Human CYP4F3s are the main catalysts in the oxidation of fatty acid epoxides

CYP4F isoforms are involved in the oxidation of important cellular mediators such as leukotriene B4 (LTB4) and prostaglandins. The proinflammatory agent LTB4 and cytotoxic leukotoxins have been associated with several inflammatory diseases. We present evidence that the hydroxylation of Z 9(10)-epoxyoctadecanoic, Z 9(10)-epoxyoctadec-Z 12-enoic, and Z 12(13)-epoxyoctadec-Z 9-enoic acids and that of monoepoxides from arachidonic acid [epoxyeicosatrienoic acid (EET)] is important in the regulation of leukotoxin and EET activity. These three epoxidized derivatives from the C18 family (C18-epoxides) were converted to 18-hydroxy-C18-epoxides by human hepatic microsomes with apparent Km values of between 27.6 and 175 microM. Among recombinant P450 enzymes, CYP4F2 and CYP4F3B catalyzed mainly the omega-hydroxylation of C18-epoxides with an apparent Vmax of between 0.84 and 15.0 min(-1), whereas the apparent Vmax displayed by CYP4F3A, the isoform found in leukocytes, ranged from 3.0 to 21.2 min(-1). The rate of omega-hydroxylation by CYP4A11 was experimentally found to be between 0.3 and 2.7 min(-1). CYP4F2 and CYP4F3 exhibited preferences for omega-hydroxylation of Z 8(9)-EET, whereas human liver microsomes preferred Z 11(12)-EET and, to a lesser extent, Z 8(9)-EET. Moreover, vicinal diol from both C18-epoxides and EETs were omega-hydroxylated by liver microsomes and by CYP4F2 and CYP4F3. These data support the hypothesis that the human CYP4F subfamily is involved in the omega-hydroxylation of fatty acid epoxides. These findings demonstrate that another pathway besides conversion to vicinal diol or chain shortening by beta-oxidation exists for fatty acid epoxide inactivation.     

cDNA cloning and expression of a novel cytochrome p450 (cyp4f12) from human small intestine

A cDNA encoding a novel human CYP4F enzyme (designated CYP4F12) was cloned by PCR from a human small intestine cDNA library. RT-PCR analysis demonstrated that CYP4F12 is expressed in human small intestine and liver. This cDNA contains an entire coding region of a 524-amino-acid protein that is 81.7, 78.3, and 78.2% identical to CYP4F2, CYP4F3, and CYP4F8, respectively. When expressed in Saccharomyces cerevisiae, the P450 catalyzes leukotriene B(4) omega-hydroxylation and arachidonic acid omega-hydroxylation, typical reactions of CYP4F isoforms. Their activity levels are, however, much lower than those of CYP4F2. Interestingly, CYP4F12 catalyzes the hydroxylation of the antihistamine ebastine with significantly higher catalytic activity relative to CYP4F2 (385 vs 5 pmol/min/nmol P450). These results indicate that CYP4F12 has a different profile of substrate specificity from other CYP4F isoforms, enzymes responsible for metabolizing endogenous autacoids, therefore suggesting that it may play an important role in xenobiotic biotransformation in the human small intestine.     

Leukotriene B4 omega-hydroxylase in rat liver microsomes: identification as a cytochrome P-450 that catalyzes prostaglandin A1 omega-hydroxylation, and participation of cytochrome b5

The omega-hydroxylation of leukotriene B4 (LTB4) by rat liver microsomes requires NADPH and molecular oxygen, suggesting that the hydroxylation is catalyzed by a cytochrome P-450 (P-450)-linked monooxygenase system. The reaction is inhibited by CO, and the inhibition is reversed by irradiation of light at 450 nm in a light-intensity-dependent manner. The extent of the reversal is strongly dependent on the wavelength of the light used, the 450-nm light is most efficient. The finding provides direct evidence for the identification of the LTB4 omega-hydroxylase as a P-450. The P-450 seems to be also responsible for prostaglandin A1 (PGA1) omega-hydroxylation, but not for lauric acid omega-hydroxylation. The LTB4 omega-hydroxylation is competitively inhibited by PGA1, but not affected by lauric acid. The Ki value for PGA1 of 38 microM agrees with the Km value for PGA1 omega-hydroxylation of 40 microM. LTB4 inhibits the PGA1 omega-hydroxylation by rat liver microsomes in a competitive manner with the Ki of 43 microM, which is consistent with the Km for the LTB4 omega-hydroxylation of 42 microM. An antiserum raised against rabbit pulmonary PG omega-hydroxylase (P-450p-2) inhibits slightly the omega-hydroxylations of LTB4 and PGA1, while it has stronger inhibitory effect on lauric acid omega-hydroxylation. In addition to NADPH-cytochrome P-450 reductase, cytochrome b5 appears to participate in the LTB4 omega-hydroxylating system, since the reaction is inhibited by an antibody raised against the cytochrome b5 as well as one raised against the reductase.     

