRNA for the generation of full-length CYP2C8 (Var_1) and Var_3 mRNAs. Ex, exon; aa, amino acids. C, DNA amplicons generated by RT-PCR of total RNAs using the common 3′- and 5′-primers were resolved on a 2% agarose gel (w/v) and stained with ethidium bromide. WT CYP2C8, Var_3 (V3), and a slow migrating minor component, Var_2 (V2), are shown for the liver samples analyzed in Fig. 1A. M, DNA marker. Relative band intensities of Var_3 and Var_1 amplicons are presented as ratios in parentheses below the gel patterns. C, panels i and ii, immunoblot analysis of mitochondrial proteins (50 μg each) from individual liver samples from Fig. 1A. Mitochondrial protein (Mito) from HepG2 cells expressing Var_3 cDNA was run alongside.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Variant Assay, Sequencing, Generated, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Marker, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: In silico analysis of WT CYP2C8 and CYP2C8 variant *3. A, three-dimensional structures of WT CYP2C8 (WT2C8; in yellow at left), Var_3 (V3-2C8; in pink at center), and Var_3 (V3) superimposed on WT (right). Heme is colored in red, and felodipine is colored blue. B, amino acid residues interacting with the heme (in red) through hydrogen-bonding interactions in WT CYP2C8 (at left) and Var_3 (at right). C, list of amino acid residues (one-letter notation) involved in anchoring the heme in the two molecular forms.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: In Silico, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Different subcellular targeting efficiencies of the three variant proteins. A, in vitro import of 35S-labeled translation products in isolated rat liver mitochondria. Panel i, 35S-labeled translation products of wild type (WT2C8), Var_2 (V2-2C8), and Var_3 (V3-2C8). Radiometric imaging of gels was performed to determine the level of import of input protein for each construct in trypsin-treated samples (T). The input protein level was considered to be 100% in each case. Panel ii, import of dihydrofolate reductase (DHFR) and SU9-dihydrofolate reductase (Su9-DHFR) proteins as negative and positive controls, respectively. The lanes marked “In” or “I” (for input) were loaded with 20% of the counts used for the import reactions. “C” 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. B, translocation of WT and variant 2 and 3 proteins in transiently transfected COS-7 cells. Panel i, total cell lysates (50 μg each) from transiently transfected COS-7 cells were resolved by 12% SDS (w/v)-PAGE and probed with antibodies to CYP2C8 and β-actin for assessing loading levels. Panel ii, mitochondria and microsomes were isolated from transfected COS-7 cells, and 50 μg of protein each was resolved by 12% SDS (w/v)-PAGE and probed with antibodies to CYP2C8, cytochrome P450 reductase (CPR), and cytochrome-c oxidase subunit I. Panel iii, relative resistance of mitochondrion-associated proteins to trypsin treatment (T). In some cases, mitochondria were lysed by treatment with 1% Triton X-100 (v/v) before trypsin treatment (TT). Proteins (50 μg each) were resolved by SDS-PAGE and probed with antibodies to CYP2C8 and TOM20 for immunoblot analysis. C, immunofluorescence microcopy of COS-7 cells transfected with WT (Var_1), Var_2, or Var_3 cDNA. Panel i, a–c, co-localization of CYP2C8 with a mitochondrial marker, cytochrome-c oxidase subunit I (CcOI). Cells were stained with a 1:1,000 dilution (v/v) of primary anti-goat antibody to CYP2C8 (green) (Abcam, Cambridge, MA) and co-stained with a 1:500 dilution (v/v) of cytochrome-c oxidase subunit I (red) (anti-mouse) antibody (Abcam). Panel ii, a–c, co-localization of CYP2C8 with microsomal membrane marker calreticulin. Cells were stained with CYP2C8 (green) as above and co-stained with a 1:500 dilution (v/v) of calreticulin (CRT) (anti-rabbit) antibody (red) (Santa Cruz Biotechnology, Santa Cruz, CA). The cells were subsequently incubated with secondary Alexa Fluor 546-conjugated anti-mouse and then anti-rabbit IgG and Alexa Fluor 488-conjugated anti-goat IgG and imaged through a confocal microscope. Numbers in the bottom panels indicate Pearson coefficients for coincidence calculated using Volocity 5.3 software.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Variant Assay, In Vitro, Labeling, Isolation, Imaging, Construct, Translocation Assay, Transfection, SDS Page, Western Blot, Immunofluorescence, Marker, Staining, Incubation, Microscopy, Software

