BBA - Molecular and Cell Biology of Lipids (v.1738, #1-3)

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Sterol Regulatory Element Binding Proteins (SREBP) are transcription factors that regulate lipid synthesis. We have shown recently that the human CASP-2 gene, encoding procaspase-2, was under the positive control of SREBP-2 that transactivates several responsive elements in the promoter region. We describe here the function of an additional SREBP-responsive element located in the first intron. Remarkably, this site is uniquely responsive to SREBP-1c that is mainly involved in fatty acids synthesis. This observation, together with our recent findings, strengthens the notion that the CASP-2 gene belongs to the SREBP-responsive gene battery in human cells.
Keywords: Human caspase-2; SREBP; Lipid; Transcriptional regulation; Intron;

Direct evidence in vivo of impaired macrophage-specific reverse cholesterol transport in ATP-binding cassette transporter A1-deficient mice by Laura Calpe-Berdiel; Noemi Rotllan; Xavier Palomer; Vicent Ribas; Francisco Blanco-Vaca; Joan Carles Escolà-Gil (6-9).
The ATP-binding cassette transporter A1 (ABCA1) is a key regulator of high-density lipoprotein (HDL) metabolism. There is strong evidence that ABCA1 is a key regulator of reverse cholesterol transport (RCT). However, this could not be proved in vivo since hepatobiliary cholesterol transport was unchanged in ABCA1-deficient mice (ABCA1−/−). We used ABCA1−/− mice to test the hypothesis that ABCA1 is a critical determinant of macrophage-specific RCT. Although this cell-specific RCT only accounts for a tiny part of total RCT, it is widely accepted that it may have a major impact on atherosclerosis susceptibility. [3H]cholesterol-labeled endogenous macrophages were injected intraperitoneally into wild-type ABCA1+/+, ABCA1+/− and ABCA1−/− mice maintained on a chow diet. A direct relationship was observed between ABCA1 gene dose and plasma [3H]cholesterol at 24 and 48 h after the injection of tracer into the mice. Forty-eight hours after this injection, ABCA1−/− mice had significantly reduced [3H]cholesterol in liver (2.8-fold), small intestine enterocytes (1.7-fold) and feces (2-fold). To our knowledge, this is the first direct in vivo quantitative evidence that ABCA1 is a critical determinant of macrophage-specific RCT.
Keywords: ATP-binding cassette transporter A1; High-density lipoprotein; Macrophage; Reverse cholesterol transport;

Previously, we have shown, in vivo, that the acyl coenzyme A: cholesterol acyltransferase (ACAT) inhibitor avasimibe decreases hepatic apolipoprotein (apo) B secretion into plasma. To test the hypothesis that avasimibe modulates postprandial triglyceride-rich lipoprotein (TRL) metabolism in vivo, an oral fat load (2 g fat/kg) containing retinol was given to 9 control miniature pigs and to 9 animals after 28 days treatment with avasimibe (10 mg/kg/day, n  = 5; 25 mg/kg/day, n  = 4). The kinetic parameters for plasma retinyl palmitate (RP) metabolism were determined by multi-compartmental modeling using SAAM II. Avasimibe decreased the 2-h TRL (d  < 1.006 g/mL; S f  > 20) triglyceride concentrations by 34%. The TRL triglyceride 0–12 h area under the curve (AUC) was decreased by 21%. In contrast, avasimibe had no effect on peak TRL RP concentrations, time to peak, or its rate of appearance into plasma, however, the TRL RP 0–12 h AUC was decreased by 17%. Analysis of the RP kinetic parameters revealed that the TRL fractional clearance rate (FCR) was increased 1.4-fold with avasimibe. The TRL RP FCR was negatively correlated with very low density lipoprotein (VLDL) apoB production rate measured in the fasting state (r  = −0.504). No significant changes in total intestinal lipid concentrations were observed. Thus, although avasimibe had no effect on intestinal TRL secretion, plasma TRL clearance was significantly increased; an effect that may relate to a decreased competition with hepatic VLDL for removal processes.
Keywords: ACAT inhibitor; Avasimibe; Triglyceride-rich lipoprotein; Tracer kinetic; Compartmental model;

