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Mitochondrial targeting of cytochrome P450 (CYP) 1B1 and its role in polycyclic aromatic hydrocarbon-induced mitochondrial dysfunction.
Bansal S, Leu AN, Gonzalez FJ, Guengerich FP, Chowdhury AR, Anandatheerthavarada HK, Avadhani NG
(2014) J Biol Chem 289: 9936-51
MeSH Terms: Adrenodoxin, Animals, Aryl Hydrocarbon Hydroxylases, Benzo(a)pyrene, COS Cells, Cell Line, Tumor, Chlorocebus aethiops, Cytochrome P-450 CYP1B1, Female, Humans, Male, Mice, Mice, Knockout, Mitochondria, Mutagenesis, Oxidation-Reduction, Oxygen Consumption, Polychlorinated Dibenzodioxins, Protein Sorting Signals, Protein Transport, Teratogens
Show Abstract · Added March 10, 2014
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.
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21 MeSH Terms
A tricistronic human adrenodoxin reductase-adrenodoxin-cytochrome P450 27A1 vector system for substrate hydroxylation in Escherichia coli.
Salamanca-Pinzón SG, Guengerich FP
(2011) Protein Expr Purif 79: 231-6
MeSH Terms: Adrenodoxin, Animals, Blotting, Western, Cattle, Cholecalciferol, Cholesterol, Chromatography, Liquid, Cytochrome P-450 Enzyme System, Escherichia coli, Ferredoxin-NADP Reductase, Genetic Vectors, Humans, Hydroxylation, Mitochondria, NADP, Protein Binding, Protein Engineering, Recombinant Fusion Proteins, Spectrometry, Mass, Electrospray Ionization, Tandem Mass Spectrometry
Show Abstract · Added March 7, 2014
Cytochrome P450 (P450) 27A1 catalyzes 27-hydroxylation of cholesterol and 25-hydroxylation of vitamin D(3), serving as an important component for the maintenance of lipid homeostasis. In eukaryotic cells P450 27A1 is a membrane-bound protein located on the inner mitochondrial membrane and requires two auxiliary reduction partners, adrenodoxin (Adx) and NADPH-adrenodoxin reductase (Adr), for catalysis in the bile acid biosynthesis pathway. A strategy was developed for the functional coexpression of P450 27A1 with Adr and Adx in a tricistronic fashion (single RNA, three proteins) in Escherichia coli, mimicking the mitochondrial P450 system. Intact bacterial cells coexpressing the P450 vector (pTC27A1) efficiently hydroxylated cholesterol at the 27 position as well as vitamin D(3) at the 25 position when supplemented with glycerol as a carbon source. Thus, E. coli containing pTC27A1 is able to hydroxylate cholesterol in a self-sufficient fashion and is suitable for further applications of protein interaction, drug discovery, and inhibitor evaluation and for the study of other mitochondrial P450s and oxysterol production in microorganisms without a need for membrane reconstitution, membrane simulation by detergents, or purification of the components.
Copyright © 2011 Elsevier Inc. All rights reserved.
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Heterologous expression, purification, and properties of human cytochrome P450 27C1.
Wu ZL, Bartleson CJ, Ham AJ, Guengerich FP
(2006) Arch Biochem Biophys 445: 138-46
MeSH Terms: Adrenodoxin, Amino Acid Sequence, Calcifediol, Catalysis, Cholecalciferol, Cholesterol, Cloning, Molecular, Cytochrome P-450 Enzyme System, Cytochrome P450 Family 27, Escherichia coli, Ferredoxin-NADP Reductase, Humans, Mass Spectrometry, Mitochondria, Molecular Sequence Data, Mutation, Organ Specificity, Peptide Fragments, RNA, Messenger, Recombinant Proteins
Show Abstract · Added March 5, 2014
Cytochrome P450 (P450) 27C1 is one of the "orphan" P450 enzymes without a known biological function. A human P450 27C1 cDNA with a nucleotide sequence modified for Escherichia coli usage was prepared and modified at the N-terminus, based on the expected mitochondrial localization. A derivative with residues 3-60 deleted was expressed at a level of 1350nmol/L E. coli culture and had the characteristic P450 spectra. The identity of the expressed protein was confirmed by mass spectrometry of proteolytic fragments. The purified P450 was in the low-spin iron state, and the spin equilibrium was not perturbed by any of the potential substrates vitamin D(3), 1alpha- or 25-hydroxy vitamin D(3), or cholesterol. P450s 27A1 and 27B1 are known to catalyze the 25-hydroxylation of vitamin D(3) and the 1alpha-hydroxylation of 25-hydroxy vitamin D(3), respectively. In the presence of recombinant human adrenodoxin and adrenodoxin reductase, recombinant P450 27C1 did not catalyze the oxidation of vitamin D(3), 1alpha- or 25-hydroxy vitamin D(3), or cholesterol at detectable rates. P450 27C1 mRNA was determined to be expressed in liver, kidney, pancreas, and several other human tissues.
