BBA - Molecular and Cell Biology of Lipids (v.1781, #8)

Genetic analyses involving interactions between the ergosterol biosynthetic enzymes, lanosterol synthase (Erg7p) and 3-ketoreductase (Erg27p), in the yeast Saccharomyces cerevisiae by B. Teske; S. Taramino; M.S.A. Bhuiyan; N.S. Kumaraswami; S.K. Randall; R. Barbuch; J. Eckstein; G. Balliano; M. Bard (359-366).
Protein–protein interaction studies in the Saccharomyces cerevisiae ergosterol biosynthetic pathway suggest that enzymes in this pathway may act as an integrated multienzyme complex. The yeast sterol 3-ketoreductase (Erg27p) required for C-4 demethylation of sterols has previously been shown to also be required for the function of the upstream oxidosqualene cyclase/lanosterol synthase (Erg7p); thus, erg27 mutants accumulate oxidosqualenes as precursors rather than 3-ketosterones. In the present study, we have created various mutations in the ERG27 gene. These mutations include 5 C-terminal truncations, 6 internal deletions, and 32 point mutants of which 14 were obtained by site-directed mutagenesis and 18 by random mutagenesis. We have characterized these ERG27 mutations by determining the following: Erg27 and Erg7 enzyme activities, presence of Erg27p as determined by western immunoblots, ability to grow on various sterol substrates and GC sterol profiles. Mutations of the predicted catalytic residues, Y202F and K206A, resulted in the endogenous accumulation of 3-ketosterones rather than oxidosqualenes suggesting retention of Erg7 enzyme activity. This novel phenotype demonstrated that the catalytic function of Erg27p can be separated from its Erg7p chaperone ability. Other erg27 mutations resulted in proteins that were present, as determined by western immunoblotting, but unable to interact with the Erg7 protein. We also classify Erg27p as belonging to the SDR (short-chain dehydrogenase/reductase) family of enzymes and demonstrate the possibility of homo- or heterodimerization of the protein. This study provides new insights into the role of Erg27p in sterol biosynthesis.
Keywords: CSM; EGS; HA; SDR; Saccharomyces cerevisiae; Ergosterol; Erg7; Erg27; 3-ketoreductase; Lanosterol synthase; Protein–protein interaction;

Lipolysis of the semi-solid self-emulsifying excipient Gelucire® 44/14 by digestive lipases by Sylvie Fernandez; Jean-David Rodier; Nicolas Ritter; Bruno Mahler; Frédéric Demarne; Frédéric Carrière; Vincent Jannin (367-375).
Gelucire® 44/14 is a semi-solid self-emulsifying excipient used for the oral delivery of poorly water-soluble drugs. It is composed of C8-C18 acylglycerols and PEG-32 esters, all of which are potential substrates for digestive lipases. Here we studied the lipolysis of Gelucire® 44/14 by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases. Human pancreatic lipase (HPL), the main lipase involved in the digestion of triacylglycerols, did not show any significant activity on Gelucire® 44/14 or on either of its individual compounds, C8-C18 acylglycerols and PEG-32 esters. Other pancreatic lipases such as human pancreatic lipase-related protein 2 (HPLRP2) showed low activity on Gelucire® 44/14 although the highest activity of HPLRP2 was that observed on the C8-C18 acylglycerol fraction, which accounts for 20% (w/w) of Gelucire® 44/14. In addition, HPLRP2 showed low activities on the PEG-32 esters, whether these were tested individually or mixed together. Carboxyl ester hydrolase (CEH) showed high activity on Gelucire® 44/14, and the highest activities of CEH were those recorded on the total PEG-32 ester fraction and on each individual PEG-32 ester, except for PEG-32 monostearate. The highest activity of all the enzymes tested was that of dog gastric lipase (DGL) on Gelucire® 44/14, although DGL showed low activity on the PEG-32 ester fraction and on each individual PEG-32 ester. We compared the lipolysis of Gelucire® 44/14 with that of Labrasol®, another self-emulsifying excipient, which is liquid at room temperature. Human pancreatic juice showed similar rates of activity on both Gelucire® 44/14 and Labrasol®. This finding means that these excipients are hydrolyzed in vivo during pancreatic digestion, mainly by CEH in the case of Gelucire® 44/14 and by both HPLRP2 and CEH in that of Labrasol®, whereas HPL showed very low activities on each of these two excipients. This is the first time the effects of PEG and acyl chain length on the lipolytic activity of digestive lipases on PEG esters have been investigated.
Keywords: Lipase; Lipolysis; Macrogolglycerides; Oral drug delivery; Semi-solid excipient, PEG esters;