CYP4F3B is induced by PGA1 in human liver cells: a regulation of the 20-HETE synthesis

The regulation of the human liver-specific cytochrome P450 4F3B (CYP4F3B) isoform, a splice variant of the CYP4F3 gene with strong substrate specificity for long chain fatty acids, is yet an unsolved question. This report provides the first evidence that CYP4F3B is uniquely induced by prostaglandin A(1) (PGA(1)) in human hepatocyte-like HepaRG cells and leads to the synthesis of 20-hydroxy-eicosatetraenoic acids (HETEs). Real time PCR, immunoblot analysis with a specific antipeptide antibody, and determination of fatty acid omega-hydroxylase activity demonstrate that PGA(1) treatment strongly increases expression of CYP4F3B. This induction drives the production of 20-HETE (19-fold increase). SiRNA-mediated-silencing of CYP4F3 suppresses both 20-HETE synthesis and PGA(1) induced 20-HETE production. Taken together, these results provide evidence that CYP4F3B is the key enzyme to produce 20-HETE by omega-hydroxylation of arachidonic acid in liver cells. Since 20-HETE is a potent activator of PPARalpha and an important inflammatory mediator, CYP4F3B may exert important functions in lipid homeostasis and in inflammatory diseases.     

Characterization of human neutrophil leukotriene B4 omega-hydroxylase as a system involving a unique cytochrome P-450 and NADPH-cytochrome P-450 reductase

Leukotriene B4 (LTB4), a potent chemotactic agent, was catabolized to 20-hydroxyleukotriene B4 (20-OH-LTB4) by the 150,000 x g pellet (microsomal fraction) of human neutrophil sonicate. The reaction required molecular oxygen and NADPH, and was significantly inhibited by carbon monoxide, suggesting that a cytochrome P-450 is involved. The neutrophil microsomal fraction showed a carbon monoxide difference spectrum with a peak at 450 nm in the presence of NADPH or dithionite, indicating the presence of a cytochrome P-450. The addition of LTB4 to the microsomal fraction gave a type-I spectral change with a peak at around 390 nm and a trough at 422 nm, indicating a direct interaction of LTB4 with the cytochrome P-450. The dissociation constant of LTB4, determined from the difference spectra, is 0.40 microM, in agreement with the kinetically determined apparent Km value for LTB4 (0.30 microM). Such a spectral change was not observed with prostaglandins A1, E1 and F2 alpha or lauric acid, none of which inhibited the LTB4 omega-hydroxylation. The inhibition of the LTB4 omega-hydroxylation by carbon monoxide was effectively reversed by irradiation with monochromatic light of 450 nm wavelength. The photochemical action spectrum of the light reversal of the inhibition corresponded remarkably well with the carbon monoxide difference spectrum. These observations provide direct evidence that the oxygen-activating component of the LTB4 omega-hydroxylase system is a cytochrome P-450. Ferricytochrome c inhibited the hydroxylation of LTB4 and the inhibition was fortified by cytochrome oxidase. An antibody raised against rat liver NADPH-cytochrome-P-450 reductase inhibited both LTB4 omega-hydroxylase activity and the NADPH-cytochrome-c reductase activity of human neutrophil microsomal fraction. These observations indicate that NADPH-cytochrome-P-450 reductase acts as an electron carrier in LTB4 omega-hydroxylase. On the other hand, an antibody raised against rat liver microsomal cytochrome b5 inhibited the NADH-cytochrome-c reductase activity but not the LTB4 omega-hydroxylase activity of human neutrophil microsomal fraction, suggesting that cytochrome b5 does not participate in the LTB4-hydroxylating system. These characteristics indicate that the isoenzyme of cytochrome P-450 in human neutrophils, LTB4 omega-hydroxylase, is different from the ones reported to be involved in omega-hydroxylation reactions of prostaglandins and fatty acids.     

