PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (994706)

Clipboard (0)
None

Related Articles

1.  CYB5D2 Requires Heme-Binding to Regulate HeLa Cell Growth and Confer Survival from Chemotherapeutic Agents 
PLoS ONE  2014;9(1):e86435.
The cytochrome b5 domain containing 2 (CYB5D2; Neuferricin) protein has been reported to bind heme, however, the critical residues responsible for heme-binding are undefined. Furthermore, the relationship between heme-binding and CYB5D2-mediated intracellular functions remains unknown. Previous studies examining heme-binding in two cytochrome b5 heme-binding domain-containing proteins, damage-associated protein 1 (Dap1; Saccharomyces cerevisiae) and human progesterone receptor membrane component 1 (PGRMC1), have revealed that conserved tyrosine (Y) 73, Y79, aspartic acid (D) 86, and Y127 residues present in human CYB5D2 may be involved in heme-binding. CYB5D2 binds to type b heme, however, only the substitution of glycine (G) at D86 (D86G) within its cytochrome b5 heme-binding (cyt-b5) domain abolished its heme-binding ability. Both CYB5D2 and CYB5D2(D86G) localize to the endoplasmic reticulum. Ectopic CYB5D2 expression inhibited cell proliferation and anchorage-independent colony growth of HeLa cells. Conversely, CYB5D2 knockdown and ectopic CYB5D2(D86G) expression increased cell proliferation and colony growth. As PGRMC1 has been reported to regulate the expression and activities of cytochrome P450 proteins (CYPs), we examined the role of CYB5D2 in regulating the activities of CYPs involved in sterol synthesis (CYP51A1) and drug metabolism (CYP3A4). CYB5D2 co-localizes with cytochrome P450 reductase (CYPOR), while CYB5D2 knockdown reduced lanosterol demethylase (CYP51A1) levels and rendered HeLa cells sensitive to mevalonate. Additionally, knockdown of CYB5D2 reduced CYP3A4 activity. Lastly, CYB5D2 expression conferred HeLa cell survival from chemotherapeutic agents (paclitaxel, cisplatin and doxorubicin), with its ability to promote survival being dependent on its heme-binding ability. Taken together, this study provides evidence that heme-binding is critical for CYB5D2 in regulating HeLa cell growth and survival, with endogenous CYB5D2 being required to modulate CYP activities.
doi:10.1371/journal.pone.0086435
PMCID: PMC3899279  PMID: 24466094
2.  Cytochrome P450 Monooxygenase CYP53 Family in Fungi: Comparative Structural and Evolutionary Analysis and Its Role as a Common Alternative Anti-Fungal Drug Target 
PLoS ONE  2014;9(9):e107209.
Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins whose role as a drug target against pathogenic microbes has been explored because of their stereo- and regio-specific oxidation activity. We aimed to assess the CYP53 family's role as a common alternative drug target against animal (including human) and plant pathogenic fungi and its role in fungal-mediated wood degradation. Genome-wide analysis of fungal species revealed the presence of CYP53 members in ascomycetes and basidiomycetes. Basidiomycetes had a higher number of CYP53 members in their genomes than ascomycetes. Only two CYP53 subfamilies were found in ascomycetes and six subfamilies in basidiomycetes, suggesting that during the divergence of phyla ascomycetes lost CYP53 P450s. According to phylogenetic and gene-structure analysis, enrichment of CYP53 P450s in basidiomycetes occurred due to the extensive duplication of CYP53 P450s in their genomes. Numerous amino acids (103) were found to be conserved in the ascomycetes CYP53 P450s, against only seven in basidiomycetes CYP53 P450s. 3D-modelling and active-site cavity mapping data revealed that the ascomycetes CYP53 P450s have a highly conserved protein structure whereby 78% amino acids in the active-site cavity were found to be conserved. Because of this rigid nature of ascomycetes CYP53 P450s' active site cavity, any inhibitor directed against this P450 family can serve as a common anti-fungal drug target, particularly toward pathogenic ascomycetes. The dynamic nature of basidiomycetes CYP53 P450s at a gene and protein level indicates that these P450s are destined to acquire novel functions. Functional analysis of CYP53 P450s strongly supported our hypothesis that the ascomycetes CYP53 P450s ability is limited for detoxification of toxic molecules, whereas basidiomycetes CYP53 P450s play an additional role, i.e. involvement in degradation of wood and its derived components. This study is the first report on genome-wide comparative structural (gene and protein structure-level) and evolutionary analysis of a fungal P450 family.
doi:10.1371/journal.pone.0107209
PMCID: PMC4164535  PMID: 25222113
3.  CYP51 structures and structure-based development of novel, pathogen-specific inhibitory scaffolds 
Graphical abstract
Highlights
► CYP51s (sterol 14alpha-demethylases) are efficient drug target enzymes. ► CYP51s have a highly rigid substrate binding cavity. ► CYP51 structure-based development of a new inhibitory scaffold is described.
CYP51 (sterol 14α-demethylase) is a cytochrome P450 enzyme essential for sterol biosynthesis and the primary target for clinical and agricultural antifungal azoles. The azoles that are currently in clinical use for systemic fungal infections represent modifications of two basic scaffolds, ketoconazole and fluconazole, all of them being selected based on their antiparasitic activity in cellular experiments. By studying direct inhibition of CYP51 activity across phylogeny including human pathogens Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum, we identified three novel protozoa-specific inhibitory scaffolds, their inhibitory potency correlating well with antiprotozoan activity. VNI scaffold (carboxamide containing β-phenyl-imidazoles) is the most promising among them: killing T. cruzi amastigotes at low nanomolar concentration, it is also easy to synthesize and nontoxic. Oral administration of VNI (up to 400 mg/kg) neither leads to mortality nor reveals significant side effects up to 48 h post treatment using an experimental mouse model of acute toxicity. Trypanosomatidae CYP51 crystal structures determined in the ligand-free state and complexed with several azole inhibitors as well as a substrate analog revealed high rigidity of the CYP51 substrate binding cavity, which must be essential for the enzyme strict substrate specificity and functional conservation. Explaining profound potency of the VNI inhibitory scaffold, the structures also outline guidelines for its further development. First steps of the VNI scaffold optimization have been undertaken; the results presented here support the notion that CYP51 structure-based rational design of more efficient, pathogen-specific inhibitors represents a highly promising direction.
doi:10.1016/j.ijpddr.2012.06.001
PMCID: PMC3596085  PMID: 23504044
Sterol 14α-demethylase; CYP51; Inhibition; Crystal structure
4.  Specific Effects of Potassium Ion Binding on Wild-Type and L358P Cytochrome P450cama 
Biochemistry  2006;45(48):14379-14388.