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20 MeSH Terms
Evolutionarily divergent electron donor proteins interact with P450MT2 through the same helical domain but different contact points.
Anandatheerthavarada HK, Amuthan G, Biswas G, Robin MA, Murali R, Waterman MR, Avadhani NG
(2001) EMBO J 20: 2394-403
MeSH Terms: Adrenodoxin, Animals, Binding Sites, Cattle, Chromosome Mapping, Cross-Linking Reagents, Cytochrome P-450 CYP1A1, Evolution, Molecular, Flavodoxin, Mammals, Mitochondria, Liver, Models, Molecular, Mutagenesis, NADPH-Ferrihemoprotein Reductase, Protein Structure, Secondary, Rats, Two-Hybrid System Techniques
Show Abstract · Added February 12, 2015
We have investigated the sites of N-terminally truncated cytochrome P4501A1 targeted to mitochondria (P450MT2) which interact with adrenodoxin (Adx), cytochrome P450 reductase (CPR) and bacterial flavodoxin (Fln). The binding site was mapped by a combination of in vitro mutagenesis, in vivo screening with a mammalian two-hybrid system, spectral analysis, reconstitution of enzyme activity and homology-based structural modeling. Our results show that part of an aqueous accessible helix (putative helix G, residues 264-279) interacts with all three electron donor proteins. Mutational studies revealed that Lys267 and Lys271 are crucial for binding to Adx, while Lys268 and Arg275 are important for binding to CPR and FLN: Additive effects of different electron donor proteins on enzyme activity and models on protein docking show that Adx and CPR bind in a non-overlapping manner to the same helical domain in P450MT2 at different angular orientations, while CPR and Fln compete for the same binding site. We demonstrate that evolutionarily divergent electron donor proteins interact with the same domain but subtly different contact points of P450MT2.
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17 MeSH Terms
Three zinc finger nuclear proteins, Sp1, Sp3, and a ZBP-89 homologue, bind to the cyclic adenosine monophosphate-responsive sequence of the bovine adrenodoxin gene and regulate transcription.
Cheng PY, Kagawa N, Takahashi Y, Waterman MR
(2000) Biochemistry 39: 4347-57
MeSH Terms: Adrenal Cortex, Adrenodoxin, Animals, Base Sequence, Cattle, Cell Line, Chelating Agents, Consensus Sequence, Cyclic AMP, DNA, DNA-Binding Proteins, Drosophila melanogaster, Gene Expression Regulation, Mice, Molecular Weight, Mutation, Nuclear Proteins, Phenanthrolines, Repressor Proteins, Response Elements, Sp1 Transcription Factor, Sp3 Transcription Factor, Transcription Factors, Transcription, Genetic, Zinc Fingers
Show Abstract · Added February 12, 2015
Adrenocorticotropin acting through cyclic adenosine monophosphate (cAMP) regulates transcription of the bovine adrenodoxin (Adx) gene in the adrenal cortex. The bovine Adx cAMP-responsive transcription sequence (CRS) has previously been found to contain two consensus GC boxes. By use of nuclear extracts from adrenocortical cells, Sp1 and Sp3 are shown here to bind to CRS. Mutations designed to enhance the identification of additional CRS binding proteins by reducing Sp protein binding showed the presence of an additional DNA-binding protein (Adx factor). Adx factor binding is inhibited by the zinc-chelating agent, 1,10-o-phenanthroline, suggesting it might be a zinc finger protein. By a fractionation/renaturation technique the Adx factor in mouse Y1 adrenocortical cells was found to be in the size range of 106-115 kDa by gel mobility shift assay. On the basis of size, the CRS sequence to which it binds, and its tentative identification as a zinc finger protein, Adx factor has been identified as a Krüppel-like zinc finger protein (a mouse ZBP-89 homologue). Further mutagenesis of CRS demonstrates that it can further be divided into two similar cAMP-responsive elements, and elimination of ZBP-89 binding does not affect cAMP responsiveness of either. Expression of these three nuclear proteins in Drosophila SL2 cells has been used to decipher the role of Adx CRS binding proteins in regulating transcription. Sp1 and Sp3 confer basal transcriptional activities, yet only Sp1 confers cAMP-responsive activity. ZBP-89 represses basal transcriptional activity.