Evaluation of cytochrome P450 4 family as mediator of phospholipase D activation in aortic vascular smooth muscle cells

Norepinephrine (NE) stimulates phospholipase D (PLD) activity via phospholipase A2-dependent arachidonic acid release in rabbit aortic vascular smooth muscle cells (VSMC). We have previously shown that exogenous 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid generated through the cytochrome P450 (CYP) 4A pathway in vivo, stimulates PLD activity. Whether endogenous CYP4-derived arachidonic acid metabolites act as intracellular mediators of NE-induced PLD activation in VSMC is not known. In rabbit aortic VSMC, prototypical hepatic/renal CYP4A inducers such as fenofibrate and Wy 14643 inhibited both basal and NE-induced PLD activity after 48 h of exposure. The level of CYP4F, and to a lesser extent CYP4A, was also decreased by these agents. The expression levels of rabbit aortic VSMC CYP4A and CYP4F isoforms were reduced by antisense oligonucleotides treatment for 48 hours as measured by RTQ-PCR or Western blotting. This reduction in CYP4A or CYP4F levels did not change NE-induced PLD activation. The corresponding CYP4A scrambled and CYP4F sense oligonucleotides did not alter CYP levels. PLD activity was increased by ~70% after 15 min of stimulation with NE, whereas lauric acid omega-hydroxylase activity, a measure of fatty acid omega-hydroxylation, was unchanged. Inhibition of omega-hydroxylation with DDMS and HET0016, selective omega-hydroxylase inhibitors, and 20-HEDE, an antagonist of 20-HETE, increased PLD activity in a concentration-dependent manner and did not alter NE-induced PLD activation. These data suggest that PLD activation by NE is independent of the CYP4A/4F enzymes in rabbit aortic VSMC.     

Identification of the cytochrome P450 enzymes responsible for the omega-hydroxylation of phytanic acid

Patients suffering from Refsum disease have a defect in the alpha-oxidation pathway which results in the accumulation of phytanic acid in plasma and tissues. Our previous studies have shown that phytanic acid is also a substrate for the omega-oxidation pathway. With the use of specific inhibitors we now show that members of the cytochrome P450 (CYP450) family 4 class are responsible for phytanic acid omega-hydroxylation. Incubations with microsomes containing human recombinant CYP450s (Supersomes) revealed that multiple CYP450 enzymes of the family 4 class are able to omega-hydroxylate phytanic acid with the following order of efficiency: CYP4F3A>CYP4F3B>CYP4F2>CYP4A11.     

omega-hydroxylation activity toward leukotriene B(4) and polyunsaturated fatty acids in the human hepatoblastoma cell line, HepG2, and human lung adenocarcinoma cell line, A549

The addition of glucose to the culture medium of HepG2 or A549 cells for 22 h caused a dose-dependent increase in leukotriene B(4) omega-hydroxylation activity in the homogenate. The addition of genistein to the culture medium of HepG2 or A549 cells for 22 h caused a dose-dependent decrease in the activity, although the number of living cells was not influenced by the addition of genistein. The inhibition by genistein was reversed by removal of genistein from the culture medium in 22 h. The specific leukotriene B(4) omega-hydroxylation activity was high in the nuclear envelope fraction of HepG2 or A549 cells, and a large portion of the activity was concentrated in the nuclear envelope fraction. In the nuclear envelope fraction, leukotriene B(4) omega-hydroxylation activity was accompanied by high polyunsaturated fatty acid omega-hydroxylation activity. The apparent K(m) values for arachidonic acid and leukotriene B(4) in the fractions of HepG2 or A549 cells were 25 and 50 microM, or 22 and 66 microM, respectively. The V(max) values were 222 and 104 pmol/min/mg protein, or 175 and 370 pmol/min/mg protein, respectively. NADPH-dependent omega-hydroxylation of LTB(4) in the nuclear envelope fraction of HepG2 or A549 cells was strongly inhibited by metyrapone and CO. The expression of cytochrome P450 4F2 mRNAs was detected in HepG2 and A549 cells, and thus the arachidonic acid and leukotriene B(4) omega-hydroxylation activities in the nuclear envelope fractions of HepG2 and A549 cells are likely due to cytochrome P450 4F2.     

Unique properties of purified, Escherichia coli-expressed constitutive cytochrome P4504A5.

