Functional Cytochrome Research Articles

Drug target prediction using elementary mode analysis in Ascaris lumbricoides energy metabolism

Ascariasis, an intestinal worm infection is caused by the parasite Ascaris lumbricoides and a report by World Health Organization (WHO) on soil transmitted helminths suggests that over one billion people are affected by Ascariasis. This disease is prevalent in developing countries, and in places of poor sanitation and unhygienic conditions. Even though anthelminthic drugs are available for the treatment of ascariasis, it is considered as a neglected tropical disease (NTD). Resistance of the parasite to the existing drugs necessitates a detailed study of its energy metabolism for identification of new drug targets. The catabolic pathway of the parasite is an evolved design well suited for parasitic life and obtains constant input from its host. Its energy metabolism is predominantly anaerobic. The parasite mitochondrion plays a key role as it lacks the functional tricarboxylic acid cycle (TCA cycle) and cytochrome oxidase activity. In adult ascarid mitochondrion, there is no external final electron acceptor and endogenously produced fumarate and 2-methyl branched-chain enoyl — CoAs function as the terminal electron acceptors instead of oxygen. In this study, elementary flux mode analysis (EFM), a metabolic pathway analysis tool has been applied to model energy metabolism of the parasite A. lumbricoides. This study identifies a set of enzymes that have been suggested to be essential for the survival of the parasite; the inhibition of these enzymes paralyzes the parasite. The key enzymes of glycolysis and the phosphoenolpyruvate carboxykinase-succinate pathway are identified as drug targets since the knock-out of any of these enzymes results in zero flux value for all EFM that have been identified.

Conversion of Corynebacterium glutamicum from an aerobic respiring to an aerobic fermenting bacterium by inactivation of the respiratory chain

In this study a comparative analysis of three Corynebacterium glutamicum ATCC 13032 respiratory chain mutants lacking either the cytochrome bd branch (ΔcydAB), or the cytochrome bc1–aa3 branch (Δqcr), or both branches was performed. The lack of cytochrome bd oxidase was inhibitory only under conditions of oxygen limitation, whereas the absence of a functional cytochrome bc1–aa3 supercomplex led to decreases in growth rate, biomass yield, respiration and proton-motive force (pmf) and a strongly increased maintenance coefficient under oxygen excess. These results show that the bc1–aa3 supercomplex is of major importance for aerobic respiration. For the first time, a C. glutamicum strain with a completely inactivated aerobic respiratory chain was obtained (ΔcydABΔqcr), named DOOR (devoid of oxygen respiration), which was able to grow aerobically in BHI (brain–heart infusion) glucose complex medium with a 70% reduced biomass yield compared to the wild type. Surprisingly, reasonable aerobic growth was also possible in glucose minimal medium after supplementation with peptone. Under these conditions, the DOOR strain displayed a fermentative type of catabolism with l-lactate as major and acetate and succinate as minor products. The DOOR strain had about 2% of the oxygen consumption rate of the wild type, showing the absence of additional terminal oxidases. The pmf of the DOOR mutant was reduced by about 30% compared to the wild type. Candidates for pmf generation in the DOOR strain are succinate:menaquinone oxidoreductase, which probably can generate pmf in the direction of fumarate reduction, and F1FO-ATP synthase, which can couple ATP hydrolysis to the export of protons.

Polycyclic Aromatic Hydrocarbons (PAHs) Mediate Transcriptional Activation of the ATP Binding Cassette Transporter ABCB6 Gene via the Aryl Hydrocarbon Receptor (AhR)

Liver is endowed with a mechanism to induce hepatic cytochromes P450 (CYP450s) in response to therapeutic drugs and environmental contaminants, leading to increased detoxification and elimination of the xenobiotics. Each CYP450 is composed of an apoprotein moiety and a heme prosthetic group, which is required for CYP450 activity. Thus, under conditions of CYP450 induction, there is a coordinate increase in heme biosynthesis to compensate for the increased expression of CYP450s. ABCB6, a mitochondrial ATP binding cassette transporter, which regulates coproporphyrinogen transport from the cytoplasm into the mitochondria to complete heme biosynthesis, represents a previously unrecognized rate-limiting step in heme biosynthesis. However, it is not known if exposure to drugs and environmental contaminants induces ABCB6 expression, to assure an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoprotein. In the present study, we demonstrate that polycyclic aromatic hydrocarbons (PAHs), the widely distributed environmental toxicants shown to induce porphyrin accumulation causing hepatic porphyria, up-regulate ABCB6 expression in both mice and humans. Using siRNA technology and Abcb6 knock-out mice, we demonstrate that PAH-mediated increase in hepatic porphyrins is compromised in the absence of ABCB6. Moreover, in vivo studies in aryl hydrocarbon receptor (AhR) knock-out mice demonstrate that PAH induction of ABCB6 is mediated by AhR. Promoter activation studies combined with electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrate direct interactions between the AhR binding sites in the ABCB6 promoter and the AhR receptor, implicating drug activation mechanisms for ABCB6 similar to those found in inducible cytochrome P450s. These studies are the first to describe direct transcriptional activation of both mouse and human ABCB6 by xenobiotics.