Effects of arachidonic acid analogs on FcεRI-mediated activation of mast cells by Nobuhiro Nakano; Atsuhito Nakao; Takafumi Uchida; Norifumi Shirasaka; Hajime Yoshizumi; Ko Okumura; Ryoji Tsuboi; Hideoki Ogawa (19-28).
Polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA) have been shown to modulate a number of inflammatory disorders. Mast cells play a critical role in the initiation and maintenance of inflammatory responses. However, the effects of PUFAs on mast cell functions have not been fully addressed. We here–in examined the effects of PUFAs on the high affinity IgE receptor (FcεRI)–mediated mast cell activation using RBL-2H3 cells, a rat mast cell line, that were cultured in the medium containing palmitic acid (PA), AA, or the AA analogs mead acid (MA) and eicosapentaenoic acid (EPA). In AA-supplemented cells, the FcεRI-mediated β-hexosamidase and TNF-α release, calcium (Ca2+) influx, and some protein tyrosine phosphorylations including Syk and linker for activation of T cells (LAT) were enhanced, whereas, in MA- or PA-supplemented cells, they were not changed when compared with cells cultured in control medium. In EPA-supplemented cells, the enhancements of β-hexosamidase release and protein tyrosine phosphorylations were observed. Furthermore, in AA- or EPA-supplemented cells, FcεRI-mediated intracellular production of reactive oxygen species (ROS) that is required for the tyrosine phosphorylation of LAT and Ca2+ influx were enhanced when compared with the other cells. Thus, preincubation of AA or EPA augmented FcεRI-mediated degranulation in mast cells by affecting early events of FcεRI signal transduction, which might be associated with the change of fatty acid composition of the cell membrane and enhanced production of ROS. The results suggest that some PUFAs can modulate FcεRI-mediated mast cell activation and might affect FcεRI/mast cell-mediated inflammation, such as allergic reaction.
Keywords: Arachidonic acid; Eicosapentaenoic acid; Mast cell; Degranulation; Signal transduction; Reactive oxygen specie;

Substrate specificity and kinetic properties of enzymes belonging to the hormone-sensitive lipase family: Comparison with non-lipolytic and lipolytic carboxylesterases by Henri Chahinian; Yassine Ben Ali; Abdelkarim Abousalham; Stefan Petry; Luigi Mandrich; Guiseppe Manco; Stephane Canaan; Louis Sarda (29-36).
We have studied the kinetics of hydrolysis of triacylglycerols, vinyl esters and p-nitrophenyl butyrate by four carboxylesterases of the HSL family, namely recombinant human hormone-sensitive lipase (HSL), EST2 from Alicyclobacillus acidocaldarius, AFEST from Archeoglobus fulgidus, and protein RV1399C from Mycobacterium tuberculosis. The kinetic properties of enzymes of the HSL family have been compared to those of a series of lipolytic and non-lipolytic carboxylesterases including human pancreatic lipase, guinea pig pancreatic lipase related protein 2, lipases from Mucor miehei and Thermomyces lanuginosus, cutinase from Fusarium solani, LipA from Bacillus subtilis, porcine liver esterase and Esterase A from Aspergilus niger. Results indicate that human HSL, together with other lipolytic carboxylesterases, are active on short chain esters and hydrolyze water insoluble trioctanoin, vinyl laurate and olive oil, whereas the action of EST2, AFEST, protein RV1399C and non-lipolytic carboxylesterases is restricted to solutions of short chain substrates. Lipolytic and non-lipolytic carboxylesterases can be differentiated by their respective value of K 0.5 (apparent K m) for the hydrolysis of short chain esters. Among lipolytic enzymes, those possessing a lid domain display higher activity on tributyrin, trioctanoin and olive oil suggesting, then, that the lid structure contributes to enzyme binding to triacylglycerols. Progress reaction curves of the hydrolysis of p-nitrophenyl butyrate by lipolytic carboxylesterases with lid domain show a latency phase which is not observed with human HSL, non-lipolytic carboxylesterases, and lipolytic enzymes devoid of a lid structure as cutinase.
Keywords: Carboxylesterase; EST2; AFEST; Hormone-sensitive lipase; HSL family; Kinetic study;