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: The mitochondrial targeting efficiency of N-terminal signals of the full-length (Var_1; WT), Var_2 (V*2), and Var_3 (V*3) proteins The mitochondrial targeting efficiency of the three proteins was analyzed using the MitoProt II-v1.101 program. aa, amino acids.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Reconstitution of catalytic activity of purified WT CYP2C8. A, reconstitution of paclitaxel 6-hydroxylation activity was done with 0.2 nmol of purified CYP protein with or without 0.5 nmol of cytochrome P450 reductase (CPR), 0.4 nmol of purified Adx, 0.04 nmol of purified AdxR, and 10 μm paclitaxel in a 0.3-ml final volume as described under “Materials and Methods.” Montelukast (Mon) (5 μm) and inhibitory antibody to CYP2C8 (2C8Ab) (10 mg/ml) were used. Control ascites fluid (CAF; 10 mg/ml) was used as a negative control. B, reconstitution of dibenzylfluorescein oxidation was carried out essentially as described above in A. The activities in all cases represent the means ± S.E. (error bars) of three to five separate assays. Purified CYP2C8 was preincubated with inhibitors and control ascites fluid as described under “Materials and Methods.” ♦ in A indicates no detectable activity.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Activity Assay, Purification, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Subcellular distribution of CYP2C8 in stable HepG2 cells. A, immunoblot analysis of mitochondrial (Mt) and microsomal (Mc) fractions isolated from stable HepG2 cells with CYP2C8 antibody (middle panel). The blots were also probed with antibodies to TOM20 (bottom panel) as a mitochondrion-specific marker and NADPH-cytochrome P450 reductase (top panel) as a microsome-specific marker. Std, standard. B, membrane-extrinsic and -intrinsic nature of wild-type CYP2C8 (WT 2C8) and Var_3 (V3 2C8) in the mitochondrial (Mito) and microsomal (Micro) fractions was analyzed by the alkaline Na2CO3 extraction method described under “Materials and Methods.” The soluble (E) and insoluble (P) protein fractions were subjected to SDS-PAGE separation and probed with CYP2C8 antibody (2C8Ab) by immunoblot analysis. C, the relative levels of viral vector DNA (puromycin acetyltransferase gene) were determined by real time PCR using total cell DNA as template and the actin gene as an internal reference. D, spectrophotometric scans of CYP heme in the mitochondrial and microsomal fractions obtained from HepG2 stable cells expressing WT and Var_3 proteins. Fe2+-CO versus Fe2+ spectra were recorded as described under “Materials and Methods.” Abs, absorbance. E, relative CYP contents of mitochondria and microsomes from mock-, wild-type CYP2C8-, and CYP2C8 Var_3-expressing HepG2 cells. CYP content was measured by CO difference spectra as described under “Materials and Methods.” F, paclitaxel 6-hydroyxlation activity reconstituted with mitochondria and microsomes from stable HepG2 cells expressing WT CYP2C8 and Var_3 CYP2C8 and mock-transfected cells. Assays were carried out as described under “Materials and Methods.” G, ROS production in isolated mitochondria from stable HepG2 cell lines with or without treatment with the antioxidant N-acetylcysteine (NAC) or the inhibitors proadifen and montelukast. Mitochondria (50 μg each) were seeded in 96-well plates for ROS measurements using the 2′,7′-dichlorodihydrofluorescein (DCF) diacetate method as described under “Materials and Methods.” Results represent means ± S.E. (error bars) of three to four separate assays. * indicates a p value <0.05, and ** represents a p value <0.001. ♦ in F indicates no detectable activity. CPR, cytochrome P450 reductase.