Rapid Detection of an ABT-737-Sensitive Primed for Death State in Cells Using Microplate-Based Respirometry

Cells that exhibit an absolute dependence on the anti-apoptotic BCL-2 protein for survival are termed “primed for death” and are killed by the BCL-2 antagonist ABT-737. Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2 expression. We show here that 1) stable BCL-2 overexpression alone can induce a primed for death state and 2) that an ABT-737-induced loss of functional cytochrome c from the electron transport chain causes a reduction in maximal respiration that is readily detectable by microplate-based respirometry. Stable BCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis. Mitochondria within permeabilized BCL-2 overexpressing cells were selectively vulnerable to ABT-737-induced cytochrome c release compared to those from control-transfected cells, consistent with a primed state. ABT-737 treatment caused a dose-dependent impairment of maximal O2 consumption in MCF10A BCL-2 overexpressing cells but not in control-transfected cells or in immortalized mouse embryonic fibroblasts lacking both BAX and BAK. This impairment was rescued by delivering exogenous cytochrome c to mitochondria via saponin-mediated plasma membrane permeabilization. An ABT-737-induced reduction in maximal O2 consumption was also detectable in SP53, JeKo-1, and WEHI-231 B-cell lymphoma cell lines, with sensitivity correlating with BCL-2:MCL-1 ratio and with susceptibility (SP53 and JeKo-1) or resistance (WEHI-231) to ABT-737-induced apoptosis. Multiplexing respirometry assays to ELISA-based determination of cytochrome c redistribution confirmed that respiratory inhibition was associated with cytochrome c release. In summary, cell-based respiration assays were able to rapidly identify a primed for death state in cells with either artificially overexpressed or high endogenous BCL-2. Rapid detection of a primed for death state in individual cancers by “bioenergetics-based profiling” may eventually help identify the subset of patients with chemoresistant but primed tumors who can benefit from treatment that incorporates a BCL-2 antagonist.

Redundant role of the cytochrome c-mediated intrinsic apoptotic pathway in pancreatic β-cells

Cytochrome c is one of the central mediators of the mitochondrial or the intrinsic apoptotic pathway. Mice harboring a 'knock-in' mutation of cytochrome c, impairing only its apoptotic function, have permitted studies on the essential role of cytochrome c-mediated apoptosis in various tissue homeostasis. To this end, we examined the role of cytochrome c in pancreatic β-cells under homeostatic conditions and in diabetes models, including those induced by streptozotocin (STZ) and c-Myc. Previous studies have shown that both STZ- and c-Myc-induced β-cell apoptosis is mediated through caspase-3 activation; however, the precise mechanism in these modes of cell death was not characterized. The results of our study show that lack of functional cytochrome c does not affect glucose homeostasis or pancreatic β-cell mass under basal conditions. Moreover, the cytochrome c-mediated intrinsic apoptotic pathway is required for neither STZ- nor c-Myc-induced β-cell death. We also observed that the extrinsic apoptotic pathway mediated through caspase-8 was not essential in c-Myc-induced β-cell destruction. These findings suggest that cytochrome c is not required for STZ-induced β-cell apoptosis and, together with the caspase-8-mediated extrinsic pathway, plays a redundant role in c-Myc-induced β-cell apoptosis.

Isotopic labeling of the heme cofactor in cytochrome p450 and other heme proteins

A recombinant bacterial expression system that generates (13)C-labeled heme or (15)N-labeled heme in functional cytochrome P450 enzymes and other heme-containing systems is reported here using a mutant strain of Escherichia coli (HU227) in which the HemA gene is inactive. By synthesizing several isotopomers of aminolevulinic acid with (13)C or (15)N at different locations, isotopes have been incorporated with high abundance into the heme cofactor of five different cytochrome P450 isoforms, along with one peroxidase. Confirmed both (13)C- and (15)N-incorporation; spectral and catalytic assays show the labeled enzymes produced in this system are functional.