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25 MeSH Terms
The tertiary structure of full-length bovine adrenodoxin suggests functional dimers.
Pikuleva IA, Tesh K, Waterman MR, Kim Y
(2000) Arch Biochem Biophys 373: 44-55
MeSH Terms: Adrenodoxin, Animals, Catalytic Domain, Cattle, Crystallization, Crystallography, X-Ray, Cytochrome P-450 Enzyme System, Dimerization, Electrochemistry, Electron Transport, Models, Molecular, Molecular Sequence Data, Peptide Fragments, Protein Structure, Quaternary, Protein Structure, Tertiary, Recombinant Proteins, Solutions
Show Abstract · Added February 12, 2015
The three-dimensional X-ray crystal structure of full-length oxidized bovine adrenodoxin (Adx) has been determined at 2.5 A resolution by molecular replacement using a structure of a truncated form as a starting model. Crystals of Adx belong to a primitive monoclinic space group P2(1) with four Adx molecules in an asymmetric unit. The unit cell dimensions are a = 59.44 A, b = 77.03 A, c = 59.68 A, and beta = 94.83 degrees. The structure has been refined to an R factor of 23.5%. Structures of the four molecules of full-length Adx (127 amino acids) in the asymmetric unit were compared with each other and also with that of the truncated Adx (4-108). The overall topology of full-length Adx remains the same as described earlier for the truncated protein. Differences that do occur are almost wholly confined to alternate side-chain conformations that reflect differing lattice contacts made by two proteins. Extensive interactions found between molecules 1 and 2 in the full-length Adx asymmetric unit may reflect the ability of Adx to form dimers in vivo and are consistent with hydrodynamic measurements which show that in solution there is an equilibrium between monomeric and dimeric forms of Adx. Dimerization of Adx could explain why the truncated form has greater affinity for the P450 redox partner than the full-length form. From these results it can be considered that the mechanism of electron transfer is not necessarily the same in different mitochondrial P450 systems.
Copyright 2000 Academic Press.
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17 MeSH Terms
Protein synthesis inhibitors and ethanol selectively enhance heterologous expression of P450s and related proteins in Escherichia coli.
