Yeast Microsomes Research Articles

Sunitinib Inhibits Cell Proliferation and Alters Steroidogenesis by Down-Regulation of HSD3B2 in Adrenocortical Carcinoma Cells.

The multi-tyrosine kinase inhibitor sunitinib is used in the treatment of several solid tumors. Animal experiments pointed to an adrenotoxic effect of sunitinib. Therefore, we evaluated the expression of key targets of sunitinib in human adrenocortical carcinoma (ACC) tumor samples and investigated its in vitro effects in ACC cell lines. We carried out immunohistochemistry for vascular endothelial growth factor (VEGF) and its receptor (VEGF-R2) in 157 ACC samples and nine normal adrenal glands. VEGF and VEGF-R2 protein were expressed in 72 and 99% of ACC samples, respectively. Using NCI-H295 and SW13 ACC cell lines, we investigated the effects of sunitinib on cell proliferation. Sunitinib reduced dose-dependently cell viability of both NCI-H295 and SW13 cells (SW13: 0.1 μM 96 ± 7%, 1 μM 90 ± 9%*, 5 μM 62 ± 6%*, controls 100 ± 9%; *p < 0.05). To determine sunitinib effects on steroidogenesis, we measured steroid hormones in cell culture supernatant by gas chromatography–mass spectrometry. We observed a pronounced decrease of cortisol secretion (1 μM 90.1 ± 1.5%*, 5 μM 57.2 ± 0.3%*, controls 100 ± 2.4%) and a concomitant increase in the DHEA/4-androstenedione and 17-hydroxypregnenolone/17-hydroxyprogesterone ratios, indicating specific inhibition of 3β-hydroxysteroid dehydrogenase (HSD3B2). In yeast microsomes transformed with HSD3B2, no direct inhibition of HSD3B2 by sunitinib was detected. Sunitinib induced down-regulation of HSD3B2 mRNA and protein in ACC cell lines (mRNA: 1 μM 44 ± 16%*; 5 μM 22 ± 2%*; 10 μM 19 ± 4%*; protein: 1 μM 82 ± 8%; 5 μM 63 ± 8%*; 10 μM 55 ± 9%*). CYP11B1 was down-regulated at mRNA but not at protein level and CYP11A1 remained unchanged. In conclusion, target molecules of sunitinib are expressed in the vast majority of ACC samples. Sunitinib exhibits anti-proliferative effects in vitro, and appears to specifically block adrenal steroidogenesis by down-regulation of HSD3B2, rendering it a promising option for treatment of ACC.

A Combined Zinc/Cadmium Sensor and Zinc/Cadmium Export Regulator in a Heavy Metal Pump

Heavy metal pumps (P1B-ATPases) are important for cellular heavy metal homeostasis. AtHMA4, an Arabidopsis thaliana heavy metal pump of importance for plant Zn(2+) nutrition, has an extended C-terminal domain containing 13 cysteine pairs and a terminal stretch of 11 histidines. Using a novel size-exclusion chromatography, inductively coupled plasma mass spectrometry approach we report that the C-terminal domain of AtHMA4 is a high affinity Zn(2+) and Cd(2+) chelator with capacity to bind 10 Zn(2+) ions per C terminus. When AtHMA4 is expressed in a Zn(2+)-sensitive zrc1 cot1 yeast strain, sequential removal of the histidine stretch and the cysteine pairs confers a gradual increase in Zn(2+) and Cd(2+) tolerance and lowered Zn(2+) and Cd(2+) content of transformed yeast cells. We conclude that the C-terminal domain of AtHMA4 serves a dual role as Zn(2+) and Cd(2+) chelator (sensor) and as a regulator of the efficiency of Zn(2+) and Cd(2+) export. The identification of a post-translational handle on Zn(2+) and Cd(2+) transport efficiency opens new perspectives for regulation of Zn(2+) nutrition and tolerance in eukaryotes.