GC-rich sequences in the 5-lipoxygenase gene promoter are required for expression in Mono Mac 6 cells, characterization of a novel Sp1 binding site by David Dishart; Nicole Schnur; Niko Klan; Oliver Werz; Dieter Steinhilber; Bengt Samuelsson; Olof Rådmark (37-47).
5-lipoxygenase (5LO) catalyzes formation of leukotrienes, mediators with roles in several inflammatory disorders, including atherosclerosis. The human 5LO gene promoter contain multiple GC-boxes. The relevance of these for expression of 5LO in the human monocytic cell line Mono Mac 6 (MM6) was studied. A downregulating effect of the GC-box binding compound mithramycin indicated that GC-rich sequences in the 5LO gene promoter are important for expression of native 5LO. In DNase I footprinting, mithramycin and Sp1 protected known GC-boxes, but also a novel Sp1 binding site was found, comprising 20 bp upstream of the major transcription initiation site, beside an Initiator-like sequence. Mutation of this site reduced Sp1 binding and expression of reporter genes in MM6 cells, compatible with a function as basal promoter element for the TATA-less 5LO gene. When differentiation was induced by TGFβ and vitamin D3, 5LO expression became prominent, but expression levels of Sp1/3 and Egr-1 were the same as for control cells. Also, 5LO reporter gene activity in transiently transfected MM6 cells was insensitive to differentiation. Thus, the GC-rich part of the 5LO gene promoter, including a novel Sp1 site, appear important for basal (rather than upregulated) transcription of 5LO in MM6 cells.
Keywords: Leukotriene; Eicosanoid; Arachidonic acid; Egr-1; Sp1;

Fenofibrate reverses the decline in HDL cholesterol in mice overexpressing human phospholipid transfer protein by Jessica Lie; Inge M. Lankhuizen; Barbara Gross; Teus van Gent; Rien van Haperen; Leo Scheek; Bart Staels; Rini de Crom; Arie van Tol (48-53).
In humans, fibrates are used to treat dyslipidemia, because these drugs lower plasma triglycerides and raise HDL cholesterol. Treatment with fibrates lowers plasma phospholipid transfer protein (PLTP) activity in humans, but increases PLTP activity in mice, without a consistent effect on HDL-cholesterol concentration. Earlier, we found that PLTP overexpression in transgenic mice results in decreased plasma HDL levels and increased diet-induced atherosclerosis. So it seems that the interplay between fibrates, PLTP and HDL is different in mice and man, which may be important for atherosclerosis development. In the present study, we measured the effects of fibrates on PLTP expression in cultured human hepatocytes and effects of fibrate treatment on human PLTP expression, plasma PLTP activity and HDL levels in human PLTP transgenic mice.Fibrate treatment did not influence PLTP mRNA levels in human hepatocytes. Hepatic human PLTP mRNA levels and PLTP activity were both moderately elevated by fenofibrate treatment in human PLTP transgenic mice. In wild-type mice, however, feeding fenofibrate resulted in a strong induction of PLTP mRNA in the liver and a more than 4-fold increase of plasma PLTP activity. Plasma triglycerides were reduced in all mice by 48% or more by fenofibrate treatment. HDL-cholesterol concentrations were substantially increased by fenofibrate in PLTP overexpressing mice (+72%), but unaffected in wild-type mice. We conclude that fenofibrate treatment reverses the HDL-lowering effect of PLTP overexpression in human PLTP transgenic mice.
Keywords: PLTP; Fenofibrate; HDL; Transgenic mouse; Human hepatocyte;