Kusano K, Waterman MR, Sakaguchi M, Omura T, Kagawa N
(1999) Arch Biochem Biophys 367: 129-36
MeSH Terms: Adrenodoxin, Aminoglycosides, Animals, Anti-Bacterial Agents, Cattle, Chloramphenicol, Cold Temperature, Cytochrome P-450 Enzyme System, Escherichia coli, Ethanol, Gene Expression, Heat-Shock Response, Isopropyl Thiogalactoside, Membrane Proteins, Microsomes, Mitochondria, Protein Synthesis Inhibitors, Rats, Recombinant Proteins, Sequence Deletion, Tetracycline
Show Abstract · Added February 12, 2015
The antibiotics chloramphenicol (Cm), tetracycline, and erythromycin, which inhibit bacterial protein synthesis and are known to induce the cold shock response, unexpectedly enhance the heterologous expression of P450s and related proteins in Escherichia coli. In contrast, antibiotics that mimic heat shock in E. coli such as puromycin, streptomycin, and kanamycin decrease the expression of the same proteins. A sublethal dose of Cm (1 microgram/ml) effectively enhances the expression of both membrane-bound proteins (microsomal and mitochondrial P450s) and a soluble mitochondrial protein (adrenodoxin) over the range of two- to eightfold. The expression level of N-terminal truncated P450c17 (1600 nmol/liter culture without Cm), for instance, reached 3500 nmol/liter culture by the addition of Cm, approximately 8.4% of the total cellular protein. Cm also enabled expression at useful levels of active P450s previously difficult to express in E. coli. In contrast, the expression of P450scc, a mitochondrial protein, is decreased by Cm but enhanced by ethanol, a powerful elicitor of heat shock response in E. coli. These results show that both the cold shock response induced by some antibiotics and the heat shock response induced by ethanol may lead to enhanced expression of certain heterologous proteins in E. coli. This study also indicates that protein synthesis inhibitors associated with the cold shock response may act as protein synthesis enhancers under certain conditions.
Copyright 1999 Academic Press.
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21 MeSH Terms
An additional electrostatic interaction between adrenodoxin and P450c27 (CYP27A1) results in tighter binding than between adrenodoxin and p450scc (CYP11A1).
Pikuleva IA, Cao C, Waterman MR
(1999) J Biol Chem 274: 2045-52
MeSH Terms: Adrenodoxin, Amino Acid Sequence, Amino Acids, Base Sequence, Cholestanetriol 26-Monooxygenase, Cholesterol Side-Chain Cleavage Enzyme, Cytochrome P-450 Enzyme System, Humans, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Binding, Recombinant Proteins, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Static Electricity, Steroid Hydroxylases
Show Abstract · Added February 12, 2015
Mitochondrial cytochrome P450c27 (product of the CYP27A1 gene) is found to have significantly higher affinity for the common redox partner adrenodoxin than another mitochondrial P450, P450scc (product of the CYP11A1 gene). To investigate the basis of the approximately 30-fold difference in adrenodoxin binding, two sets of P450c27 mutants were generated, expressed in Escherichia coli, and purified. Mutations of one set were within the putative adrenodoxin-binding site containing conserved lysine residues also crucial in P450scc for binding adrenodoxin. The second set included mutations within a sequence aligning with the "meander region" of P450BM-3 proposed to be a site of redox-partner interactions in P450s (Hasemann, C. A., Kurumbail, R. G., Boddupalli, S. S., Peterson, J. A., and Deisenhofer, J. (1995) Structure 3, 41-62). Mutation of the P450c27 conserved lysines (K354A and K358A) led to a approximately 20-fold increase in apparent Ks for adrenodoxin, confirming that these two positively charged residues conserved in mitochondrial P450s are important for adrenodoxin binding. Mutation of Arg-418, conserved in the CYP27A1 family, to serine also decreased the affinity for adrenodoxin approximately 20-fold. This residue is predicted to be located in the meander region. A triple K354A/K358A/R418S mutation profoundly reduced adrenodoxin binding. Thus, in contrast to P450scc, where mutation of the two conserved positively charged residues results in virtually complete inhibition of adrenodoxin binding, in P450c27 there are three of such residues (Lys-354, Lys-358, and Arg-418) important for adrenodoxin interaction.
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15 MeSH Terms
Preimplantation mouse blastocysts fail to express CYP genes required for estrogen biosynthesis.