Inhibitor and substrate activities of sesquiterpene olefins toward (+)-δ-cadinene-8-hydroxylase, a cytochrome P450 monooxygenase (CYP706B1)

Several lines of evidence indicate that (+)-δ-cadinene-8-hydroxylase (CYP706B1) plays an important role in biosynthesis of gossypol in Gossypium arboreum L. ( Luo et al., 2001; Wang et al., 2003). The catalytically active enzyme has been expressed in yeast microsomes. Some microsomal preparations conjugated the hydroxylated (+)-δ-cadinene to a moiety that has not yet been identified. However, when microsomes were treated with n-octyl-β- d-glucoside (OG), a non-ionic detergent, (+)-δ-cadinene was reproducibly converted to the free alcohol, 8-hydroxy-(+)-δ-cadinene. OG had little effect on K m and slightly stimulated apparent V max. Enzymic activity was more than 10-fold more sensitive to inhibition by the N-substituted imidazole clotrimazole than to miconazole. Sesquiterpene olefins (−)-δ-cadinene, (−)-α-cubebene, (−)-α-muurolene, α-humulene, and a mixture of (−)- and (+)-α-copaene were inhibitory to hydroxylation of (+)-δ-cadinene. In addition, (−)-α-cubebene, (−)-α-muurolene, α-humulene, and, to a smaller extent, (−)-δ-cadinene served as alternative substrates for (+)-δ-cadinene-8-hydroxylase and were converted to mono-hydroxylated products. Of the five olefins tested, α-humulene and α-copaene are found in lysigenous glands of cotton ( Elzen et al., 1985), which are also the site of gossypol accumulation ( Bell et al., 1978; Mace et al., 1976) and the probable site of its biosynthesis.

A distinct DGAT with sn -3 acetyltransferase activity that synthesizes unusual, reduced-viscosity oils in Euonymus and transgenic seeds

Endosperm and embryo tissues from the seeds of Euonymus alatus (Burning Bush) accumulate high levels of 3-acetyl-1,2-diacyl-sn-glycerols (acTAGs) as their major storage lipids. In contrast, the aril tissue surrounding the seed produces long-chain triacylglycerols (lcTAGs) typical of most other organisms. The presence of the sn-3 acetyl group imparts acTAGs with different physical and chemical properties, such as a 30% reduction in viscosity, compared to lcTAGs. Comparative transcriptome analysis of developing endosperm and aril tissues using pyrosequencing technology was performed to isolate the enzyme necessary for the synthesis of acTAGs. An uncharacterized membrane-bound O-acyltransferase (MBOAT) family member was the most abundant acyltransferase in the endosperm but was absent from the aril. Expression of this MBOAT in yeast resulted in the accumulation of acTAGs but not lcTAG; hence, the enzyme was named EaDAcT (Euonymus alatus diacylglycerol acetyltransferase). Yeast microsomes expressing EaDAcT possessed acetyl-CoA diacylglycerol acetyltransferase activity but lacked long-chain acyl-CoA diacylglycerol acyltransferase activity. Expression of EaDAcT under the control of a strong, seed-specific promoter in Arabidopsis resulted in the accumulation of acTAGs, up to 40 mol % of total TAG in the seed oil. These results demonstrate the utility of deep transcriptional profiling with multiple tissues as a gene discovery strategy for low-abundance proteins. They also show that EaDAcT is the acetyltransferase necessary and sufficient for the production of acTAGs in Euonymus seeds, and that this activity can be introduced into the seeds of other plants, allowing the evaluation of these unusual TAGs for biofuel and other applications.

Cloning and Expression in Pichia pastoris of a New Cytochrome P450 Gene from a Dandruff-causing Malassezia globosa

The Malassezia fungi are responsible for various human skin disorders including dandruff and seborrheic dermatitis. Of the Malassezia fungi, Malassezia globosa (M. globosa) is one of the most common in human scalp. The completed genome sequence of M. globosa contains four putative cytochrome P450 genes. To determine the roles of Malassezia P450 enzymes in the biosynthesis of ergosterol, we isolated MGL3996 gene from M. globosa chromosomal DNA by PCR. The MGL3996 gene encodes an enzyme of 616 amino acids, which shows strong similarity with known CYP52s of other species. MGL3996 gene was cloned and expressed in Pichia pastoris (P. pastoris) heterologous yeast expression system. Using the yeast microsomes expressing MGL3996 protein, a typical P450 CO-difference spectrum was shown with absorption maximum at 448 nm. SDS-PAGE analysis revealed a protein band of apparent molecular weight 69 kDa and Western blot with anti-histidine tag antibody showed that MGL3996 was successfully expressed in P. pastoris. Cloning and expression of a new P450 gene is an important step to study the P450 monooxygenase system of M. globosa and to understand the role of P450 enzymes in pathophysiology of dandruff.