The camphor monoxygenase cytochrome P450cam (CYP101) requires potassium ion (K+) to drive formation of the characteristic high-spin state of the heme Fe+3 upon substrate binding. Amide 1H, 15N correlations in perdeuterated [U-15N] CYP101 were monitored as a function of K+ concentration by 2D-TROSY-HSQC in both camphor-bound oxidized (CYP-S) and camphor-and CO-bound reduced CYP101 (CYP-S-CO). In both forms, K+-induced spectral perturbations are detected in the vicinity of the K+ binding site proposed from crystallographic structures, but are larger and more widespread structurally in CYP-S than in CYP-S-CO. In CYP-S-CO, K+-induced perturbations occur primarily near the proposed K+ binding site in the B-B’ loop and B’ helix, which are also perturbed by binding of effector, putidaredoxin (Pdx). The spectral effects of K+ binding in CYP-S-CO oppose those observed upon Pdxr titration. However, Pdxr titration of CYP-S-CO in the absence of K+ results in multiple conformations. The spin-state equilibrium in the L358P mutant of CYP101 is more sensitive to K+ concentration than WT CYP101, consistent with a hypothesis that L358P preferentially populates conformations enforced by Pdx binding in WT CYP101. Thallium (I), a K+ mimic, minimizes the effects of Pdx titration on the NMR spectrum of CYP-S-CO, but is competent to replace K+ in driving the formation of high-spin CYP-S. These observations suggest that the role of K+ is to stabilize conformers of CYP-S that drive the spin state change prior to the first electron transfer, and that K+ stabilizes the CYP-S-CO conformer that interacts with Pdx. However, upon binding of Pdx, further conformational changes occur that disfavor K+ binding.
doi:10.1021/bi0617355
PMCID: PMC1764623  PMID: 17128977
CYP101; putidaredoxin; NMR; HSQC; electron transfer complex; TROSY; thallium; CYP101, cytochrome P450cam; CYP-S, oxidized camphor-bound CYP101; CYP-S-CO, reduced camphor- and carbonmonoxy-bound CYP101; IPTGm, isopropyl β-D-thiogalactoside; LB, Luria-Bertani media; NMR, nuclear magnetic resonance; HSQC, heteronuclear single-quantum correlation; M9, minimal growth medium; OD600, optical density at 600 nm; PdR, putidaredoxin reductase; Pdx, putidaredoxin; Pdxr, reduced putidaredoxin; TROSY, transverse relaxation optimized spectroscopy
5.  Purification, Reconstitution, and Inhibition of Cytochrome P-450 Sterol Δ22-Desaturase from the Pathogenic Fungus Candida glabrata 
Sterol Δ22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14α-demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol Δ22-desaturase activity in a reconstituted system with NADPH–cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 μM and a Vmax of 0.59 nmol of this substrate metabolized/min/nmol of P-450. This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans and Schizosaccharomyces pombe genome projects. Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme. The azole antifungal compounds inhibited reconstituted sterol Δ22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added. These results reveal the potential for sterol Δ22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell.
PMCID: PMC89351  PMID: 10390230
6.  Genetic Basis for Differential Activities of Fluconazole and Voriconazole against Candida krusei 
Invasive infections caused by Candida krusei are a significant concern because this organism is intrinsically resistant to fluconazole. Voriconazole is more active than fluconazole against C. krusei in vitro. One mechanism of fluconazole resistance in C. krusei is diminished sensitivity of the target enzyme, cytochrome P450 sterol 14α-demethylase (CYP51), to inhibition by this drug. We investigated the interactions of fluconazole and voriconazole with the CYP51s of C. krusei (ckCYP51) and fluconazole-susceptible Candida albicans (caCYP51). We found that voriconazole was a more potent inhibitor of both ckCYP51 and caCYP51 in cell extracts than was fluconazole. Also, the ckCYP51 was less sensitive to inhibition by both drugs than was caCYP51. These results were confirmed by expressing the CYP51 genes from C. krusei and C. albicans in Saccharomyces cerevisiae and determining the susceptibility of the transformants to voriconazole and fluconazole. We constructed homology models of the CYP51s of C. albicans and C. krusei based on the crystal structure of CYP51 from Mycobacterium tuberculosis. These models predicted that voriconazole is a more potent inhibitor of both caCYP51 and ckCYP51 than is fluconazole, because the extra methyl group of voriconazole results in a stronger hydrophobic interaction with the aromatic amino acids in the substrate binding site and more extensive filling of this site. Although there are multiple differences in the predicted amino acid sequence of caCYP51 and ckCYP51, the models of the two enzymes were quite similar and the mechanism for the relative resistance of ckCYP51 to the azoles was not apparent.
doi:10.1128/AAC.47.4.1213-1219.2003
PMCID: PMC152512  PMID: 12654649
7.  Identification of Two Different 14-α Sterol Demethylase-Related Genes (cyp51A and cyp51B) in Aspergillus fumigatus and Other Aspergillus species 
Journal of Clinical Microbiology  2001;39(7):2431-2438.
Two cyp51-related genes (cyp51A and cyp51B) encoding 14-α sterol demethylase-like enzymes were identified in the opportunistic human pathogen Aspergillus fumigatus. PCR amplification using degenerate oligonucleotides based on conserved areas of cytochrome P450 demethylases of other filamentous fungi and yeasts allowed the cloning and sequencing of two different homologue genes in A. fumigatus. Southern analysis confirmed that both genes hybridized to distinct genomic loci and that both are represented as single copies in the genome. Comparison of the deduced Cyp51A and Cyp51B proteins with the CYP51 proteins from Penicillium italicum, Aspergillus nidulans, Erysiphe graminis, Uncinula necator, Botrytis cinerea, Ustilago maydis, Cryptococcus neoformans, Candida albicans, Saccharomyces cerevisiae, Candida tropicalis, and Candida glabrata showed that the percentages of identity of the amino acid sequences (range, 40 to 70%) were high enough to consider Cyp51A and Cyp51B to be members of the fungal CYP51 family. Fragments from both genes were also cloned from other Aspergillus spp. (A. flavus, A. nidulans, and A. terreus). Phylogenetic analysis showed that, at least in the most pathogenic species of Aspergillus, there are two fungal CYP51 proteins. This is the first report of the existence of two homologue genes coding for 14-α sterol demethylase in the fungal kingdom. This finding could provide insights into the azole resistance mechanisms operating in fungi. The primers used here may be useful molecular tools for facilitating the cloning of novel 14-α sterol demethylase genes in other filamentous fungi.
doi:10.1128/JCM.39.7.2431-2438.2001
PMCID: PMC88166  PMID: 11427550
8.  Trypanosoma cruzi CYP51 Inhibitor Derived from a Mycobacterium tuberculosis Screen Hit 
Background
The two front-line drugs for chronic Trypanosoma cruzi infections are limited by adverse side-effects and declining efficacy. One potential new target for Chagas' disease chemotherapy is sterol 14α-demethylase (CYP51), a cytochrome P450 enzyme involved in biosynthesis of membrane sterols.