Transport by vesicles of glycine- and taurine-conjugated bile salts and taurolithocholate 3-sulfate: A comparison of human BSEP with rat Bsep by Hisamitsu Hayashi; Tappei Takada; Hiroshi Suzuki; Reiko Onuki; Alan F. Hofmann; Yuichi Sugiyama (54-62).
The bile salt export pump (BSEP) of hepatocyte secretes conjugated bile salts across the canalicular membrane in an ATP-dependent manner. The biliary bile salts of human differ from those of rat in containing a greater proportion of glycine conjugates and taurolithocholate 3-sulfate (TLC-S). In the present study, the transport properties of hBSEP and rBsep were investigated using membrane vesicles from HEK293 cells infected with recombinant adenoviruses containing hBSEP or rBsep cDNA. ATP-dependent uptake of radiolabeled glycine-, taurine-conjugated bile salts, and [3H]cholate was observed when hBSEP or rBsep was expressed. Comparison of initial uptake rates indicated that for both transporters, taurine-conjugated bile salts were transported more rapidly than glycine-conjugated bile salts, however, hBSEP transported glycine conjugates to an extent that was approximately 2-fold greater than rBsep. In addition, [3H]TLC-S was significantly transported by hBSEP, and hardly transported by rBsep. The mean K m value for the uptake of [3H]TLC-S by hBSEP was 9.5 ± 1.5 μM, a value similar to that for hMRP2 (8.2 ± 1.3 μM). In conclusion, both hBSEP and rBsep transport taurine-conjugated bile salts better than glycine-conjugated bile salts, but hBSEP transports glycine conjugates to a greater extent as compared to rBsep. TLC-S, which is present in human bile but not rodent bile, is more avidly transported by hBSEP compared with rBsep.
Keywords: Bile salt; ABC transporter; hBSEP/rBsep;

The microalga Parietochloris incisa (Trebouxiophyceae, Chlorophyta) was isolated from an alpine environment. It was found to accumulate unusually high amounts of arachidonic acid (AA)-rich TAG. We have hypothesized that microalgal PUFA-rich TAG might have a role as a depot of PUFA, which could be mobilized for the construction of chloroplastic membranes under sudden changes in environmental conditions. We have thus studied the changes in lipid and fatty acid composition during recovery from nitrogen starvation at 24 and 12 °C. At both temperatures, TAG was mainly consumed to support growth, however, there was a significant increase in the content of AA in the chloroplastic lipids, predominantly, monogalactosyldiacylglycerol (MGDG) at 24 °C, but much less so at 12 °C. Similar results were obtained using radiolabeled precursors. These and other findings point to the existence of three modes of operation for the construction of chloroplastic lipids that the alga can utilize to support growth under changing environmental conditions. When environmental conditions do not support growth, the prokaryotic pathway predominates. When sudden changes occur, the eukaryotic pathway is enhanced and can be even further augmented by influx of acyl moieties from TAG to maximize the exploitation of growth conditions that may possibly be transitory.
Keywords: Arachidonic acid; Microalgae; Parietochloris incisa; PUFA biosynthesis; Triacylglycerol;

Sphingosine 1-phosphate (S1P) the product of sphingosine kinase (SK) action plays an important role in various pathological conditions like inflammation and cancer. In this study, we show that in the human breast cancer cell line MCF7, epidermal growth factor (EGF) stimulates SK-1 activity in a biphasic manner with a first peak after 15 min and a second delayed activation occurring after 1 h up to 18 h and thereafter declining again. This delayed activation is accompanied by increased mRNA and protein expression of SK-1, but not SK-2. Mechanistically, the transcriptional upregulation is dependent on the classical mitogen-activated protein kinase, protein kinase C (PKC) and the phosphoinositide 3-kinase, since specific inhibitors of these enzymes all abolish the EGF-induced mRNA upregulation and activity of SK-1. Moreover, dexamethasone also suppressed EGF-induced SK-1 mRNA expression and activity which is reversed by the glucocorticoid receptor antagonist RU486. To see whether EGF-induced upregulation of SK-1 is of relevance for tumor progression, we investigated two hallmarks of carcinogenesis, i.e., cell proliferation and migration. Stimulation of cells with EGF leads to enhanced [3H]thymidine incorporation into DNA and also to stimulated migration in a modified Boyden chamber assay. When cells are depleted of SK-1, but not SK-2, by siRNA transfection or by dexamethasone treatment, EGF-induced proliferation and migration are drastically reduced. In summary, these data show that EGF causes an acute stimulation of SK-1 activity and, moreover, triggers a delayed SK activation which is due to increased gene transcription and de novo synthesis of SK-1, which in turn directs cells towards growth and increased motility. Thus, the sphingosine kinase-1 may represent a novel attractive target for cancer therapy.
Keywords: Epidermal growth factor; Sphingosine kinase-1; Sphingosine 1-phosphate; MCF7;