Nanoridges that characterize the surface morphology of flowers require the synthesis of cutin polyester.

Distinctive nanoridges on the surface of flowers have puzzled plant biologists ever since their discovery over 75 years ago. Although postulated to help attract insect pollinators, the function, chemical nature, and ontogeny of these surface nanostructures remain uncertain. Studies have been hampered by the fact that no ridgeless mutants have been identified. Here, we describe two mutants lacking nanoridges and define the biosynthetic pathway for 10,16-dihydroxypalmitate, a major cutin monomer in nature. Using gene expression profiling, two candidates for the formation of floral cutin were identified in the model plant Arabidopsis thaliana: the glycerol-3-phosphate acyltransferase 6 (GPAT6) and a member of a cytochrome P450 family with unknown biological function (CYP77A6). Plants carrying null mutations in either gene produced petals with no nanoridges and no cuticle could be observed by either scanning or transmission electron microscopy. A strong reduction in cutin content was found in flowers of both mutants. In planta overexpression suggested GPAT6 preferentially uses palmitate derivatives in cutin synthesis. Comparison of cutin monomer profiles in knockouts for CYP77A6 and the fatty acid omega-hydroxylase CYP86A4 provided genetic evidence that CYP77A6 is an in-chain hydroxylase acting subsequently to CYP86A4 in the synthesis of 10,16-dihydroxypalmitate. Biochemical activity of CYP77A6 was demonstrated by production of dihydroxypalmitates from 16-hydroxypalmitate, using CYP77A6-expressing yeast microsomes. These results define the biosynthetic pathway for an abundant and widespread monomer of the cutin polyester, show that the morphology of floral surfaces depends on the synthesis of cutin, and identify target genes to investigate the function of nanoridges in flower biology.

Nicastrin Is Dispensable for γ-Secretase Protease Activity in the Presence of Specific Presenilin Mutations

γ-Secretase is a multisubunit membrane protein complex consisting of presenilin (PS1), nicastrin (NCT), anterior pharynx-1, and presenilin enhancer 2. To analyze the activity of familial Alzheimer disease mutants and to understand the roles of the subunits, we established a yeast transcriptional activator Gal4p system with artificial γ-secretase substrates containing amyloid precursor protein or Notch fragments. The γ-secretase activities were evaluated by transcriptional activation of reporter genes upon Gal4p release from the membrane-bound substrates, i.e. growth of yeast on histidine and adenine, or β-galactosidase assay. We screened and evaluated γ-secretase mutants using this reconstitution system in yeast, which does not possess endogenous γ-secretase activity. When we introduced familial Alzheimer mutants of PS1 in this system, their activities were shown to be loss of function. Although the protease activity of wild type PS1 depends on the other three subunits introduced, we obtained 15 new PS1 mutants, which are active in the absence of NCT. They possessed a S438P mutation at the ninth transmembrane domain (TM9) together with one missense mutation distributed through transmembrane and loop regions. These mutations were not related to familial Alzheimer mutations of PS1 as identified so far. The S438P mutant was partially active but required other mutations for full activation. Results of the β-galactosidase assay suggested that they have wild type protease activities, which were further confirmed by the endoproteolysis of PS1, amyloid β peptides, and Notch intracellular domain production in mammalian cells. These results suggest that NCT is dispensable for the protease activity of γ-secretase.