High-yield production, purification and characterization of functional human duodenal cytochrome b in an Escherichia coli system

Human duodenal cytochrome b (Dcytb) is a transmembrane hemoprotein found in the duodenal brush border membrane and in erythrocytes. Dcytb has been linked to uptake of dietary iron and to ascorbate recycling in erythrocytes. Detailed biophysical and biochemical characterization of Dcytb has been limited by difficulties in expressing sufficient amounts of functional recombinant protein in yeast and insect cell systems. We have developed an Escherichia coli Rosetta-gami B(DE3) cell system for production of recombinant His-tagged human Dcytb with a yield of ∼26 mg of purified, ascorbate-reducible cytochrome per liter of culture. The recombinant protein is readily solubilized with n-dodecyl-β- d-maltoside and purified to electrophoretic homogeneity by one-step chromatography on cobalt affinity resin. The purified recombinant Dcytb has a heme to protein ratio very close to the theoretical value of 2 and retains functional reactivity with ascorbate, as assessed by spectroscopic and kinetic measurements. Ascorbate showed a marked kinetic selectivity for the high-potential heme center over the low-potential heme center in purified Dcytb. This new E. coli expression system for Dcytb offers ∼7-fold improvement in yield and other substantial advantages over existing expression systems for reliable production of functional Dcytb at levels suitable for biochemical, biophysical and structural characterization.

Corrigendum: Cloning and Characterization of the Biosynthetic Gene Cluster of 16‐Membered Macrolide Antibiotic FD‐891: Involvement of a Dual Functional Cytochrome P450 Monooxygenase Catalyzing Epoxidation and Hydroxylation

A few changes were missed when the proofs of this manuscript were corrected. The genes given in Table 1 should be as follows: Gene Size[a] Homologue (biosynthetic product, identity [%]/similarity [%]) Predicted function in FD-891 biosynthesis gfsA 7480 polyketide synthase PimS1 (pimaricin, 54/66) PKS loading, module 1–4 gfsB 5571 polyketide synthase NysJ (nystatin, 51/62) PKS modules 5–7 gfsC 2228 polyketide synthase Orf17 (ECO-02301, 63/74) PKS module 8 gfsD 3639 polyketide synthase PteA2 (filipin, 53/65) PKS modules 9–10 gfsE 3799 polyketide synthase Orf17 (ECO-02301, 51/63) PKS modules 11–12 gfsF 414 P450 monooxygenase Mflv2418 (57/70) P450 monooxygenase gfsG 278 Methyltransferase AtM (AT2433, 57/70) methyltransferase gfsR 243 NarL family two-component response regulator transcriptional regulator The caption to Figure 5 should read “HPLC analysis of GfsF reaction with 25-O-methyl-FD-892 coupled with putidaredoxin and putidaredoxin reductase.” and the bottom of p. 1581 should read: “The amplified PCR product was subcloned into the dephosphorylated EcoRV site of LITMUS28. After digestion of LT28 with EcoRV, we dephosphorylated the vector with BAP.” Finally, the upper hydroxy group in the Table of Contents image should not have been colored red.1

Cloning and Characterization of the Biosynthetic Gene Cluster of 16‐Membered Macrolide Antibiotic FD‐891: Involvement of a Dual Functional Cytochrome P450 Monooxygenase Catalyzing Epoxidation and Hydroxylation

FD-891 is a 16-membered cytotoxic antibiotic macrolide that is especially active against human leukemia such as HL-60 and Jurkat cells. We identified the FD-891 biosynthetic (gfs) gene cluster from the producer Streptomyces graminofaciens A-8890 by using typical modular type I polyketide synthase (PKS) genes as probes. The gfs gene cluster contained five typical modular type I PKS genes (gfsA, B, C, D, and E), a cytochrome P450 gene (gfsF), a methyltransferase gene (gfsG), and a regulator gene (gfsR). The gene organization of PKSs agreed well with the basic polyketide skeleton of FD-891 including the oxidation states and alpha-alkyl substituent determined by the substrate specificities of the acyltransferase (AT) domains. To clarify the involvement of the gfs genes in the FD-891 biosynthesis, the P450 gfsF gene was inactivated; this resulted in the loss of FD-891 production. Instead, the gfsF gene-disrupted mutant accumulated a novel FD-891 analogue 25-O-methyl-FD-892, which lacked the epoxide and the hydroxyl group of FD-891. Furthermore, the recombinant GfsF enzyme coexpressed with putidaredoxin and putidaredoxin reductase converted 25-O-methyl-FD-892 into FD-891. In the course of the GfsF reaction, 10-deoxy-FD-891 was isolated as an enzymatic reaction intermediate, which was also converted into FD-891 by GfsF. Therefore, it was clearly found that the cytochrome P450 GfsF catalyzes epoxidation and hydroxylation in a stepwise manner in the FD-891 biosynthesis. These results clearly confirmed that the identified gfs genes are responsible for the biosynthesis of FD-891 in S. graminofaciens.