Methodology/Principal Finding
In a screening effort targeting Mycobacterium tuberculosis CYP51 (CYP51Mt), we previously identified the N-[4-pyridyl]-formamide moiety as a building block capable of delivering a variety of chemotypes into the CYP51 active site. In that work, the binding modes of several second generation compounds carrying this scaffold were determined by high-resolution co-crystal structures with CYP51Mt. Subsequent assays against the CYP51 orthologue in T. cruzi, CYP51Tc, demonstrated that two of the compounds tested in the earlier effort bound tightly to this enzyme. Both were tested in vitro for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness. One of the compounds had potent, selective anti–T. cruzi activity in infected mouse macrophages. Cure of treated host cells was confirmed by prolonged incubation in the absence of the inhibiting compound. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability (phenylalanine versus isoleucine) of a single residue at a critical position in the active site.
Conclusions/Significance
CYP51Mt-based crystal structure analysis revealed that the functional groups of the two tightly bound compounds are likely to occupy different spaces in the CYP51 active site, suggesting the possibility of combining the beneficial features of both inhibitors in a third generation of compounds to achieve more potent and selective inhibition of CYP51Tc.
Author Summary
Enzyme sterol 14α-demethylase (CYP51) is a well-established target for anti-fungal therapy and is a prospective target for Chagas' disease therapy. We previously identified a chemical scaffold capable of delivering a variety of chemical structures into the CYP51 active site. In this work the binding modes of several second generation compounds carrying this scaffold were determined in high-resolution co-crystal structures with CYP51 of Mycobacterium tuberculosis. Subsequent assays against CYP51 in Trypanosoma cruzi, the agent of Chagas' disease, demonstrated that two of the compounds bound tightly to the enzyme. Both were tested for inhibitory effects against T. cruzi and the related protozoan parasite Trypanosoma brucei. One of the compounds had potent, selective anti–T. cruzi activity in infected mouse macrophages. This compound is currently being evaluated in animal models of Chagas' disease. Discrimination between T. cruzi and T. brucei CYP51 by the inhibitor was largely based on the variability of a single amino acid residue at a critical position in the active site. Our work is aimed at rational design of potent and highly selective CYP51 inhibitors with potential to become therapeutic drugs. Drug selectivity to prevent host–pathogen cross-reactivity is pharmacologically important, because CYP51 is present in human host.
doi:10.1371/journal.pntd.0000372
PMCID: PMC2629123  PMID: 19190730
9.  Computational identification and binding analysis of orphan human cytochrome P450 4X1 enzyme with substrates 
BMC Research Notes  2015;8:9.
Background
Cytochrome P450s (CYPs) are important heme-containing proteins, well known for their monooxygenase reaction. The human cytochrome P450 4X1 (CYP4X1) is categorized as “orphan” CYP because of its unknown function. In recent studies it is found that this enzyme is expressed in neurovascular functions of the brain. Also, various studies have found the expression and activity of orphan human cytochrome P450 4X1 in cancer. It is found to be a potential drug target for cancer therapy. However, three-dimensional structure, the active site topology and substrate specificity of CYP4X1 remain unclear.
Methods
In the present study, the three-dimensional structure of orphan human cytochrome P450 4X1 was generated by homology modeling using Modeller 9v8. The generated structure was accessed for geometrical errors and energy stability using PROCHECK, VERFIY 3D and PROSA. A molecular docking analysis was carried out against substrates arachidonic acid and anandamide and the docked substrates were predicted for drug-likeness, ADME-Tox parameters and biological spectrum activity.
Results
The three-dimensional model of orphan human cytochrome P450 4X1 was generated and assessed with various structural validation programmes. Docking of orphan human cytochrome P450 4X1 with arachidonic acid revealed that TYR 112, ALA 126, ILE 222, ILE 223, THR 312, LEU 315, ALA 316, ASP 319, THR 320, PHE 491 and ILE 492 residues were actively participating in the interaction, while docking of CYP4X1 with anandamide showed that TYR 112, GLN 114, PRO 118, ALA 126, ILE 222, ILE 223, SER 251, LEU 315, ALA 316 and PHE 491 key residues were involved in strong interaction.
Conclusion
From this study, several key residues were identified to be responsible for the binding of arachidonic acid and anandamide with orphan human cytochrome P450 4X1. Both substrates obeyed Lipinski rule of five in drug-likeness test and biological spectrum prediction showed anticarcinogenic activity. Compared to anandamide, arachidonic acid showed strong interaction with cytochrome P450 4X1 and also less health effect in certain human system in ADME-Tox prediction. These findings provide useful information on the biological role and structure-based drug design of orphan human cytochrome P450 4X1.
doi:10.1186/s13104-015-0976-4
PMCID: PMC4322450  PMID: 25595103
Homology modeling; Human cytochrome; CYP4X1; Molecular docking; Arachidonic acid; Anandamide
10.  Structure and Dynamics of the Membrane-Bound Cytochrome P450 2C9 
PLoS Computational Biology  2011;7(8):e1002152.
The microsomal, membrane-bound, human cytochrome P450 (CYP) 2C9 is a liver-specific monooxygenase essential for drug metabolism. CYPs require electron transfer from the membrane-bound CYP reductase (CPR) for catalysis. The structural details and functional relevance of the CYP-membrane interaction are not understood. From multiple coarse grained molecular simulations started with arbitrary configurations of protein-membrane complexes, we found two predominant orientations of CYP2C9 in the membrane, both consistent with experiments and conserved in atomic-resolution simulations. The dynamics of membrane-bound and soluble CYP2C9 revealed correlations between opening and closing of different tunnels from the enzyme's buried active site. The membrane facilitated the opening of a tunnel leading into it by stabilizing the open state of an internal aromatic gate. Other tunnels opened selectively in the simulations of product-bound CYP2C9. We propose that the membrane promotes binding of liposoluble substrates by stabilizing protein conformations with an open access tunnel and provide evidence for selective substrate access and product release routes in mammalian CYPs. The models derived here are suitable for extension to incorporate other CYPs for oligomerization studies or the CYP reductase for studies of the electron transfer mechanism, whereas the modeling procedure is generally applicable to study proteins anchored in the bilayer by a single transmembrane helix.