Molecular Cloning and Characterization of a cDNA for Pterocarpan 4-Dimethylallyltransferase Catalyzing the Key Prenylation Step in the Biosynthesis of Glyceollin, a Soybean Phytoalexin

Glyceollins are soybean (Glycine max) phytoalexins possessing pterocarpanoid skeletons with cyclic ether decoration originating from a C5 prenyl moiety. Enzymes involved in glyceollin biosynthesis have been thoroughly characterized during the early era of modern plant biochemistry, and many genes encoding enzymes of isoflavonoid biosynthesis have been cloned, but some genes for later biosynthetic steps are still unidentified. In particular, the prenyltransferase responsible for the addition of the dimethylallyl chain to pterocarpan has drawn a large amount of attention from many researchers due to the crucial coupling process of the polyphenol core and isoprenoid moiety. This study narrowed down the candidate genes to three soybean expressed sequence tag sequences homologous to genes encoding homogentisate phytyltransferase of the tocopherol biosynthetic pathway and identified among them a cDNA encoding dimethylallyl diphosphate: (6aS, 11aS)-3,9,6a-trihydroxypterocarpan [(-)-glycinol] 4-dimethylallyltransferase (G4DT) yielding the direct precursor of glyceollin I. The full-length cDNA encoding a protein led by a plastid targeting signal sequence was isolated from young soybean seedlings, and the catalytic function of the gene product was verified using recombinant yeast microsomes. Expression of the G4DT gene was strongly up-regulated in 5 to 24 h after elicitation of phytoalexin biosynthesis in cultured soybean cells similarly to genes associated with isoflavonoid pathway. The prenyl part of glyceollin I was demonstrated to originate from the methylerythritol pathway by a tracer experiment using [1-(13)C]Glc and nuclear magnetic resonance measurement, which coincided with the presumed plastid localization of G4DT. The first identification of a pterocarpan-specific prenyltransferase provides new insights into plant secondary metabolism and in particular those reactions involved in the disease resistance mechanism of soybean as the penultimate gene of glyceollin biosynthesis.

CDNA cloning and functional characterisation of CYP98A14 and NADPH:cytochrome P450 reductase from Coleus blumei involved in rosmarinic acid biosynthesis

The final reactions of rosmarinic acid biosynthesis, the introduction of the aromatic 3- and 3'-hydroxyl groups, are catalysed by cytochrome P450-dependent hydroxylases. The cDNAs encoding CYP98A14 as well as a NADPH:cytochrome P450 reductase (CPR) were isolated from Coleus blumei and actively expressed in Saccharomyces cerevisiae. The CYP98A14-cDNA showed an open reading frame of 1521 nucleotides with high similarities to 4-coumaroylshikimate/quinate 3-hydroxylases. Yeast microsomes harbouring the CYP98A14 protein catalysed the 3-hydroxylation of 4-coumaroyl-3',4'-dihydroxyphenyllactate and the 3'-hydroxylation of caffeoyl-4'-hydroxyphenyllactate, in both cases forming rosmarinic acid. Apparent K (m)-values for 4-coumaroyl-3',4'-dihydroxyphenyllactate and caffeoyl-4'-hydroxyphenyllactate were determined to be at 5 microM and 40 microM, respectively. CYP98A14 differs from CYP98s from other plants, since 4-coumaroylshikimate or -quinate were not accepted as substrates. Coexpression of the Coleus blumei CPR and CYP98A14 in the same yeast cells increased the hydroxylation activity up to sevenfold. CYP98A14 from Coleus blumei is a novel bifunctional cytochrome P450 specialised for rosmarinic acid biosynthesis.

In vitro reconstitution of γ-secretase activity using yeast microsomes

γ-Secretase is composed of at least four transmembrane proteins, presenilin (PS) 1/2, nicastrin, anterior pharynx-1 (Aph-1) and presenilin enhancer-2 (Pen-2), and cleaves amyloid precursor protein (APP) to produce amyloid β peptides (Aβ) that is deposited in the brains of Alzheimer disease. However, the mechanism of γ-secretase-mediated cleavage remains unclear. To examine the enzymatic properties of γ-secretase, we established an in vitro assay system using Saccharomyces cerevisiae, which does not possess homologs of human PS1/2, nicastrin, Aph-1, or Pen-2. We transformed these subunits and the substrate in pep4Δ cells with vacuole proteases inactivated, and microsome was isolated for in vitro assay. In the assay, Aβ40, Aβ42, and Aβ43 were produced with an optimal pH of ∼7.0. We also detected Aβ-production by yeast endogenous protease(s), which was abolished by the addition of phosphatidyl choline. This novel system will facilitate the analysis of substrate recognition by γ-secretase.