Inhibition of Electrocatalytic O2 Reduction of Functional CcO Models by Competitive, Non-Competitive, and Mixed Inhibitors

Electrocatalytic reduction of O(2) by functional cytochrome C Oxidase (CcO) models is studied in the presence of several known inhibitors like CO, N(3)(-), CN(-), and NO(2)(-). These models successfully reproduce the inhibitions observed in CcO at similar concentrations reported for these inhibitors. Importantly, the data show very different electrochemical responses depending on the nature of the inhibitor, that is, competitive, non-competitive and mixed. Chemical models have been provided for these observed differences in the electrochemical behavior. Using the benchmark electrochemical behaviors for known inhibitors, the inhibition by NO(2)(-) is investigated. Electrochemical data suggests that NO(2)(-) acts as a competitive inhibitor at high concentrations. Spectroscopic data suggests that NO released during oxidation of the reduced catalyst in presence of excess NO(2)(-) is the source of the competitive inhibition by NO(2)(-). Presence of the distal Cu(B) lowers the inhibitory effect of CN(-) and NO(2)(-). While for CN(-) it weakens its binding affinity to the reduced complex by approximately 4.5 times, for NO(2)(-), it allows regeneration of the active catalyst from a catalytically inactive, air stable ferrous nitrosyl complex via a proposed superoxide mediated pathway.

High molecular weight FGF-2 promotes postconditioning-like cardioprotection linked to activation of the protein kinase C isoforms Akt and p70 S6 kinaseThis article is one of a selection of papers published in a special issue celebrating the 125th anniversary of the Faculty of Medicine at the University of Manitoba.

Fibroblast growth factor 2 (FGF-2) is a multifunctional protein translated as high and low molecular weight isoforms (hi- and lo-FGF-2, respectively). Although the postconditioning cardioprotective effect of lo-FGF-2 (18 kDa) has been documented, hi-FGF-2 is less well studied. We used an isolated perfused rat heart model of ischemia-reperfusion to study the effects of postischemic (during reperfusion) administration of hi-FGF-2 on recovery of contractile function and tissue salvage, as indicated by decreased cytosolic cytochrome c levels. Compared with the vehicle-treated group, hi-FGF-2-treated hearts had significantly improved recovery of systolic pressure, developed pressure, rates of contraction and relaxation, and coronary flow, as well as decreased relative levels of cytosolic cytochrome c. The effects of hi-FGF-2 on functional recovery and cytosolic cytochrome c were indistinguishable from those induced by lo-FGF-2. Both hi- and lo-FGF-2 upregulated relative levels of phosphorylated (activated) Akt and p70 S6 kinase, and they both promoted translocation of alpha, epsilon, and zeta isoforms of protein kinase C (PKC) to the particulate fraction of reperfused hearts. The magnitude of the effect on PKCzeta and p70 S6 kinases, however, was significantly more potent in the hi-FGF-2 than in the lo-FGF-2 group. We conclude that acute postischemic cardioprotection by hi- or lo-FGF-2 is isoform nonspecific and likely to be mediated by PKC and Akt. Nevertheless, isoform-specific functions are suggested by the augmented sensitivity of p70 S6 and PKCzeta to hi-FGF-2.

Induction of functional cytochrome P450 and its involvement in degradation of benzoic acid by Phanerochaete chrysosporium