Suppression of mast cell degranulation by a novel ceramide kinase inhibitor, the F-12509A olefin isomer K1 by Jin-Wook Kim; Yuichi Inagaki; Susumu Mitsutake; Nobuhiro Maezawa; Shigeo Katsumura; Yeon-Woo Ryu; Chang-Seo Park; Masaru Taniguchi; Yasuyuki Igarashi (82-90).
Antigen-induced degranulation of mast cells plays a pivotal role in allergic and inflammatory responses. Recently, ceramide kinase (CERK) and its phosphorylated product ceramide 1-phosphate (C1P) have emerged as important players in mast cell degranulation. Here, we describe the synthesis of a novel F-12509A olefin isomer, K1, as an effective CERK inhibitor. In vitro kinase assays demonstrated that K1 effectively inhibits CERK without inhibiting sphingosine kinase and diacylglycerol kinase. Treating RBL-2H3 cells with K1 reduced cellular C1P levels to 40% yet had no effect on cell growth. Furthermore, treatment with K1 significantly suppressed both calcium ionophore- and IgE/antigen-induced degranulation, indicating that K1 interferes with signals that happen downstream of Ca2+ mobilization. Finally, we show that K1 affects neither IgE/antigen-induced global tyrosine phosphorylation nor subsequent Ca2+ elevation, suggesting a specificity for CERK-mediated signals. Our novel CERK inhibitor provides a useful tool for studying the biological functions of CERK and C1P. Moreover, to our knowledge, this is the first report demonstrating that inhibition of CERK suppresses IgE/antigen-induced mast cell degranulation. This finding suggests that CERK inhibitors might be a potential therapeutic tool in the treatment of allergic diseases.
Keywords: Ceramide 1-phosphate; Ceramide kinase; Inhibitor; Degranulation; Mast cell;

We investigated the effects of dietary administration of docosahexaenoic acid (DHA; C22:6n-3) on the levels of amyloid β (Aβ) peptide (1–40) and cholesterol in the nonionic detergent Triton 100×-insoluble membrane fractions (DIFs) of the cerebral cortex and, also, on learning-related memory in an animal model of Alzheimer's disease (AD) rats infused with Aβ peptide (1–40) into the cerebral ventricle. The infusion increased the levels of Aβ peptide and cholesterol in the DIFs concurrently with a significant increase in reference memory errors (measured by eight-arm radial-maze tasks) compared with those of vehicle rats. Conversely, the dietary administration of DHA to AD-model rats decreased the levels of Aβ peptide and cholesterol in the DIFs, with the decrease being more prominent in the DHA-administered rats. Regression analysis revealed a significant positive correlation between Aβ peptide and each of cholesterol, palmitic acid and stearic acid, and between the number of reference memory errors and each of cholesterol, palmitic, stearic and oleic acid; moreover, a significant negative correlation was observed between the number of reference memory errors and the molar ratio of DHA to palmitic plus stearic acid. These results suggest that DHA-induced protection of memory deficits in AD-model rats is related to the interactions of cholesterol, palmitic acid or stearic acid with Aβ peptides in DIFs where DHA ameliorates these interactions.
Keywords: Amyloid β peptide; Detergent-insoluble membrane fraction; Docosahexaenoic acid; Learning ability; Alzheimer's disease model rats;