Identification of a novel GPCAT activity and a new pathway for phosphatidylcholine biosynthesis in S. cerevisiae

Turnover of phospholipids in the yeast Saccharomyces cerevisiae generates intracellular glycerophosphocholine (GPC). Here we show that GPC can be reacylated in an acyl-CoA-dependent reaction by yeast microsomal membranes. The lysophosphatidylcholine that is formed in this reaction is efficiently further acylated to phosphatidylcholine (PC) by yeast microsomes, thus providing a new pathway for PC biosynthesis that can either recycle endogenously generated GPC or utilize externally provided GPC. Genetic and biochemical evidence suggests that this new enzymatic activity, which we call GPC acyltransferase (GPCAT), is not mediated by any of the previously known acyltransferases in yeast. The GPCAT activity has an apparent V(max) of 8.7 nmol/min/mg protein and an apparent K(m) of 2.5 mM. It has a neutral pH optimum, similar to yeast glycerol-3-phosphate acyltransferase, but differs from the latter in being more heat stable. The GPCAT activity is sensitive to N-ethylmaleimide, phenanthroline, and Zn(2+) ions. In vivo experiments showed that PC is efficiently labeled when yeast cells are fed with [(3)H]choline-GPC, and that this reaction occurs also in pct1 knockout strains, where de novo synthesis of PC by the CDP-choline pathway is blocked. This suggests that GPCAT can provide an alternative pathway for PC biosynthesis in vivo.

Molecular cloning of CYP76A3, a novel cytochrome P450 from Petunia hybrida catalyzing the ω-hydroxylation of myristic acid

In higher plants, fatty acid hydroperoxides are intermediates in the synthesis of a diverse group of bioactive compounds. We used the reverse-transcriptase polymerase chain reaction to isolate a gene responsible for the oxidization of fatty acids from Petunia hybrida. A P450 cDNA that has not previously been isolated (CYP76A3) contained an open reading frame predicted to encode a polypeptide consisting of 507 amino acid residues. The cyp76A3 cDNA was expressed in Saccharomyces cerevisiae AH22 cells under the control of an alcohol dehydrogenase promoter and terminator. The recombinant yeast microsome containing the CYP76A3 hemoprotein was found to specifically catalyze ω-hydroxylation of myristic acid. A high level of the transcripts of the cyp76A3 gene was found in the leaves and roots of P. hybrida, but not in the stems and flowers.

Characterization of a methyl jasmonate and wounding‐responsive cytochrome P450 of Arabidopsis thaliana catalyzing dicarboxylic fatty acid formation in vitro

A fatty-acid-metabolizing enzyme from Arabidopsis thaliana, CYP94C1, belonging to the cytochrome P450 family was cloned and characterized. CYP94C1 was heterologously expressed in a Saccharomyces cerevisiae strain (WAT11) engineered for P450 expression. When recombinant yeast microsomes were incubated with lauric acid (C12:0) for 15 min, one major metabolite was formed. The product was purified and identified by GC/MS as 12-hydroxylauric acid. Longer incubation (40 min) led to the formation of an additional metabolite identified by GC/MS as dodecadioic acid. This diacid was also produced by incubation with 12-hydroxylauric acid. These compounds were not produced by incubating microsomes from yeast transformed with a void plasmid, demonstrating the involvement of CYP94C1. This new enzyme also metabolized fatty acids of varying aliphatic chain lengths (C12 to C18) and in-chain modifications, for example, degree of unsaturation or the presence of an epoxide as an additional polar functional group. Transcription of the gene encoding CYP94C1 is enhanced by stress, treatment with the hormone methyl jasmonate and wounding. Treatment with methyl jasmonate also induced lauric acid metabolism in microsomes prepared from Arabidopsis. The induction of hydroxylase activity was dose dependent and increased with exposure time, reaching 16x higher in microsomes from 24-h treated Arabidopsis compared with control plants. Analysis of the metabolites showed a mixture of 12-, 11- and 10-hydroxylauric acids, revealing for the first time the presence of fatty acid in-chain hydroxylase in Arabidopsis.