The white rot fungus Phanerochaete chrysosporium has the largest cytochrome P450 contingent known to date in fungi, but the study on the function of these P450s is limited. In this study, induction of functional P450 in P. chrysosporium was first shown and P450-mediate degradation of benzoic acid was demonstrated in this fungus. Carbon monoxide difference spectra indicated significant induction of P450 by benzoic acid, m-chlorobenzoic acid, p-chlorobenzoic acid and n-hexane, and showed the effect of inducer concentration and nutrient condition on the induction of P450. The high contents of P450 in the microsomal fractions facilitated the study on the function of P450. While the n-hexane-induced P450 could not interact with benzoic acid, the microsomal P450 induced by benzoic acid produced type I substrate binding spectra upon the addition of benzoic acid. The benzoic acid degradation by the microsomal P450 was NADPH-dependent at a specific rate of 194 +/- 14 min(-1), and significantly inhibited by piperonyl butoxide (a P450 inhibitor). However, inhibition of benzoic acid degradation by piperonyl butoxide was slight or not detectable in the cultures of this fungus, suggesting presumable involvement of other enzyme in benzoic acid degradation. The extracellular ligninolytic enzymes, lignin peroxidase and manganese-dependent peroxidase, were not involved in initial metabolism of benzoic acid under the test conditions.

Structure of the open conformation of a functional chimeric NADPH cytochrome P450 reductase

Two catalytic domains, bearing FMN and FAD cofactors, joined by a connecting domain, compose the core of the NADPH cytochrome P450 reductase (CPR). The FMN domain of CPR mediates electron shuttling from the FAD domain to cytochromes P450. Together, both enzymes form the main mixed-function oxidase system that participates in the metabolism of endo- and xenobiotic compounds in mammals. Available CPR structures show a closed conformation, with the two cofactors in tight proximity, which is consistent with FAD-to-FMN, but not FMN-to-P450, electron transfer. Here, we report the 2.5 A resolution crystal structure of a functionally competent yeast-human chimeric CPR in an open conformation, compatible with FMN-to-P450 electron transfer. Comparison with closed structures shows a major conformational change separating the FMN and FAD cofactors from 86 A.

Genome-wide identification and characterization of cytochrome P450 monooxygenase genes in the ciliate Tetrahymena thermophila

BackgroundCytochrome P450 monooxygenases play key roles in the metabolism of a wide variety of substrates and they are closely associated with endocellular physiological processes or detoxification metabolism under environmental exposure. To date, however, none has been systematically characterized in the phylum Ciliophora. T. thermophila possess many advantages as a eukaryotic model organism and it exhibits rapid and sensitive responses to xenobiotics, making it an ideal model system to study the evolutionary and functional diversity of the P450 monooxygenase gene family.ResultsA total of 44 putative functional cytochrome P450 genes were identified and could be classified into 13 families and 21 sub-families according to standard nomenclature. The characteristics of both the conserved intron-exon organization and scaffold localization of tandem repeats within each P450 family clade suggested that the enlargement of T. thermophila P450 families probably resulted from recent separate small duplication events. Gene expression patterns of all T. thermophila P450s during three important cell physiological stages (vegetative growth, starvation and conjugation) were analyzed based on EST and microarray data, and three main categories of expression patterns were postulated. Evolutionary analysis including codon usage preference, site-specific selection and gene-expression evolution patterns were investigated and the results indicated remarkable divergences among the T. thermophila P450 genes.ConclusionThe characterization, expression and evolutionary analysis of T. thermophila P450 monooxygenase genes in the current study provides useful information for understanding the characteristics and diversities of the P450 genes in the Ciliophora, and provides the baseline for functional analyses of individual P450 isoforms in this model ciliate species.

Combining chemical genomics screens in yeast to reveal spectrum of effects of chemical inhibition of sphingolipid biosynthesis.

BackgroundSingle genome-wide screens for the effect of altered gene dosage on drug sensitivity in the model organism Saccharomyces cerevisiae provide only a partial picture of the mechanism of action of a drug.ResultsUsing the example of the tumor cell invasion inhibitor dihydromotuporamine C, we show that a more complete picture of drug action can be obtained by combining different chemical genomics approaches – analysis of the sensitivity of ρ0 cells lacking mitochondrial DNA, drug-induced haploinsufficiency, suppression of drug sensitivity by gene overexpression and chemical-genetic synthetic lethality screening using strains deleted of nonessential genes. Killing of yeast by this chemical requires a functional mitochondrial electron-transport chain and cytochrome c heme lyase function. However, we find that it does not require genes associated with programmed cell death in yeast. The chemical also inhibits endocytosis and intracellular vesicle trafficking and interferes with vacuolar acidification in yeast and in human cancer cells. These effects can all be ascribed to inhibition of sphingolipid biosynthesis by dihydromotuporamine C.ConclusionDespite their similar conceptual basis, namely altering drug sensitivity by modifying gene dosage, each of the screening approaches provided a distinct set of information that, when integrated, revealed a more complete picture of the mechanism of action of a drug on cells.