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Western Blot, Isolation, Marker, SDS Page, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Oxidation of arachidonic acid by mitochondria and microsomes isolated from stable HepG2 cells expressing Var_1 (WT) CYP2C8 and Var_3 proteins. A–D, the mitochondrial (Mt) and microsomal (MIC) proteins (300–500 μg) were assayed for arachidonic acid metabolism as described under “Materials and Methods” using 70 μm arachidonic acid as substrate. Inhibition studies were performed by preincubating enzymes with 5 μm montelukast at 37 °C for 20 min. Reactions were initiated by the addition of 1 mm NADPH and continued for 5 min at 37 °C in a shaking water bath, and the metabolites were extracted and analyzed as described under “Materials and Methods.” Four major arachidonate products (11,12-EET, 14,15-EET, 8,9-EET, and 20-HETE) were quantified as described under “Materials and Methods.” E, mitochondrial (Mito) proteins from Var_3 (V3)-expressing cells were reconstituted with or without added Adx/AdxR or added montelukast using arachidonic acid as substrate. In one case, purified Var_1 (V1) CYP2C8 was reconstituted with Adx/AdxR as described in Fig. 5. The total EET metabolites were quantified using the LC-MS method to ascertain the dependence of the enzyme on Adx/AdxR. The results represent means ± S.E. (error bars) of three independent assays. * indicates p < 0.05, and ** indicates p < 0.001. CPR, cytochrome P450 reductase.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Isolation, Expressing, Inhibition, Purification, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Respiratory dysfunction and ROS generation in cells treated with arachidonic acid. A, panels i–iii, the effect of arachidonic acid on respiration profile was measured using a Seahorse Bioscience XF24 extracellular flux analyzer. All parameters were analyzed using XF software and are displayed as oxygen consumption rates (pmol of O2/min/100 μg of protein) after normalizing for the protein concentration of each well. Panel i, basal OCR accounts for baseline rates of oxygen consumption. Panel ii, 2,4-dinitrophenol-mediated uncoupling generates maximal OCR. Panel iii, inhibition by oligomycin corresponds to ATP-linked OCR. Mean values ± S.E. (error bars) were calculated based on three separate measurements. B, effects of arachidonic acid (AA) on ROS production in stable HepG2 cells. Cells were grown with or without arachidonic acid for 24 h in 6-well plates, and the culture fluids were used for assaying the levels of H2O2 produced using the Amplex Red method as described under “Materials and Methods.” Results represent means ± S.E. (error bars) of three separate readings. * indicates a p value <0.05, and ** indicates a p value <0.001. ♦ in A (panel iii) indicates no detectable activity. V3, Var_3; 2C8, CYP2C8.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Software, Protein Concentration, Inhibition, Produced, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Different molecular forms of CYP2C8 in human liver samples. A, panels i–iii, representative immunoblots of mitochondrial and microsomal proteins (50 μg each) from human liver samples (Mt, mitochondrial fraction; Mc, microsomal fraction; HL, human liver used in labeling of some of the samples). Blots were developed with polyclonal antibodies to CYP2C8 (1:500 dilution, v/v) and TOM20 (1:2,000 dilution, v/v) and monoclonal antibody to cytochrome P450 reductase (CPR) (1:1,500 dilution, v/v). In addition to the full-length CYP2C8, a smaller form of 44 kDa (Var_3 (V3)) was seen predominantly in the mitochondrial fraction. The numbers in parentheses below the CYP2C8 immunoblot represent the ratios of Var_3 and full-length (Var_1) proteins in terms of band intensities. B, relative distribution of full-length CYP2C8 in mitochondria and microsomes of the liver samples analyzed in A. The percent distribution was calculated based on the densitometry of band intensities in A. Results represent averages from two blots.