Author Summary
We describe the first atomic-detail models and simulations of a full-length, membrane-bound mammalian cytochrome P450. To date, all the structural studies of microsomal, drug-metabolizing cytochrome P450s have been performed using engineered, solubilized forms of the enzymes and it is not yet understood how the membrane influences their structure, dynamics, and ability to bind substrates. We focused on CYP2C9, the second most abundant cytochrome P450 in the human liver which oxidizes 20% of all marketed drugs. Here, we have derived models of CYP2C9-membrane complexes with a modeling procedure based on molecular dynamics simulations started with arbitrary configurations of the protein in the membrane and performed using both coarse grained and atomic-detail molecular representations. This procedure is expected to be generally applicable to proteins that are anchored in the membrane with a single transmembrane helix. The models and simulations provide evidence for selective substrate access and product release routes in membrane-bound CYPs. This observation may contribute to new strategies to manipulate the activity of CYPs and other enzymes with buried active sites. We anticipate that this study will bring about a paradigm shift towards studying microsomal CYPs as dynamic structures in their natural, lipid environment rather than in artificially solubilized forms.
doi:10.1371/journal.pcbi.1002152
PMCID: PMC3154944  PMID: 21852944
11.  The Cytochrome P450 Superfamily Complement (CYPome) in the Annelid Capitella teleta 
PLoS ONE  2014;9(11):e107728.
The Cytochrome P450 super family (CYP) is responsible for a wide range of functions in metazoans, having roles in both exogenous and endogenous substrate metabolism. Annelids are known to metabolize polycyclic aromatic hydrocarbons (PAHs) and produce estrogen. CYPs are postulated to be key enzymes in these processes in annelids. In this study, the CYP complement (CYPome) of the annelid Capitella teleta has been robustly identified and annotated with the genome assembly available. Phylogenetic analyses were performed to understand the evolutionary relationships between CYPs in C. teleta and other species. Predictions of which CYPs are potentially involved in both PAH metabolism and steroidogensis were made based on phylogeny. Annotation of 84 full length and 12 partial CYP sequences predicted a total of 96 functional CYPs in C. teleta. A further 13 CYP fragments were found but these may be pseudogenes. The C. teleta CYPome contained 24 novel CYP families and seven novel CYP subfamilies within existing families. A phylogenetic analysis identified that the C. teleta sequences were found in 9 of the 11 metazoan CYP clans. Two CYPs, CYP3071A1 and CYP3072A1, did not cluster with any metazoan CYP clans. We found xenobiotic response elements (XREs) upstream of C. teleta CYPs related to vertebrate CYP1 (CYP3060A1, CYP3061A1) and from families with reported transcriptional upregulation in response to PAH exposure (CYP4, CYP331). C. teleta had a CYP51A1 with ∼65% identity to vertebrate CYP51A1 sequences and has been predicted to have lanosterol 14 α-demethylase activity. CYP376A1, CYP3068A1, CYP3069A1, and CYP3070A1 were the most appropriate candidates for steroidogenesis genes based on their phylogeny and warrant further analyses, though no specific aromatase (estrogen synthesis) candidates were found. Presence of XREs upstream of C. teleta CYPs may indicate a functional aryl hydrocarbon receptor in C. teleta and candidate CYPs for studies of PAH metabolism.
doi:10.1371/journal.pone.0107728
PMCID: PMC4229089  PMID: 25390889
12.  Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3 
Cytochrome P450 monooxygenases (P450s), which constitute a superfamily of heme-containing proteins, catalyze the direct oxidation of a variety of compounds in a regio- and stereospecific manner; therefore, they are promising catalysts for use in the oxyfunctionalization of chemicals. In the course of our comprehensive substrate screening for all 27 putative P450s encoded by the Streptomyces griseus genome, we found that Escherichia coli cells producing an S. griseus P450 (CYP154C3), which was fused C terminally with the P450 reductase domain (RED) of a self-sufficient P450 from Rhodococcus sp., could transform various steroids (testosterone, progesterone, Δ4-androstene-3,17-dione, adrenosterone, 1,4-androstadiene-3,17-dione, dehydroepiandrosterone, 4-pregnane-3,11,20-trione, and deoxycorticosterone) into their 16α-hydroxy derivatives as determined by nuclear magnetic resonance and high-resolution mass spectrometry analyses. The purified CYP154C3, which was not fused with RED, also catalyzed the regio- and stereospecific hydroxylation of these steroids at the same position with the aid of ferredoxin and ferredoxin reductase from spinach. The apparent equilibrium dissociation constant (Kd) values of the binding between CYP154C3 and these steroids were less than 8 μM as determined by the heme spectral change, indicating that CYP154C3 strongly binds to these steroids. Furthermore, kinetic parameters of the CYP154C3-catalyzed hydroxylation of Δ4-androstene-3,17-dione were determined (Km, 31.9 ± 9.1 μM; kcat, 181 ± 4.5 s−1). We concluded that CYP154C3 is a steroid D-ring 16α-specific hydroxylase which has considerable potential for industrial applications. This is the first detailed enzymatic characterization of a P450 enzyme that has a steroid D-ring 16α-specific hydroxylation activity.
doi:10.1128/AEM.03504-13
PMCID: PMC3911049  PMID: 24334658
13.  Rapid Birth–Death Evolution Specific to Xenobiotic Cytochrome P450 Genes in Vertebrates 
PLoS Genetics  2007;3(5):e67.