Fatty acid ethyl esters, nonoxidative ethanol metabolites, synthesis, uptake, and hydrolysis by human platelets by Raneem O. Salem; Joanne E. Cluette-Brown; Michael Laposata (99-104).
The consumption of alcohol is known to have both positive and negative effects on the functioning of the cardiovascular system in general, and on platelet function in particular. Fatty acid ethyl esters (FAEEs) are non-oxidative metabolite of ethanol that may mediate the ethanol effect on platelet function leading to either bleeding or clotting. The aim of the current study was to investigate the synthesis, uptake, and hydrolysis of FAEEs by human platelets. Isolated platelets were incubated with ethanol for various times, and FAEE synthesis were measured by gas chromatography mass-spectrometry (GC-MS). In addition, platelets were incubated with 14C-ethyl oleate, and FAEE uptake and hydrolysis were measured. There was significant synthesis of FAEEs by human platelets within 30 min of exposure to ethanol. The major FAEE species formed by human platelets exposed to ethanol were ethyl palmitate and ethyl stearate. FAEE uptake by human platelets showed maximum uptake by 60 s. The majority of FAEEs (50–80%) incorporated into platelets remained intact for up to 10 min. FAEE hydrolysis led to an increase in free fatty acids, with minimal subsequent esterification of the free fatty acids into phospholipids, triglycerides, and cholesterol esters. These studies show that FAEEs, non-oxidative metabolite of ethanol, can be incorporated into, synthesized, and hydrolyzed by human platelets.
Keywords: Alcohol; Fatty acids; Fatty acid ethyl esters; Lipids; Platelets;

Production of (10E,12Z)-conjugated linoleic acid in yeast and tobacco seeds by Ellen Hornung; Claudia Krueger; Christian Pernstich; Martijn Gipmans; Andrea Porzel; Ivo Feussner (105-114).
The polyenoic fatty acid isomerase from Propioniumbacterium acnes (PAI) was expressed in E. coli and biochemically characterized. PAI catalyzes the isomerization of a methylene-interrupted double bond system to a conjugated double bond system, creating (10E,12Z)-conjugated linoleic acid (CLA). PAI accepted a wide range of free polyunsaturated fatty acids as substrates ranging from 18:2 fatty acids to 22:6, converting them to fatty acids with two or three conjugated double bonds. For expression of PAI in yeast the PAI-sequence encoding 20 N-terminal amino acid residues was altered for optimal codon usage, yielding codon optimized PAI (coPAI). The percentage of 10,12-CLA of total esterified fatty acids was 8 times higher in yeast transformed with coPAI than in cells transformed with PAI. CLA was detected in amounts up to 5.7% of total free fatty acids in yeast transformed with coPAI but none was detected in yeast transformed with PAI. PAI or coPAI under the control of the constitutive CaMV 35S promoter or the seed-specific USP promoter was transformed into tobacco plants. CLA was only detected in seeds in coPAI-transgenic plants. The amount of CLA detected in esterified fatty acids was up to 0.3%, in free fatty acids up to 15%.
Keywords: Conjugated linoleic acid isomer; Polyenoic fatty acid isomerase; Propioniumbacterium acne; Seed-specific expression; Substrate specificity;