Molecular Characterization of Specifically Active Recombinant Fused Enzymes Consisting of CYP3A4, NADPH-Cytochrome P450 Oxidoreductase, and Cytochrome b5

Microsomal cytochrome P450 3A4 (CYP3A4) catalyzes monooxygenase reactions toward a diverse group of exogenous and endogenous substrates and requires cytochrome b5 (b5) in the oxidation of the typical substrate testosterone. To analyze the molecular interaction among CYP3A4, NADPH-cytochrome P450 oxidoreductase (P450 reductase), and b5, we constructed several fused enzyme genes and expressed them in Saccharomyces cerevisiae. The recombinant fused enzymes CYP3A4-truncated (t)-P450 reductase-t-b5 (3RB) and CYP3A4-t-b5-t-P450 reductase (3BR) in yeast microsomes showed a higher specific activity in 6beta-hydroxylation of testosterone than did the reconstitution premixes of CYP3A4, P450 reductase, and b5. The purified fused enzymes exhibited lower Km values and substantially increased Vmax values in 6beta-hydroxylation of testosterone and oxidation of nifedipine. Moreover, the fused enzymes showed significantly higher activities in cytochrome c reduction than the reconstitution premixes. Although the affinity of 3RB toward cytochrome c was twice as high as that of 3BR, 3BR and 3RB showed nearly the same affinity toward NADPH/NADH. In addition, the heme of the CYP3A4 moiety of 3RB was reduced preferentially and more rapidly than that of 3BR, whereas the heme of the b5 moiety of 3BR was selectively reduced compared with that of 3RB. These results suggest that the conformation of the 3RB molecule was the most suitable for high activity because of appropriate ordering of the CYP3A4, P450 reductase, and b5 moieties for efficient electron flow. Thus, we believe that the b5 moiety plays an important role in the efficient transfer of the second electron in the vicinity of the CYP3A4 moiety.

Variety of Nucleotide Sugar Transporters with Respect to the Interaction with Nucleoside Mono- and Diphosphates

Nucleotide sugar transporters have long been assumed to be antiporters that exclusively use nucleoside monophosphates as antiport substrates. Here we present evidence indicating that two other types of nucleotide sugar transporters exist that differ in their antiport substrate specificity. Biochemical studies using microsomes derived from Saccharomyces cerevisiae cells expressing either human (h) UGTrel7 or the Drosophila (d) FRC (Fringe connection) transporter revealed that (i) efflux of preloaded UDP-glucuronic acid from the yeast microsomes expressing hUGTrel7 was strongly enhanced by UDP-GlcNAc added in the external medium, but not by UMP or UDP, suggesting that hUGTrel7 may be described as a UDP-sugar/UDP-sugar antiporter, and (ii) addition of UDP-sugars, UDP, or UMP in the external medium stimulated the efflux of preloaded UDP-GlcNAc from the yeast microsomes expressing dFRC to a comparable extent, suggesting that UDP, as well as UMP, may serve as an antiport substrate of dFRC. Antiport of UDP-sugars with these specific substrates was reproduced and definitively confirmed using proteoliposomes reconstituted from solubilized and purified transporters. Possible physiological implications of these observations are discussed.