Genes vary greatly in their long-term phylogenetic stability and there exists no general explanation for these differences. The cytochrome P450 (CYP450) gene superfamily is well suited to investigating this problem because it is large and well studied, and it includes both stable and unstable genes. CYP450 genes encode oxidase enzymes that function in metabolism of endogenous small molecules and in detoxification of xenobiotic compounds. Both types of enzymes have been intensively studied. My analysis of ten nearly complete vertebrate genomes indicates that each genome contains 50–80 CYP450 genes, which are about evenly divided between phylogenetically stable and unstable genes. The stable genes are characterized by few or no gene duplications or losses in species ranging from bony fish to mammals, whereas unstable genes are characterized by frequent gene duplications and losses (birth–death evolution) even among closely related species. All of the CYP450 genes that encode enzymes with known endogenous substrates are phylogenetically stable. In contrast, most of the unstable genes encode enzymes that function as xenobiotic detoxifiers. Nearly all unstable CYP450 genes in the mouse and human genomes reside in a few dense gene clusters, forming unstable gene islands that arose by recurrent local gene duplication. Evidence for positive selection in amino acid sequence is restricted to these unstable CYP450 genes, and sites of selection are associated with substrate-binding regions in the protein structure. These results can be explained by a general model in which phylogenetically stable genes have core functions in development and physiology, whereas unstable genes have accessory functions associated with unstable environmental interactions such as toxin and pathogen exposure. Unstable gene islands in vertebrates share some functional properties with bacterial genomic islands, though they arise by local gene duplication rather than horizontal gene transfer.
Author Summary
Genes vary greatly in their long-term phylogenetic stability, and there exists no general explanation for these differences. Stable genes persist as a single copy over a wide range of distantly related species, whereas unstable genes undergo frequent duplication and loss in a process called birth-death evolution. The vertebrate cytochrome P450 (CYP450) gene superfamily includes many genes that are present in a single copy in species ranging from teleost fish to mammals and other groups of genes that undergo active birth-death evolution across the same species. The author found that nearly all stable CYP450 genes encode enzymes known to function in the synthesis and degradation of steroid and retinoid hormones (and related molecules). These hormones function in core developmental pathways in vertebrates. In contrast, most unstable CYP450 genes encode enzymes that detoxify foreign small molecules (called xenobiotics—foreign biochemicals). In addition, many of the unstable CYP450 genes are subject to natural selection to change their amino acid sequence over time (positive selection), probably in response to changes in xenobiotic exposure. These findings suggest that stable and unstable genes differ in their rates of birth-death evolution, because stable genes have core endogenous functions whereas unstable genes respond to changing environmental conditions.
doi:10.1371/journal.pgen.0030067
PMCID: PMC1866355  PMID: 17500592
14.  Hydrogen-Deuterium Exchange Mass Spectrometry for Investigation of Backbone Dynamics of Oxidized and Reduced Cytochrome P450cam 
Journal of inorganic biochemistry  2007;102(2):364-370.
Backbone dynamics of the camphor monoxygenase cytochrome P450cam (CYP101) as a function of oxidation/ligation state of the heme iron were investigated via hydrogen/deuterium exchange (H/D exchange) as monitored by mass spectrometry. Main chain amide NH hydrogens can exchange readily with solvent and the rate of this exchange depends upon, among other things, dynamic fluctuations in local structural elements. A fluxional region of the polypeptide will exchange more quickly with solvent than one that is more constrained. In most regions of the enzyme, exchange rates were similar between oxidized high-spin camphor-bound and reduced camphor-and CO-bound CYP101 (CYP-S and CYP-S-CO, respectively.) However, in regions of the protein that have previously been implicated in substrate access by structural and molecular dynamics investigations, the reduced enzyme shows significantly slower exchange rates than the oxidized CYP-S. This observation corresponds to increased flexibility of the oxidized enzyme relative to the reduced form. Structural features previously found to be perturbed in CYP-S-CO upon binding of the biologically relevant effector and reductant putidaredoxin (Pdx) as determined by nuclear magnetic resonance are also more protected from exchange in the reduced state. To our knowledge, this study represents the first experimental investigation of backbone dynamics within the P450 family using this methodology.
doi:10.1016/j.jinorgbio.2007.10.001
PMCID: PMC2279175  PMID: 18023482
redox-dependent dynamics; electron transfer; tandem mass spectrometry; hydrogen/deuterium exchange
15.  CYP3A4-Mediated Carbamazepine (CBZ) Metabolism: Formation of a Covalent CBZ-CYP3A4 Adduct and Alteration of the Enzyme Kinetic Profile 
Carbamazepine (CBZ) is a widely prescribed anticonvulsant whose use is often associated with idiosyncratic hypersensitivity. Sera of CBZ-hypersensitive patients often contain anti-CYP3A antibodies, including those to a CYP3A23 K-helix peptide that is also modified during peroxidative CYP3A4 heme-fragmentation. We explored the possibility that cytochromes P450 (P450s) such as CYP3A4 bioactivate CBZ to reactive metabolite(s) that irreversibly modify the P450 protein. Such CBZ-P450 adducts, if stable in vivo, could engender corresponding serum P450 autoantibodies. Incubation with CBZ not only failed to inactivate functionally reconstituted, purified recombinant CYP3A4 or CYP3A4 Supersomes in a time-dependent manner, but the inclusion of CBZ (0–1 mM) also afforded a concentration-dependent protection to CYP3A4 from inactivation by NADPH-induced oxidative uncoupling. Incubation of CYP3A4 Supersomes with 3H-CBZ resulted in its irreversible binding to CYP3A4 protein with a stoichiometry of 1.58 ± 0.15 pmol 3H-CBZ bound/pmol CYP3A4. Inclusion of glutathione (1.5 mM) in the incubation reduced this level to 1.09. Similar binding (1.0 ± 0.4 pmol 3H-CBZ bound/pmol CYP3A4) was observed after 3H-CBZ incubation with functionally reconstituted, purified recombinant CYP3A4(His)6. The CBZ-modified CYP3A4 retained its functional activity albeit at a reduced level, but its testosterone 6β-hydroxylase kinetics were altered from sigmoidal (a characteristic profile of substrate cooperativity) to near-hyperbolic (Michaelis-Menten) type, suggesting that CBZ may have modified CYP3A4 within its active site.
doi:10.1124/dmd.107.016501
PMCID: PMC2881839  PMID: 18096676
16.  Alkane-induced expression, substrate binding profile, and immunolocalization of a cytochrome P450 encoded on the nifD excision element of Anabaena 7120 
BMC Microbiology  2005;5:16.
Background
Alkanes have been hypothesized to act as universal inducers of bacterial cytochrome P450 gene expression. We tested this hypothesis on an unusual P450 gene (cyp110) found on a conserved 11 kilobase episomal DNA element of unknown function found in filamentous cyanobacteria. We also monitored the binding of potential substrates to the P450 protein and explored the distribution of P450 protein in vegetative cells and nitrogen-fixing heterocysts using immuno-electron microscopy.