Strömstedt M, Keeney DS, Waterman MR, Paria BC, Conley AJ, Dey SK
(1996) Mol Reprod Dev 43: 428-36
MeSH Terms: Adrenodoxin, Animals, Aromatase, Base Sequence, Blastocyst, Cholestanetriol 26-Monooxygenase, Cytochrome P-450 Enzyme System, DNA Primers, Embryonic Development, Estrogens, Female, Gene Expression, Liver, Male, Mice, Molecular Sequence Data, NADH, NADPH Oxidoreductases, NADPH-Ferrihemoprotein Reductase, Ovary, Oxidoreductases, Pregnancy, Steroid 17-alpha-Hydroxylase, Steroid Hydroxylases, Sterol 14-Demethylase, Swine
Show Abstract · Added February 12, 2015
Implantation is initiated on day 4 in the mouse and on day 13 in the pig. The preimplantation pig blastocyst synthesizes steroid hormones, but whether preimplantation rodent embryos also have this ability has remained unresolved for the last two decades. In this study, the mRNAs encoding NADPH-cytochrome P450 reductase (P450-reductase), adrenodoxin, lanosterol 14-demethylase P450 (CYP51), 17 alpha-hydroxylase P450 (CYP17), cholesterol side-chain cleavage P450 (CYP11A1), sterol 27-hydroxylase P450 (CYP27), and aromatase P450 (CYP19) were examined in day 4 mouse blastocysts (day 1 = vaginal plug) and in day 13 and 16 pig blastocysts using reverse transcription-polymerase chain reaction (RT-PCR). In mouse blastocysts, mRNAs of P450-reductase, adrenodoxin, and CYP51, but not CYP17, CYP11A1, CYP27, and CYP19, were detected. In agreement with this finding, no aromatase protein could be detected by immunohistochemistry. By contrast, all these mRNAs were detected in the pig blastocyst. Furthermore, both the ovarian and placental types of aromatase (CYP19) mRNAs were detected in the pig blastocyst on days 13 and 16 of pregnancy, although the ovarian form was more abundant. Both forms of aromatase were much higher in day 13 than in day 16 pig blastocysts. The results provide definitive evidence that the preimplantation mouse blastocyst, as opposed to the pig blastocyst, has no ability to synthesize estrogen and no steroidogenic capacity. Maternal estrogen synthesis is essential for implantation of the mouse blastocyst.
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25 MeSH Terms
Messenger RNAs encoding steroidogenic enzymes are expressed in rodent brain.
Strömstedt M, Waterman MR
(1995) Brain Res Mol Brain Res 34: 75-88
MeSH Terms: Adrenodoxin, Animals, Base Sequence, Brain Chemistry, Cytochrome P-450 Enzyme System, DNA-Binding Proteins, Fushi Tarazu Transcription Factors, Genetic Code, Homeodomain Proteins, Male, Mice, Mice, Inbred ICR, Molecular Sequence Data, NADPH-Ferrihemoprotein Reductase, Nerve Tissue Proteins, RNA, Messenger, Rats, Rats, Sprague-Dawley, Receptors, Cytoplasmic and Nuclear, Steroidogenic Factor 1, Transcription Factors
Show Abstract · Added February 12, 2015
Using the reverse transcription polymerase chain reaction, mRNAs encoding steroidogenic P450s as well as NADPH-cytochrome P450 reductase (P450 reductase), adrenodoxin and the transcription factor steroidogenic factor 1 (SF-1) were all detected in rodent brain, but their distribution between brain regions varied. Adrenodoxin and P450 reductase were detected in all regions, suggesting the presence of both mitochondrial and microsomal P450s throughout the brain. Messenger RNAs encoding P450scc (CYP11A1) and P45017 alpha (CYP17) were also detected in all brain regions, this being the first report of CYP17 in the brain. P450c21 (CYP21) was detected only in the brain stem. P45011 beta (CYP11B1) and P450aldo (CYP11B2) are expressed in rat brain, but not in mouse brain; CYP11B1 primarily in the cerebrum, whereas CYP11B2 was detected in all brain regions. In both species, highest levels of aromatase P450 (CYP19) mRNA were detected in the cerebrum. SF-1 expression was restricted to the cerebrum minus cortex. Thus, although SF-1 is required for high level expression of the steroidogenic enzymes in adrenals and gonads, other factors may influence the expression of these genes in the brain. If the mRNAs detected by RT-PCR are indeed translated into functional enzymes, these studies suggest that different brain regions have different capacities for local steroid hormone production and metabolism. This raises the technical challenge of locating the specific sites of synthesis as well as the function of such locally produced ligands.
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21 MeSH Terms