Cytochromes P450 of the 4A family metabolize a variety of fatty acids, prostaglandins, and eicosanoids mainly at the terminal carbon (omega-hydroxylation) and, to a lesser extent, at the penultimate carbon [(omega-1)-hydroxylation]. In the present study, cytochrome P4504A5 (4A5) has been successfully expressed in Escherichia coli, with an average yield of enzyme of approximately 80 nmol/liter of cells. Spectroscopic characterization of the purified enzyme, using electron paramagnetic resonance and absolute and substrate-perturbed optical difference spectroscopy, showed that the heme of resting 4A5 is primarily low spin, but is converted primarily to high spin by substrate binding. The kcat and Km values for laurate omega-hydroxylation were 41 min-1 and 8.5 microM, respectively, in the absence of cytochrome b5, and 138 min-1 and 38 microM, respectively, in the presence of cytochrome b5. Hydroxylation of palmitate was dependent on the presence of cytochrome b5; kcat and Km values were 48 min-1 and 122 microM, respectively. Hydroxylation of arachidonic acid was barely detectable and was unchanged by the addition of cytochrome b5.     

Biochemical characterization of hepatic microsomal leukotriene B4 hydroxylases

omega-Hydroxylation of leukotriene B4 (LTB4) has been reported in human and rodent polymorphonuclear leukocytes; preliminary information indicates that this metabolism is cytochrome P-450 dependent. Therefore, these studies were initiated to characterize the cytochrome P-450-dependent metabolism of LTB4 in other tissues. LTB4 was metabolized by rat hepatic microsomes to two products, 20-hydroxy(omega)-LTB4 and 19-hydroxy(omega-1)-LTB4. The formation of these metabolites was both oxygen and NADPH dependent indicating that a monooxygenase(s) was responsible for these reactions. The apparent Km and Vmax for LTB4 omega-hydroxylase were 40.28 microM and 1202 pmol/min/mg of protein, respectively. In contrast, the apparent Km and Vmax for LTB4 (omega-1)-hydroxylase were 61.52 microM and 73.50 pmol/min/mg of protein, respectively. Both LTB4 omega- and (omega-1)-hydroxylases were inhibited by metyrapone in a concentration-dependent fashion. However, SK&F 525A inhibited LTB4 (omega-1)- but not omega-hydroxylase. In contrast, alpha-naphthoflavone decreased LTB4 omega- but not (omega-1)-hydroxylase activities. The differences in the Km apparent for substrate as well as the differential inhibition by inhibitors of cytochrome P-450 suggest that the omega- and (omega-1)-hydroxylations of LTB4 in hepatic microsomes are mediated by different isozymes of P-450. Furthermore, several additional characteristics of LTB4 hydroxylases indicate that these isozymes of P-450 may be different from those which catalyze similar reactions on medium-chain fatty acids, such as laurate and prostaglandins.     

Influence of major structural features of tocopherols and tocotrienols on their omega-oxidation by tocopherol-omega-hydroxylase

Human cytochrome P450 4F2 (CYP4F2) catalyzes the initial omega-hydroxylation reaction in the metabolism of tocopherols and tocotrienols to carboxychromanols and is, to date, the only enzyme shown to metabolize vitamin E. The objective of this study was to characterize this activity, particularly the influence of key features of tocochromanol substrate structure. The influence of the number and positions of methyl groups on the chromanol ring, and of stereochemistry and saturation of the side chain, were explored using HepG2 cultures and microsomal reaction systems. Human liver microsomes and microsomes selectively expressing recombinant human CYP4F2 exhibited substrate activity patterns similar to those of HepG2 cells. Although activity was strongly associated with substrate accumulation by cells or microsomes, substantial differences in specific activities between substrates remained under conditions of similar microsomal membrane substrate concentration. Methylation at C5 of the chromanol ring was associated with markedly low activity. Tocotrienols exhibited much higher Vmax values than their tocopherol counterparts. Side chain stereochemistry had no effect on omega-hydroxylation of alpha-tocopherol (alpha-TOH) by any system. Kinetic analysis of microsomal CYP4F2 activity revealed Michaelis-Menten kinetics for alpha-TOH but allosteric cooperativity for other vitamers, especially tocotrienols. Additionally, alpha-TOH was a positive effector of omega-hydroxylation of other vitamers. These results indicate that CYP4F2-mediated tocopherol-omega-hydroxylation is a central feature underlying the different biological half-lives, and therefore biopotencies, of the tocopherols and tocotrienols.     