Determination of plasma genistein fatty acid esters following administration of genistein or genistein 4′7-O-dioleate in monkeys by Maija Badeau; Matti J. Tikkanen; Susan E. Appt; Herman Adlercreutz; Thomas B. Clarkson; Antti Hoikkala; Kristiina Wähälä; Tomi S. Mikkola (115-120).
Soy-derived isoflavone phytoestrogens, such as genistein (4′,5,7-trihydroxyisoflavone), have been shown to protect low-density lipoprotein from oxidation. In addition, human plasma was previously shown to be capable of converting genistein into lipophilic fatty acid esters that accumulate in lipoproteins in vitro. We developed a method for the quantitation of genistein fatty acid esters in plasma. Furthermore, the method was utilized to measure genistein ester concentrations in monkey plasma following administration of genistein or genistein 4′,7-O-dioleate. After extraction from plasma, genistein fatty acid esters were separated from unesterified genistein by Sephadex LH-20 column chromatography. The genistein ester fraction was hydrolyzed by saponification and purified by a second chromatography on Sephadex LH-20. The hydrolyzed genistein esters were measured by time-resolved fluoroimmunoassay. Adult female rhesus monkeys (n  = 10) received a subcutaneous injection of genistein (24 mg, n  = 2) or genistein 4′,7-O-dioleate (71 mg, n  = 3) or an oral dose of genistein (24 mg, n  = 2) or genistein 4′,7-O-dioleate (71 mg, n  = 3). Plasma was collected at 4, 8, and 24 h post-dosing. Following subcutaneous administration of genistein 4′,7-O-dioleate, the plasma concentrations of genistein esters became elevated in two out of three monkeys with 8-h values exceeding 7.5 nmol/L and 24-h values above 12 nmol/L. Other treatments resulted in lower plasma values ranging between 2.7 and 6.1 nmol/L. The lower limit of detection for the method was 1.44 nmol/L. Subcutaneously administered genistein 4′,7-O-dioleate was also converted to water-soluble conjugates, but oral administration did not elevate plasma genistein fatty acid ester levels. The results suggest that it may be possible to introduce intact genistein ester molecules into plasma by parenteral but not oral administration.
Keywords: Genistein; Fatty acid; Genistein 4′7-O-dioleate; Time-resolved fluoroimmunoassay; Antioxidant; Atherosclerosis;

Characterization of heme environment and mechanism of peroxide bond cleavage in human prostacyclin synthase by Hui-Chun Yeh; Pei-Yung Hsu; Jinn-Shyan Wang; Ah-Lim Tsai; Lee-Ho Wang (121-132).
Prostacyclin is a potent mediator of vasodilation and anti-platelet aggregation. It is synthesized from prostaglandin H2 by prostacyclin synthase (PGIS), a member of Family 8 in the cytochrome P450 superfamily. Unlike most P450s, which require exogenous reducing equivalents and an oxygen molecule for mono-oxygenation, PGIS catalyzes an isomerization with an initial step of endoperoxide bond cleavage of prostaglandin H2 (PGH2). The low abundance of PGIS in natural tissues necessitates heterologous expression for studies of structure/function relationships and reaction mechanism. We report here a high-yield prokaryotic system for expression of enzymatically active human PGIS. The PGIS cDNA is modified by replacing the hydrophobic amino-terminal sequence with the more hydrophilic amino-terminal sequence from P450 2C5 and by adding a four-histidine tag at the carboxyl terminus. The resulting recombinant PGIS associates with host cell membranes and was purified to electrophoretic homogeneity by nickel affinity, hydroxyapatite and CM Sepharose column chromatography. The recombinant PGIS, with a heme:protein ratio of 0.9:1, catalyzes prostacyclin formation at a K m of 13.3 μM PGH2 and a V max of 980 per min. The dithionite-reduced PGIS binds CO with an on-rate of 5.6 × 105 M−1 s−1 and an off-rate of 15 s−1. The ferrous–CO complex of PGIS is very short-lived and decays at a rate of 0.7 s−1. Spectral binding assays showed that imidazole binds weakly to PGIS (K d ∼0.5 mM,) but clotrimazole, a bulky and rigid imidazole derivative, binds strongly (K d ∼1 μM). The transient nature of the CO complex and the weak imidazole binding seem to support an earlier proposal that PGIS active site has a limited space, but the tight binding of clotrimazole argues against this view. It appears that the heme distal pocket of PGIS is fairly adaptable to ligands of various structures. UV-visible absorption, magnetic circular dichroism and electron paramagnetic resonance spectra indicate that PGIS has a typical low-spin heme with a hydrophobic active site. PGIS catalyzes homolytic scission of the peroxide bond of a test substrate, 10-hydroperoxyoctadeca-8,12-dienoic acid, accompanied by formation of a heme intermediate with a Compound II-like optical spectrum.
Keywords: Cytochrome P450; Prostanoid; Heterologous expression; Homolysis of peroxide bond; Active site;