Clustering and dynamics of cytochrome bd‐I complexes in the Escherichia coli plasma membrane in vivo

The cytochrome bd-I complex of Escherichia coli is a respiratory terminal oxidase and an integral component of the cytoplasmic membrane. As with other respiratory components, the organization and dynamics of this complex in living membranes is unknown. We set out to visualize the distribution and dynamics of this complex in vivo. By exchanging cydB for cydB-gfpgcn4 on the E. coli chromosome, we produced a strain (YTL01) that expresses functional GFP-tagged cytochrome bd-I terminal oxidase complexes under wild-type genetic control. We imaged live YTL01 cells using video-rate epifluorescence and total internal reflection fluorescence (TIRF) microscopy in combination with fluorescence recovery after photobleaching (FRAP) and saw mobile spots of GFP fluorescence in plasma membranes. Numbers of GFP molecules per spot were quantified by step-wise photobleaching giving a broad distribution with a mean of approximately 76, indicating that cytochrome bd-I is concentrated in mobile patches in the E. coli plasma membrane. We hypothesize that respiration occurs in mobile membrane patches which we call 'respirazones'.

S11.23 Synthesis of functional Paracoccusdenitrificanscytochrome c oxidase by Escherichia colicell-free coupled transcription/translation system

S11.23 Synthesis of functional Paracoccusdenitrificanscytochrome c oxidase by Escherichia colicell-free coupled transcription/translation system

Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and non-functional cytochrome b proteins

We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc 1 complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036–36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome ( ARG8 m ). Subsequently replacing ARG8 m with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit −). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.

Association of a Functional Cytochrome P450 4F2 Haplotype with Urinary 20-HETE and Hypertension

Cytochrome P450 4F2 (CYP4F2) catalyzes the omega-hydroxylation of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a natriuretic and vasoactive eicosanoid that participates in the development of hypertension. The relationship among CYP4F2 genetic variants in the regulatory region, formation of renal 20-HETE, and hypertension is unknown. Here are reported seven genetic variants around the CYP4F2 intronic regulatory region. Four of these variants made up two common haplotypes, Hap I (c.-91T/c.-48G/c.-13T/c.+34T) and Hap II (c.-91C/c.-48C/c.-13C/c.+34G). Hap I included a major functional variant, c.-91T-->C, which was identified by reporter assay and electrophoretic mobility shift assay. Transfected into HEK293 cells, the Hap I construct showed a trend toward higher basal transcriptional activity and exhibited significantly greater LPS-stimulated activity than Hap II; these findings were the result of different NF-kappaB binding affinity between the two constructs. In vivo, a case-control study demonstrated that homozygosity for Hap I doubled the risk for hypertension in a Chinese population, even after adjustment for risk factors including age, gender, and body mass index. This association was confirmed in a family-based association study. In addition, Hap I was associated with elevated urinary 20-HETE. These results indicate that a functional variant of the CYP4F2 regulatory region, which increases the binding affinity of NF-kappaB, increases the risk for hypertension, likely by modulating the production of 20-HETE.

Inhibition of acyl-coenzyme A:cholesterol acyltransferase stimulates cholesterol efflux from macrophages and stimulates farnesoid X receptor in hepatocytes

We investigated the mechanism of spontaneous cholesterol efflux induced by acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibition, and how an alteration of cholesterol metabolism in macrophages impacts on that in HepG2 cells. Oleic acid anilide (OAA), a known ACAT inhibitor reduced lipid storage substantially by promotion of cholesterol catabolism and repression of cholesteryl ester accumulation without further increase of cytotoxicity in acetylated low-density lipoprotein-loaded THP-1 macrophages. Analysis of expressed mRNA and protein revealed that cholesterol 7alpha-hydroxylase (CYP7A1), oxysterol 7alpha- hydroxylase (CYP7B1), and cholesterol 27-hydroxylase (CYP27) were highly induced by ACAT inhibition. The presence of a functional cytochrome P450 pathway was confirmed by quantification of the biliary cholesterol mass in cell monolayers and extracelluar medium. Notably, massively secreted biliary cholesterol from macrophages suppressed the expression of CYP7 proteins in a farnesoid X receptor (FXR)-dependent manner in HepG2 cells. The findings reported here provide new insight into mechanisms of spontaneous cholesterol efflux, and suggest that ACAT inhibition may stimulate cholesterol-catabolic (cytochrome P450) pathway in lesion-macrophages, in contrast, suppress it in hepatocyte via FXR induced by biliary cholesterol (BC).