Results
Hexadecane treatments resulted in a two-fold increase in mRNA, and a four-fold increase in P450 protein levels relative to control cultures. Hexane, octane and dodecane were toxic and induced substantial changes in membrane morphology. Long-chain saturated and unsaturated fatty acids were shown to bind the CYP110 protein using a spectroscopic spin-shift assay, but alkanes did not bind. CYP110 protein was detected in vegetative cells but not in differentiated heterocysts where nitrogen fixation occurs.
Conclusion
Hexadecane treatment was an effective inducer of CYP110 expression in cyanobacteria. Based on substrate binding profiles and amino acid sequence similarities it is hypothesized that CYP110 is a fatty acid ω-hydroxylase in photosynthetic cells. CYP110 was found associated with membrane fractions unlike other soluble microbial P450 proteins, and in this regard CYP110 more closely resembles eukarytotic P450s. Substrate stablization is an unlikely mechanism for alkane induction because alkanes did not bind to purified CYP110 protein.
doi:10.1186/1471-2180-5-16
PMCID: PMC1079853  PMID: 15790415
17.  STRUCTURAL DIFFERENCES BETWEEN SOLUBLE AND MEMBRANE BOUND CYTOCHROMES P450 
The superfamily of cytochromes P450 forms a large class of heme monooxygenases with more than 13000 enzymes represented in organisms from all biological kingdoms. Despite impressive variability in sizes, sequences, location, and function, all cytochromes P450 from various organisms have very similar tertiary structures within the same fold. Here we show that systematic comparison of all available X-ray structures of cytochromes P450 reveal the presence of two distinct structural classes of cytochromes P450. For all membrane bound enzymes, except the CYP51 family, the beta-domain and the A-propionate heme side chain are shifted towards the proximal side of the heme plane, which results in an increase of the volume of the substrate binding pocket and an opening of a the potential channel for the substrate access and/or product escape directly into the membrane. This structural feature is also observed in several soluble cytochromes P450, such as CYP108, CYP151, and CYP158A2, which catalyze transformations of bulky substrates. Alternatively, both beta-domains and the A-propionate side chains in the soluble isozymes extend towards the distal site of the heme. This difference between the structures of soluble and membrane bound cytochromes P450 can be rationalized through the presence of several amino acids inserts in the latter class which are involved in direct interactions with the membrane, namely the F’ - and G’ – helices. Molecular dynamics using the most abundant human cytochrome P450, CYP3A4, incorporated into a model POPC bilayer reveals the facile conservation of a substrate access channel, directed into the membrane between the B-C loop and the beta domain, and the closure of the peripheral substrate access channel directed through the B-C loop. This is in contrast to the case when the same simulation is run in buffer, where no such channel closing occurs. Taken together, these results reveal a key structural difference between membrane bound and soluble cytochromes P450 with important functional implications induced by the lipid bilayer.
doi:10.1016/j.jinorgbio.2011.11.026
PMCID: PMC4190058  PMID: 22244217
18.  The cytochrome P450 2AA gene cluster in zebrafish (Danio rerio); expression of CYP2AA1 and CYP2AA2 and response to phenobarbital-type inducers 
Toxicology and applied pharmacology  2013;272(1):10.1016/j.taap.2013.05.017.
The cytochrome P450 (CYP) 2 gene family is the largest and most diverse CYP gene family in vertebrates. In zebrafish, we have identified 10 genes in a new subfamily CYP2AA, which does not show orthology to any human or other mammalian CYP genes. Here we report evolutionary and structural relationships of the 10 CYP2AA genes and expression of the first two genes, CYP2AA1 and CYP2AA2. Parsimony reconstruction of the tandem duplication pattern for the CYP2AA cluster suggests that CYP2AA1, CYP2AA2 and CYP2AA3 likely arose in the earlier duplication events and thus are most diverged in function from the other CYP2AAs. On the other hand, CYP2AA8 and CYP2AA9 are genes that arose in the latest duplication event, implying functional similarity between these two CYPs. A molecular model of CYP2AA1 showing the sequence conservation across the CYP2AA cluster reveals that the regions with the highest variability within the cluster map into CYP2AA1 near the substrate access channels, suggesting differing substrate specificity. Zebrafish CYP2AA1 transcript was expressed predominantly in intestine, while CYP2AA2 was most highly expressed in kidney, suggesting differing roles in physiology. In liver CYP2AA2 expression but not that of CYP2AA1, was increased by 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and, to a lesser extent, by phenobarbital (PB). In contrast, pregnenolone 16α-carbonitrile (PCN) increased CYP2AA1, but not CYP2AA2 in liver. The results identify a CYP2 subfamily in zebrafish that includes genes apparently induced by PB-type chemicals and PXR agonists, the first concrete in vivo evidence for a PB-type response in fish.
doi:10.1016/j.taap.2013.05.017
PMCID: PMC3846545  PMID: 23726801
Cytochrome P450; CYP2; Zebrafish; Molecular evolution; Homology modeling; Phenobarbital
19.  The R467K Amino Acid Substitution in Candida albicans Sterol 14α-Demethylase Causes Drug Resistance through Reduced Affinity 
The cytochrome P450 sterol 14α-demethylase (CYP51) of Candida albicans is involved in an essential step of ergosterol biosynthesis and is the target for azole antifungal compounds. We have undertaken site-directed mutation of C. albicans CYP51 to produce a recombinant mutant protein with the amino acid substitution R467K corresponding to a mutation observed clinically. This alteration perturbed the heme environment causing an altered reduced-carbon monoxide difference spectrum with a maximum at 452 nm and reduced the affinity of the enzyme for fluconazole, as shown by ligand binding studies. The specific activity of CYP51(R467K) for the release of formic acid from 3β-[32-3H]hydroxylanost-7-en-32-ol was 70 pmol/nmol of P450/min for microsomal protein compared to 240 pmol/nmol of P450/min for microsomal fractions expressing wild-type CYP51. Furthermore, inhibition of activity by fluconazole revealed a 7.5-fold-greater azole resistance of the recombinant protein than that of the wild type. This study demonstrates that resistance observed clinically can result from the altered azole affinity of the fungal CYP51 enzyme.
PMCID: PMC89629  PMID: 10602724
20.  The discovery of new coding alleles of human CYP26A1 which are potentially defective in the metabolism of all-trans retinoic acid and their assessment in a recombinant cDNA expression system 
Pharmacogenetics and genomics  2007;17(3):169-180.