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Western Blot, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Identification of the 44-kDa species as a splice variant. A, protein sequence alignment of full-length (WT) CYP2C8 (marked 1) and its two splice variants (marked 2 and 3, respectively). Conserved residues in all three sequences are marked with asterisks. The reported Var_2 and Var_3 lack the N-terminal stretch of amino acids from the full-length protein. B, schematic representation of differential splicing of pre-mRNA for the generation of full-length CYP2C8 (Var_1) and Var_3 mRNAs. Ex, exon; aa, amino acids. C, DNA amplicons generated by RT-PCR of total RNAs using the common 3′- and 5′-primers were resolved on a 2% agarose gel (w/v) and stained with ethidium bromide. WT CYP2C8, Var_3 (V3), and a slow migrating minor component, Var_2 (V2), are shown for the liver samples analyzed in Fig. 1A. M, DNA marker. Relative band intensities of Var_3 and Var_1 amplicons are presented as ratios in parentheses below the gel patterns. C, panels i and ii, immunoblot analysis of mitochondrial proteins (50 μg each) from individual liver samples from Fig. 1A. Mitochondrial protein (Mito) from HepG2 cells expressing Var_3 cDNA was run alongside.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Variant Assay, Sequencing, Generated, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Marker, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: In silico analysis of WT CYP2C8 and CYP2C8 variant *3. A, three-dimensional structures of WT CYP2C8 (WT2C8; in yellow at left), Var_3 (V3-2C8; in pink at center), and Var_3 (V3) superimposed on WT (right). Heme is colored in red, and felodipine is colored blue. B, amino acid residues interacting with the heme (in red) through hydrogen-bonding interactions in WT CYP2C8 (at left) and Var_3 (at right). C, list of amino acid residues (one-letter notation) involved in anchoring the heme in the two molecular forms.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: In Silico, Variant Assay

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Different subcellular targeting efficiencies of the three variant proteins. A, in vitro import of 35S-labeled translation products in isolated rat liver mitochondria. Panel i, 35S-labeled translation products of wild type (WT2C8), Var_2 (V2-2C8), and Var_3 (V3-2C8). Radiometric imaging of gels was performed to determine the level of import of input protein for each construct in trypsin-treated samples (T). The input protein level was considered to be 100% in each case. Panel ii, import of dihydrofolate reductase (DHFR) and SU9-dihydrofolate reductase (Su9-DHFR) proteins as negative and positive controls, respectively. The lanes marked “In” or “I” (for input) were loaded with 20% of the counts used for the import reactions. “C” 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. B, translocation of WT and variant 2 and 3 proteins in transiently transfected COS-7 cells. Panel i, total cell lysates (50 μg each) from transiently transfected COS-7 cells were resolved by 12% SDS (w/v)-PAGE and probed with antibodies to CYP2C8 and β-actin for assessing loading levels. Panel ii, mitochondria and microsomes were isolated from transfected COS-7 cells, and 50 μg of protein each was resolved by 12% SDS (w/v)-PAGE and probed with antibodies to CYP2C8, cytochrome P450 reductase (CPR), and cytochrome-c oxidase subunit I. Panel iii, relative resistance of mitochondrion-associated proteins to trypsin treatment (T). In some cases, mitochondria were lysed by treatment with 1% Triton X-100 (v/v) before trypsin treatment (TT). Proteins (50 μg each) were resolved by SDS-PAGE and probed with antibodies to CYP2C8 and TOM20 for immunoblot analysis. C, immunofluorescence microcopy of COS-7 cells transfected with WT (Var_1), Var_2, or Var_3 