Cytochrome P450 omega-hydroxylase pathway of tocopherol catabolism. Novel mechanism of regulation of vitamin E status

Postabsorptive elimination of the various forms of vitamin E appears to play a key role in regulation of tissue tocopherol concentrations, but mechanisms of tocopherol metabolism have not been elucidated. Here we describe a pathway involving cytochrome P450-mediated omega-hydroxylation of the tocopherol phytyl side chain followed by stepwise removal of two- or three-carbon moieties, ultimately yielding the 3'-carboxychromanol metabolite that is excreted in urine. All key intermediates of gamma-tocopherol metabolism via this pathway were identified in hepatocyte cultures using gas chromatography-mass spectrometry. NADPH-dependent synthesis of the initial gamma- and alpha-tocopherol 13'-hydroxy and -carboxy metabolites was demonstrated in rat and human liver microsomes. Functional analysis of several recombinant human liver P450 enzymes revealed that tocopherol-omega-hydroxylase activity was associated only with CYP4F2, which also catalyzes omega-hydroxylation of leukotriene B(4) and arachidonic acid. Tocopherol-omega-hydroxylase exhibited similar binding affinities but markedly higher catalytic activities for gamma-tocopherol than alpha-tocopherol, suggesting a role for this pathway in the preferential physiological retention of alpha-tocopherol and elimination of gamma-tocopherol. Sesamin potently inhibited tocopherol-omega-hydroxylase activity exhibited by CYP4F2 and rat or human liver microsomes. Since dietary sesamin also results in elevated tocopherol levels in vivo, this pathway appears to represent a functionally significant means of regulating vitamin E status.     

Stereochemical requirements for substrate specificity of LTB4 20-hydroxylase

LTB4 20-hydroxylase (P-450LTB) is the cytochrome P-450 in the microsomes of human polymorphonuclear leukocytes that catalyzes the omega-oxidation of leukotriene B4 (LTB4) to 20-OH LTB4. The activity of P-450LTB for LTB4 compared to isomers and analogs of LTB4 at a concentration of 0.3 microM revealed a preference of P-450LTB for both the triene bond configuration of LTB4 and for the chirality of the 5S and 12R hydroxyl groups. 15S-Hydroxyeicosatetraenoic acid, 8(R/S), 15S-dihydroxy-5-cis-9,11,13-trans-eicosatetraenoic acid, 8R,15S-dihydroxy-5,13-cis-9,11-trans-eicosatetraenoic acid, and 5S,15S-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid were each not subject to omega-oxidation, indicating a negative effect of the presence of a 15-hydroxyl group on substrate recognition. At a concentration of 1.5 microM, 12R- and 12S-hydroxyeicosatetraenoic acid were converted to their respective 20-OH derivatives at rates that were 34.2 +/- 11.6% (mean +/- S.D., n = 3) and 3.5 +/- 4.3% (mean +/- S.D., n = 4), respectively, of that of LTB4 to 20-OH LTB4, further indicating that P-450LTB can distinguish the chirality of the 12-hydroxyl group. The lower Km of LTB4 (2.0 microM), as compared to those of its 6-trans-12-epi isomer (3.8 microM) and 5-epi-LTB4 (6.6 microM) confirmed the preference of P-450LTB for the specific triene bond structure of LTB4 and its preference for the chirality of the hydroxyl groups of LTB4 within this structurally related class of molecules. At equal 1.5-microM concentrations, LTB4 completely inhibited the omega-oxidation of all other substrates and partially suppressed that of leukotriene B5, consistent with the lower Km of LTB4 and indicating that P-450LTB catalyzed the omega-oxidation of all substrates. Thus, P-450LTB is a novel cytochrome P-450 of human polymorphonuclear leukocytes with substrate recognition determined by the triene bond configuration and the chirality of the hydroxyl groups.     

Leukotriene B4 omega-side chain hydroxylation by CYP4F5 and CYP4F6

Leukotriene B(4) (LTB(4)) is a lipid mediator that plays an important role in inflammation. Metabolism of LTB(4) by cytochrome P450 (CYP) enzymes belonging to the CYP4F subfamily is considered to be of importance for the regulation of inflammation. This study investigates LTB(4) metabolism by recombinant rat CYP4F5 and CYP4F6 expressed in a yeast system and by microsomes isolated from rat organs expressing CYP4F mRNA. CYP4F6 was found to convert LTB(4) into 19-hydoxy- and 18-hydroxy-LTB(4) with an apparent K(m) of 26 microM, and CYP4F5 was found to convert LTB(4) primarily into 18-hydroxy-LTB(4) with an apparent K(m) of 9.7 microM. The rate of formation of 18-hydroxy-LTB(4) by CYP4F5 was surprisingly high. At a substrate concentration of 30 microM, the rate of formation was about 15 nmol/min/mg microsomal protein, approximately 30 times faster than the reaction catalyzed by CYP4F6. Analysis of LTB(4) metabolism by microsomes isolated from various tissues from the rat suggests that CYP4F5 and CYP4F6 are active in the lung and to some extent in the brain, kidney, and testis. CYP4F5 and CYP4F6, due to their capacities to metabolize LTB(4), may play important roles in modulating inflammatory response in these organs.     