Retinoic acid (RA) is a critical regulator of gene expression during embryonic development and in the maintenance of adult epithelial tissues. Genetic polymorphisms of CYP26A1 could cause inter-individual variation in the metabolism of retinoic acid, thus altering signaling during embryonic development. A total of 13 single nucleotide polymorphisms (SNPs) were identified in CYP26A1 in 92 racially diverse individuals (24 Caucasians, 24 African-Americans, 24 Asians and 20 individuals of unknown racial origin). Three of the 13 SNPS produced coding changes: R173S, F186L and C358R. These alleles were termed CYP26A1*2, CYP26A1*3, and CYP26A1*4, respectively, by the Human Cytochrome P450 (CYP) Allele Nomenclature Committee at http://www.cypalleles.ki.se/. cDNA constructs for wild-type and mutant alleles of CYP26A1 were constructed in a pcDNA3.1 expression vector containing a FLAG tag at the C-terminal end, which was used to identify and quantitate the CYP26A1 allelic proteins when expressed in COS-1 cells. Wild type CYP26A1 protein metabolized all-trans-retinoic acid (at-RA) to 4-oxo-RA, 4-OH-RA and 18-OH-RA as well as water-soluble metabolites. CYP26A1.3 (F186L) and CYP26A1.4 (C358R) allelic proteins exhibited significantly lower metabolism (40-80%) of at-RA than wild-type CYP26A1.1 protein. This study identifies two CYP26A1 coding alleles, CYP26A1*3 and CYP26A1*4, which are predicted to be defective in retinoic acid metabolism based on the metabolism of at-RA by the recombinant proteins. This is the first study to identify coding alleles of CYP26A1. The in vitro characterization of the recombinant allelic proteins suggests the need for future clinical studies of genotype/phenotype relationships of CYP26A1 in embryonic development.
doi:10.1097/FPC.0b013e32801152d6
PMCID: PMC2393551  PMID: 17460545
P450; CYP26A1; retinoic acid; polymorphisms; retinoic acid metabolism
21.  Magic-Angle Spinning Solid-State NMR Spectroscopy of Nanodisc– Embedded Human CYP3A4† 
Biochemistry  2007;46(48):13696-13703.
Cytochrome P450 (CYP) 3A4 contributes to the metabolism of approximately 50% of commercial drugs by oxidizing a large number of structurally diverse substrates. Like other endoplasmic reticulum-localized P450s, CYP3A4 contains a membrane-anchoring N-terminal helix and a significant number of hydrophobic domains, important for the interaction between CYP3A4 and the membrane. Although the membrane affects specificity of CYP3A4 ligand binding, the structural details of the interaction have not been revealed so far because x-ray crystallography studies are available only for the soluble domain of CYP3A4. Here we report sample preparation and initial magic-angle spinning (MAS) solid-state NMR (SSNMR) of CYP3A4 (Δ3−12) embedded in a nanoscale membrane bilayer, or Nanodisc. The growth protocol yields ∼2.5 mg of the enzymatically active, uniformly 13C, 15N-enriched CYP3A4 from a liter of growth medium. Polyethylene glycol 3350-precipitated CYP3A4 in Nanodiscs yields spectra of high resolution and sensitivity, consistent with a folded, homogeneous protein. CYP3A4 in Nanodiscs remains enzymatically active throughout the precipitation protocol as monitored by bromocriptine binding. The 13C line widths measured from 13C-13C 2D chemical shift correlation spectra are ∼0.5 ppm. The secondary structure distribution within several amino acid types determined from 13C chemical shifts is consistent with the ligand-free x-ray structures. These results demonstrate that MAS SSNMR can be performed on Nanodisc-embedded membrane proteins in a folded, active state. The combination of SSNMR and Nanodisc methodologies opens up new possibilities for obtaining structural information on CYP3A4 and other integral membrane proteins with full retention of functionality.
doi:10.1021/bi701411g
PMCID: PMC2571072  PMID: 17985934
22.  Systematic and searchable classification of cytochrome P450 proteins encoded by fungal and oomycete genomes 
BMC Genomics  2012;13:525.
Background
Cytochrome P450 proteins (CYPs) play diverse and pivotal roles in fungal metabolism and adaptation to specific ecological niches. Fungal genomes encode extremely variable “CYPomes” ranging from one to more than 300 CYPs. Despite the rapid growth of sequenced fungal and oomycete genomes and the resulting influx of predicted CYPs, the vast majority of CYPs remain functionally uncharacterized. To facilitate the curation and functional and evolutionary studies of CYPs, we previously developed Fungal Cytochrome P450 Database (FCPD), which included CYPs from 70 fungal and oomycete species. Here we present a new version of FCPD (1.2) with more data and an improved classification scheme.
Results
The new database contains 22,940 CYPs from 213 species divided into 2,579 clusters and 115 clans. By optimizing the clustering pipeline, we were able to uncover 36 novel clans and to assign 153 orphan CYP families to specific clans. To augment their functional annotation, CYP clusters were mapped to David Nelson’s P450 databases, which archive a total of 12,500 manually curated CYPs. Additionally, over 150 clusters were functionally classified based on sequence similarity to experimentally characterized CYPs. Comparative analysis of fungal and oomycete CYPomes revealed cases of both extreme expansion and contraction. The most dramatic expansions in fungi were observed in clans CYP58 and CYP68 (Pezizomycotina), clans CYP5150 and CYP63 (Agaricomycotina), and family CYP509 (Mucoromycotina). Although much of the extraordinary diversity of the pan-fungal CYPome can be attributed to gene duplication and adaptive divergence, our analysis also suggests a few potential horizontal gene transfer events. Updated families and clans can be accessed through the new version of the FCPD database.
Conclusions
FCPD version 1.2 provides a systematic and searchable catalogue of 9,550 fungal CYP sequences (292 families) encoded by 108 fungal species and 147 CYP sequences (9 families) encoded by five oomycete species. In comparison to the first version, it offers a more comprehensive clan classification, is fully compatible with Nelson’s P450 databases, and has expanded functional categorization. These features will facilitate functional annotation and classification of CYPs encoded by newly sequenced fungal and oomycete genomes. Additionally, the classification system will aid in studying the roles of CYPs in the evolution of fungal adaptation to specific ecological niches.
doi:10.1186/1471-2164-13-525
PMCID: PMC3505482  PMID: 23033934
Cytochrome P450; Genome annotation; Clustering; Fungi; Evolution; Phylogenetics; Mycotoxin
23.  Gene and protein expression and cellular localisation of cytochrome P450 enzymes of the 1A, 2A, 2C, 2D and 2E subfamilies in equine intestine and liver 
Background
Among the cytochrome P450 enzymes (CYP), families 1–3 constitute almost half of total CYPs in mammals and play a central role in metabolism of a wide range of pharmaceuticals. This study investigated gene and protein expression and cellular localisation of CYP1A, CYP2A, CYP2C, CYP2D and CYP2E in equine intestine and liver. Real-time polymerase chain reaction (RT-PCR) was used to analyse gene expression, western blot to examine protein expression and immunohistochemical analyses to investigate cellular localisation.