Flip-Flop of Fluorescently Labeled Phospholipids in Proteoliposomes Reconstituted with Saccharomyces cerevisiae Microsomal Proteins

A phospholipid flippase activity from the endoplasmic reticulum (ER) of the model organism Saccharomyces cerevisiae has been characterized and functionally reconstituted into proteoliposomes. Analysis of the transbilayer movement of acyl-7-nitrobenz-2-oxa-1,3-diazol-4-yl (acyl-NBD)-labeled phosphatidylcholine in yeast microsomes using a fluorescence stopped-flow back exchange assay revealed a rapid, ATP-independent flip-flop (half-time, <2 min). Proteoliposomes prepared from a Triton X-100 extract of yeast microsomal membranes were also capable of flipping NBD-labeled phospholipid analogues rapidly in an ATP-independent fashion. Flippase activity was sensitive to the protein modification reagents N-ethylmaleimide and diethylpyrocarbonate. Resolution of the Triton X-100 extract by velocity gradient centrifugation resulted in the identification of a approximately 4S protein fraction enriched in flippase activity as well as of other fractions where flippase activity was depleted or undetectable. We estimate that flippase activity is due to a protein(s) representing approximately 2% (wt/wt) of proteins in the Triton X-100 extract. These results indicate that specific proteins are required to facilitate ATP-independent phospholipid flip-flop in the ER and that their identification is feasible. The architecture of the ER protein translocon suggests that it could account for the flippase activity in the ER. We tested this hypothesis using microsomes prepared from a temperature-sensitive yeast mutant in which the major translocon component, Sec61p, was quantitatively depleted. We found that the protein translocon is not required for transbilayer movement of phospholipids across the ER. Our work defines yeast as a promising model system for future attempts to identify the ER phospholipid flippase and to test and purify candidate flippases.

Inactivation of CYP2D6 by methylenedioxymethamphetamine in different recombinant expression systems

Recombinantly expressed CYP450 systems (rCYPs) are often used to screen for irreversible/quasi-irreversible enzyme inhibitors during drug development. The concentration- and time-dependent inactivation of CYP2D6 by methylenedioxymethamphetamine (MDMA) was compared in three different rCYP2D6 systems (yeast microsomes, Supersomes™ and Bactosomes™) under the conditions of the most commonly used protocols in assessing mechanism-based inactivation (MBI). MDMA (2–20 μM) was pre-incubated with enzyme for 0, 2.5 and 5 min followed by a five-fold dilution and further incubation with dextromethorpan (DEX) (50 μM). The formation of dextrorphan (DOR) from DEX was used as a specific marker of CYP2D6 activity. Concentration- and time-dependent inactivation of CYP2D6 by MDMA was observed with each rCYP system. However, the apparent kinetic parameters for MBI ( k inact, the maximum inactivation rate constant and K I, the inhibitor concentration associated with half maximal rate of inactivation) were significantly greater ( p < 0.05) for Bactosomes™ (0.95 ± 0.33 min −1, 42.9 ± 20.1 μM) than those found using yeast microsomes (0.28 ± 0.04 min −1, 2.86 ± 1.18 μM) and Supersomes™ (0.38 ± 0.05 min −1, 3.66 ± 0.10 μM). After correction for depletion of MDMA during pre-incubation, k inact and K I values determined using Bactosomes™ decreased significantly but remained higher than for the other rCYP systems ( p < 0.05). Substantial metabolism of DOR after its formation from DEX was also observed using Supersomes™ and Bactosomes™. Sub-optimal study design when investigating MBI may compromise the quantitative characterization of inhibitory characteristics using some rCYP systems.

Selective Substrate Supply in the Regulation of Yeast de Novo Sphingolipid Synthesis