cDNA. Panel i, a–c, co-localization of CYP2C8 with a mitochondrial marker, cytochrome-c oxidase subunit I (CcOI). Cells were stained with a 1:1,000 dilution (v/v) of primary anti-goat antibody to CYP2C8 (green) (Abcam, Cambridge, MA) and co-stained with a 1:500 dilution (v/v) of cytochrome-c oxidase subunit I (red) (anti-mouse) antibody (Abcam). Panel ii, a–c, co-localization of CYP2C8 with microsomal membrane marker calreticulin. Cells were stained with CYP2C8 (green) as above and co-stained with a 1:500 dilution (v/v) of calreticulin (CRT) (anti-rabbit) antibody (red) (Santa Cruz Biotechnology, Santa Cruz, CA). The cells were subsequently incubated with secondary Alexa Fluor 546-conjugated anti-mouse and then anti-rabbit IgG and Alexa Fluor 488-conjugated anti-goat IgG and imaged through a confocal microscope. Numbers in the bottom panels indicate Pearson coefficients for coincidence calculated using Volocity 5.3 software.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Variant Assay, In Vitro, Labeling, Isolation, Imaging, Construct, Translocation Assay, Transfection, SDS Page, Western Blot, Immunofluorescence, Marker, Staining, Incubation, Microscopy, Software

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: The mitochondrial targeting efficiency of N-terminal signals of the full-length (Var_1; WT), Var_2 (V*2), and Var_3 (V*3) proteins The mitochondrial targeting efficiency of the three proteins was analyzed using the MitoProt II-v1.101 program. aa, amino acids.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Reconstitution of catalytic activity of purified WT CYP2C8. A, reconstitution of paclitaxel 6-hydroxylation activity was done with 0.2 nmol of purified CYP protein with or without 0.5 nmol of cytochrome P450 reductase (CPR), 0.4 nmol of purified Adx, 0.04 nmol of purified AdxR, and 10 μm paclitaxel in a 0.3-ml final volume as described under “Materials and Methods.” Montelukast (Mon) (5 μm) and inhibitory antibody to CYP2C8 (2C8Ab) (10 mg/ml) were used. Control ascites fluid (CAF; 10 mg/ml) was used as a negative control. B, reconstitution of dibenzylfluorescein oxidation was carried out essentially as described above in A. The activities in all cases represent the means ± S.E. (error bars) of three to five separate assays. Purified CYP2C8 was preincubated with inhibitors and control ascites fluid as described under “Materials and Methods.” ♦ in A indicates no detectable activity.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Activity Assay, Purification, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Subcellular distribution of CYP2C8 in stable HepG2 cells. A, immunoblot analysis of mitochondrial (Mt) and microsomal (Mc) fractions isolated from stable HepG2 cells with CYP2C8 antibody (middle panel). The blots were also probed with antibodies to TOM20 (bottom panel) as a mitochondrion-specific marker and NADPH-cytochrome P450 reductase (top panel) as a microsome-specific marker. Std, standard. B, membrane-extrinsic and -intrinsic nature of wild-type CYP2C8 (WT 2C8) and Var_3 (V3 2C8) in the mitochondrial (Mito) and microsomal (Micro) fractions was analyzed by the alkaline Na2CO3 extraction method described under “Materials and Methods.” The soluble (E) and insoluble (P) protein fractions were subjected to SDS-PAGE separation and probed with CYP2C8 antibody (2C8Ab) by immunoblot analysis. C, the relative levels of viral vector DNA (puromycin acetyltransferase gene) were determined by real time PCR using total cell DNA as template and the actin gene as an internal reference. D, spectrophotometric scans of CYP heme in the mitochondrial and microsomal fractions obtained from HepG2 stable cells expressing WT and Var_3 proteins. Fe2+-CO versus Fe2+ spectra were recorded as described under “Materials and Methods.” Abs, absorbance. E, relative CYP contents of mitochondria and microsomes from mock-, wild-type CYP2C8-, and CYP2C8 Var_3-expressing HepG2 cells. CYP content was measured by CO difference spectra as described under “Materials and Methods.” F, paclitaxel 6-hydroyxlation activity reconstituted with mitochondria and microsomes from stable HepG2 cells expressing WT CYP2C8 and Var_3 CYP2C8 and mock-transfected cells. Assays were carried out as described under “Materials and Methods.” G, ROS production in isolated mitochondria from stable HepG2 cell lines with or without treatment with the antioxidant N-acetylcysteine (NAC) or the inhibitors proadifen and montelukast. Mitochondria (50 μg each) were seeded in 96-well plates for ROS measurements using the 2′,7′-dichlorodihydrofluorescein (DCF) diacetate method as described under “Materials and Methods.” Results represent means ± S.E. (error bars) of three to four separate assays. * indicates a p value <0.05, and ** represents a p value <0.001. ♦ in F indicates no detectable activity. CPR, cytochrome P450 reductase.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Western Blot, Isolation, Marker, SDS Page, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Oxidation of arachidonic acid by mitochondria and microsomes isolated from stable HepG2 cells expressing Var_1 (WT) CYP2C8 and Var_3 proteins. A–D, the mitochondrial (Mt) and microsomal (MIC) proteins (300–500 μg) were assayed for arachidonic acid metabolism as described under “Materials and Methods” using 70 μm arachidonic acid as substrate. Inhibition studies were performed by preincubating enzymes with 5 μm montelukast at 37 °C for 20 min. Reactions were initiated by the addition of 1 mm NADPH and continued for 5 min at 37 °C in a shaking water bath, and the metabolites were extracted and analyzed as described under “Materials and Methods.” Four major arachidonate products (11,12-EET, 14,15-EET, 8,9-EET, and 20-HETE) were quantified as described under “Materials and Methods.” E, mitochondrial (Mito) proteins from Var_3 (V3)-expressing cells were reconstituted with or without added Adx/AdxR or added montelukast using arachidonic acid as substrate. In one case, purified Var_1 (V1) CYP2C8 was reconstituted with Adx/AdxR as described in Fig. 5. The total EET metabolites were quantified using the LC-MS method to ascertain the dependence of the enzyme on Adx/AdxR. The results represent means ± S.E. (error bars) of three independent assays. * indicates p < 0.05, and ** indicates p < 0.001. CPR, cytochrome P450 reductase.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Isolation, Expressing, Inhibition, Purification, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction *

doi: 10.1074/jbc.M114.583062

Figure Lengend Snippet: Respiratory dysfunction and ROS generation in cells treated with arachidonic acid. A, panels i–iii, the effect of arachidonic acid on respiration profile was measured using a Seahorse Bioscience XF24 extracellular flux analyzer. All parameters were analyzed using XF software and are displayed as oxygen consumption rates (pmol of O2/min/100 μg of protein) after normalizing for the protein concentration of each well. Panel i, basal OCR accounts for baseline rates of oxygen consumption. Panel ii, 2,4-dinitrophenol-mediated uncoupling generates maximal OCR. Panel iii, inhibition by oligomycin corresponds to ATP-linked OCR. Mean values ± S.E. (error bars) were calculated based on three separate measurements. B, effects of arachidonic acid (AA) on ROS production in stable HepG2 cells. Cells were grown with or without arachidonic acid for 24 h in 6-well plates, and the culture fluids were used for assaying the levels of H2O2 produced using the Amplex Red method as described under “Materials and Methods.” Results represent means ± S.E. (error bars) of three separate readings. * indicates a p value <0.05, and ** indicates a p value <0.001. ♦ in A (panel iii) indicates no detectable activity. V3, Var_3; 2C8, CYP2C8.

Article Snippet: Construction of WT and Variant CYP2C8 cDNAs The ORF clone of human CYP2C8 (RG204605) was purchased from Origene Technologies, Rockville, MD.

Techniques: Software, Protein Concentration, Inhibition, Produced, Activity Assay