Results
CYP1A and CYP2C were the CYPs with the highest gene expression in the intestine and also showed considerable gene expression in the liver. CYP2E and CYP2A showed the highest gene expression in the liver. CYP2E showed moderate intestinal gene expression, whereas that of CYP2A was very low or undetectable. For CYP2D, rather low gene expression levels were found in both intestine and the liver. In the intestine, CYP gene expression levels, except for CYP2E, exhibited patterns resembling those of the proteins, indicating that intestinal protein expression of these CYPs is regulated at the transcriptional level. For CYP2E, the results showed that the intestinal gene expression did not correlate to any visible protein expression, indicating that intestinal protein expression of this CYP is regulated at the post-transcriptional level. Immunostaining of intestine tissue samples showed preferential CYP staining in enterocytes at the tips of intestinal villi in the small intestine. In the liver, all CYPs showed preferential localisation in the centrilobular hepatocytes.
Conclusions
Overall, different gene expression profiles were displayed by the CYPs examined in equine intestine and liver. The CYPs present in the intestine may act in concert with those in the liver to affect the oral bioavailability and therapeutic efficiency of substrate drugs. In addition, they may play a role in first-pass metabolism of feed constituents and of herbal supplements used in equine practice.
doi:10.1186/s13028-014-0069-8
PMCID: PMC4192735  PMID: 25288196
CYP; Horse; Gene expression; Protein expression; Cellular localisation; Liver; Intestine
24.  CYP51 structures and structure-based development of novel, pathogen-specific inhibitory scaffolds 
CYP51 (sterol 14α-demethylase) is a cytochrome P450 enzyme essential for sterol biosynthesis and the primary target for clinical and agricultural antifungal azoles. The azoles that are currently in clinical use for systemic fungal infections represent modifications of two basic scaffolds, ketoconazole and fluconazole, all of them being selected based on their antiparasitic activity in cellular experiments. By studying direct inhibition of CYP51 activity across phylogeny including human pathogens Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum, we identified three novel protozoa-specific inhibitory scaffolds, their inhibitory potency correlating well with antiprotozoan activity. VNI scaffold (carboxamide containing β-phenyl-imidazoles) is the most promising among them: killing T. cruzi amastigotes at low nanomolar concentration, it is also easy to synthesize and nontoxic. Oral administration of VNI (up to 400 mg/kg) neither leads to mortality nor reveals significant side effects up to 48 h post treatment using an experimental mouse model of acute toxicity. Trypanosomatidae CYP51 crystal structures determined in the ligand-free state and complexed with several azole inhibitors as well as a substrate analog revealed high rigidity of the CYP51 substrate binding cavity, which must be essential for the enzyme strict substrate specificity and functional conservation. Explaining profound potency of the VNI inhibitory scaffold, the structures also outline guidelines for its further development. First steps of the VNI scaffold optimization have been undertaken; the results presented here support the notion that CYP51 structure-based rational design of more efficient, pathogen-specific inhibitors represents a highly promising direction.
PMCID: PMC3596085  PMID: 23504044
Sterol 14α-demethylase; CYP51; Inhibition; Crystal structure
25.  A transcriptomic approach to elucidate the physiological significance of human cytochrome P450 2S1 in bronchial epithelial cells 
BMC Genomics  2013;14:833.
Background
Cytochrome P450 2S1 (CYP2S1) is an orphan P450 with an unknown biological function. Data from our laboratory and others suggest that CYP2S1 may have an important physiological role in modulating the synthesis and metabolism of bioactive lipids including prostaglandins and retinoids. CYP2S1 expression is elevated in multiple epithelial-derived cancers as well as in the chronic hyperproliferative disease psoriasis. Whether CYP2S1 expression in proliferative disease is protective, detrimental, or neutral to disease progression remains to be determined. Two human bronchial epithelial cells (BEAS-2B) were constructed to represent chronic depletion of CYP2S1 using short-hairpin RNA (shRNA) silencing directed toward the 3’UTR (759) and exon 3 (984) of the CYP2S1 gene and compared with a non-targeting shRNA control (SCRAM). Both CYP2S1 mRNA and protein were depleted by approximately 75% in stable cell lines derived from both targeted shRNA constructs (759 and 984). To elucidate the biological significance of CYP2S1, we analyzed transcriptome alterations in response to CYP2S1 depletion in human lung cells.
Results
RNA-sequencing (RNA-seq) analysis was performed to compare the transcriptome of the control (SCRAM) and the CYP2S1-depleted (759) BEAS-2B cell lines. Transcriptomes of the replicates from the two cell lines were found to be distinct populations as determined using Principal Component Analysis and hierarchical clustering. Approximately 1000 genes were differentially expressed in response to CYP2S1 depletion. Consistent with our previous phenotypes, DAVID analysis revealed altered regulation in key pathways implicated in cell proliferation and migration. Transcriptomic profiles were also consistent with the metabolism of proposed endogenous substrates. Pathway analysis also revealed significant expression changes within mTOR signaling, a critical pathway in cell growth. To determine whether these changes manifest as altered cell size, cell diameter and volume were calculated, revealing that CYP2S1 depletion promotes cell growth in BEAS-2B cells.
Conclusions
These data suggest that pathway analysis of sequence-based gene expression is a powerful method to identify pathways and phenotypic alterations in response to changes in orphan enzyme expression. Our results suggest a novel role for CYP2S1-mediated metabolism in modulating BEAS-2B cell size. These findings warrant further studies on CYP2S1 regulated pathways to elucidate potential substrates of CYP2S1.
doi:10.1186/1471-2164-14-833
PMCID: PMC3884200  PMID: 24279958
CYP2S1; BEAS-2B; Retinoic acid; Arachidonic acid; RNA Seq; Orphan; shRNA; PGE2

Results 1-25 (994706)