The heat stress response of Saccharomyces cerevisiae is characterized by transient cell cycle arrest, altered gene expression, degradation of nutrient permeases, trehalose accumulation, and translation initiation of heat shock proteins. Importantly heat stress also induces de novo sphingolipid synthesis upon which many of these subprograms of the heat stress response depend. Despite extensive data addressing the roles for sphingolipids in heat stress, the mechanism(s) by which heat induces sphingolipid synthesis remains unknown. This study was undertaken to determine the events and/or factors required for heat stress-induced sphingolipid synthesis. Data presented indicate that heat does not directly alter the in vitro activity of serine palmitoyltransferase (SPT), the enzyme responsible for initiating de novo sphingolipid synthesis. Moreover deletion of the small peptide Tsc3p, which is thought to maximize SPT activity, specifically reduced production of C(20) sphingolipid species by over 70% but did not significantly decrease overall sphingoid base production. In contrast, the fatty-acid synthase inhibitor cerulenin nearly completely blocked sphingoid base production after heat, indicating a requirement for endogenous fatty acids for heat-mediated sphingoid base synthesis. Consistent with this, genetic studies show that fatty acid import does not contribute to heat-induced de novo synthesis under normal conditions. Interestingly the absence of medium serine also ameliorated heat-induced sphingoid base production, indicating a requirement for exogenous serine for the response, and consistent with this finding, disruption of synthesis of endogenous serine did not affect heat-induced sphingolipid synthesis. Serine uptake assays indicated that heat increased serine uptake from medium by 100% during the first 10 min of heat stress. Moreover treatments that increase serine uptake in the absence of heat including acute medium acidification and glucose treatment also enhanced de novo sphingoid base synthesis equivalent to that induced by heat stress. These data agree with findings from mammalian systems that availability of substrates is a key determinant of flux through sphingolipid synthesis. Moreover data presented here indicate that SPT activity can be driven by several factors that increase serine uptake in the absence of heat. These findings may provide insights into the many systems in which de novo synthesis is increased in the absence of elevated in vitro SPT activity.

Characterization of the Novel DefectiveCYP2C9*24Allele

CYP2C9 is one of the major drug-metabolizing enzymes, and it is involved in the oxidative metabolism of approximately 10% of clinically important drugs, among which some, such as the anticoagulant warfarin, have a narrow therapeutic index. The human CYP2C9 gene is highly polymorphic. We found a new sequence variation in exon 7 of the CYP2C9 gene (1060G>A) resulting in a substitution of acidic amino acid glutamate to basic lysine (E354K) when translated. The allele, designated CYP2C9(*)24, was present in heterozygous state in one warfarin-treated patient. To characterize the CYP2C9(*)24 allele, we expressed the wild-type and CYP2C9.24 protein in a recombinant yeast expression system and a human embryonic kidney (HEK)-293 cell system. Carbon monoxide difference spectra were recorded on dithionite-reduced microsomes, and protein was determined by Western blotting. Transfection with CYP2C9.1 cDNA resulted in detectable CYP2C9 protein in yeast or HEK-293 cells, whereas only small amounts of the protein were detected in yeast transfected with CYP2C9.24 cDNA. A strong differential absorption peak at 450 nm was observed with microsomes of yeast transfected with CYP2C9.1 cDNA, whereas no peak was detected with microsomes of yeast transfected with CYP2C9.24 cDNA or empty pYeDP60 plasmid. These results suggest that CYP2C9.24 may be improperly folded, both in yeast and mammalian cells, resulting in improper heme incorporation and rapid intracellular degradation. The data obtained in the expression systems are consistent with our findings in vivo. In conclusion, we have identified a novel defective CYP2C9 variant allele of potential importance for drug metabolism in vivo.

Molecular Cloning of CYP76B9, a Cytochrome P450 fromPetunia hybrida, Catalyzing the ω-Hydroxylation of Capric Acid and Lauric Acid

A cDNA encoding a cytochrome P450 (CYP76B9) was isolated from Petunia hybrida. Northern blot analysis revealed preferential expression of the gene in flowers and leaves. The recombinant yeast microsomes expressing CYP76B9 was allowed to react with capric acid and lauric acid as substrates. One major metabolite was produced from each fatty acid after incubation with yeast microsomes expressing CYP76B9. The metabolites were identified by gas chromatography-mass spectrometry (GC-MS) as omega-hydroxy capric acid and omega-hydroxy lauric acid. The kinetic parameters of the reactions were Km=9.4 microM and Vmax=13.6 mol min(-1) per mol of P450 for capric acid, and Km=5.7 microM and Vmax=19.1 mol min(-1) per mol of P450 for lauric acid. We found that the omega-hydroxy metabolites of capric acid and lauric acid can affect the plant growth of Arabidopsis thaliana. Plants grown in the presence of omega-hydroxy fatty acids exhibited shorter root length than control plants with the corresponding non-hydroxylated fatty acids.