Omega-oxidation of very long-chain fatty acids in human liver microsomes. Implications for X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disorder biochemically characterized by elevated levels of very long-chain fatty acids (VLCFA). Excess levels of VLCFAs are thought to play an important role in the pathogenesis of X-ALD. Therefore, therapeutic approaches for X-ALD are focused on the reduction or normalization of VLCFAs. In this study, we investigated an alternative oxidation route for VLCFAs, namely omega-oxidation. The results described in this study show that VLCFAs are substrates for the omega-oxidation system in human liver microsomes. Moreover, VLCFAs were not only converted into omega-hydroxy fatty acids, but they were also further oxidized to dicarboxylic acids via cytochrome P450-mediated reactions. High sensitivity toward the specific P450 inhibitor 17-octadecynoic acid suggested that omega-hydroxylation of VLCFAs is catalyzed by P450 enzymes belonging to the CYP4A/F subfamilies. Studies with individually expressed human recombinant P450 enzymes revealed that two P450 enzymes, i.e. CYP4F2 and CYP4F3B, participate in the omega-hydroxylation of VLCFAs. Both enzymes belong to the cytochrome P450 4F subfamily and have a high affinity for VLCFAs. In summary, this study demonstrates that VLCFAs are substrates for the human omega-oxidation system, and for this reason, stimulation of the in vivo VLCFA omega-oxidation pathway may provide an alternative mode of treatment to reduce the levels of VLCFAs in patients with X-ALD.     

Cytochrome P-450 4F18 is the leukotriene B4 omega-1/omega-2 hydroxylase in mouse polymorphonuclear leukocytes: identification as the functional orthologue of human polymorphonuclear leukocyte CYP4F3A in the down-regulation of responses to LTB4

Leukotriene B(4) (LTB(4)) is a potent chemoattractant for polymorphonuclear leukocytes (PMN) and other cells. Human PMN inactivate LTB(4) by omega-oxidation catalyzed by cytochrome P-450 (CYP) 4F3A. The contribution of the enzymatic inactivation of LTB(4) by CYP4Fs to down-regulating functional responses of cells to LTB(4) is unknown. To elucidate the role of CYP4F-mediated inactivation of LTB(4) in terminating the responses of PMN to LTB(4) and to identify a target for future genetic studies in mice, we have identified the enzyme that catalyzes the omega-1 and omega-2 oxidation of LTB(4) in mouse myeloid cells as CYP4F18. As determined by mass spectrometry, this enzyme catalyzes the conversion of LTB(4) to 19-OH LTB(4) and to a lesser extent 18-OH LTB(4). Inhibition of CYP4F18 resulted in a marked increase in calcium flux and a 220% increase in the chemotactic response of mouse PMN to LTB(4). CYP4F18 expression was induced in bone marrow-derived dendritic cells by bacterial lipopolysaccharide, a ligand for TLR4, and by poly(I.C), a ligand for TLR3. However, when bone marrow-derived myeloid dendritic cells trafficked to popliteal lymph nodes from paw pads, the expression of CYP4F18 was down-regulated. The results identify CYP4F18 as a critical protein in the regulation of LTB(4) metabolism and functional responses in mouse PMN and identify it as the functional orthologue of human PMN CYP4F3A.     

Molecular modeling and identification of substrate binding site of orphan human cytochrome P450 4F22

Cytochrome P450s are superfamily of heme proteins which generally monooxygenate hydrophobic compounds. The human cytochrome P450 4F22 (CYP4F22) was categorized into "orphan" CYPs because of its unknown function. CYP4F22 is a potential drug target for cancer therapy. However, three-dimensional structure, the active site topology and substrate specificity of CYP4F22 remain unclear. In this study, a three-dimensional model of human P450 4F22 was constructed by comparative modeling using Modeller 9v5. The resulting model was refined by energy minimization subjected to the quality assessment from both geometric and energetic aspects and was found to be of reasonable quality. Docking approach was employed to dock arachidonic acid into the active site of CYP4F22 in order to probe the ligand-binding modes. As a result, several key residues were identified to be responsible for the binding of arachidonic acid with CYP4F22. These findings provide useful information for understanding the biological roles of CYP4F22 and structure-based drug design.