Phospholipase D (PLD) has been implicated in mediating vesicular transport, mitosis, differentiation and apoptosis. The product of PLD activity, phosphatidic acid (PA) has mitogenic potential and elevated PLD expression has been detected in many tumor cell lines. Several reports have demonstrated that distinct PLD domains regulate its activity and that truncated forms of PLD retain enzymatic activity. We hypothesized that during apoptosis caspase cleavage of PLDs could result in modification of their activities. To test this idea, we have used in vitro translation of PLD1 and PLD2 which generated active enzymes exhibiting properties mimicking those of the endogenous proteins. Here we demonstrate that PLD1 was rapidly cleaved in vitro by caspases-8, -3 and -7. In contrast, PLD2 cleavage was delayed and its activity was unaffected by incubation with caspase-3. Significantly, following caspase cleavage the response of PLD1 to regulatory stimuli was altered; it was no longer activated by PKC and instead exhibited an increased activity in response to small GTPases. Notably, this enhanced activity was due to cleavage of PLD1 in the “loop” domain, a region previously associated with negative regulatory function. Thus our data have identified a novel regulatory domain in PLD1.
Keywords: Phospholipase D; Phosphatidic acid; Regulatory domain; Caspase; Apoptosis;

Metabolism of a novel side chain modified Δ8(14)-15-ketosterol, a potential cholesterol lowering drug: 28-hydroxylation by CYP27A1 by Hanna Pettersson; Maria Norlin; Ulla Andersson; Irina Pikuleva; Ingemar Björkhem; Alexander Yu. Misharin; Kjell Wikvall (383-390).
The synthetic inhibitors of sterol biosynthesis, 3β-hydroxy-5α-cholest-8(14)-en-15-one and 3β-hydroxy-24S-methyl-5α-cholesta-8(14),22-dien-15-one, are of interest as potential cholesterol lowering drugs. Rapid metabolism of synthetic 15-ketosterols may lead to a decrease, or loss, of their potency to affect lipid metabolism. 3β-Hydroxy-5α-cholest-8(14)-en-15-one is reported to be rapidly side chain oxygenated by rat liver mitochondria. In an attempt to reduce this metabolism, the novel side chain modified 15-ketosterol 3β-Hydroxy-24S-methyl-5α-cholesta-8(14),22-dien-15-one was synthesized. We have examined the metabolism by recombinant human CYP27A1 of this novel side chain modified 3β-hydroxy-24S-methyl-5α-cholesta-8(14),22-dien-15-one and compared the rate of metabolism with that of the previously described 3β-hydroxy-5α-cholest-8(14)-en-15-one. Both sterols were found to be efficiently metabolized by recombinant human CYP27A1. None of the two 15-ketosterols was significantly metabolized by microsomal 7α-hydroxylation. Interestingly, CYP27A1-mediated product formation was much lower with the side chain modified 3β-hydroxy-24S-methyl-5α-cholesta-8(14),22-dien-15-one than with the previously described 3β-hydroxy-5α-cholest-8(14)-en-15-one. A surprising finding was that this novel side chain modified sterol was metabolized mainly in the C-28 position by CYP27A1. The data on 28-hydroxylation by human CYP27A1 provide new insights on the catalytic properties and substrate specificity of this enzyme. The finding that 3β-hydroxy-24S-methyl-5α-cholesta-8(14),22-dien-15-one with a modified side chain is metabolized at a dramatically slower rate than the previously described 15-ketosterol with unmodified side chain may be important for future development of synthetic cholesterol lowering sterols.
Keywords: Human CYP27A1; 27-Hydroxylation; 28-Hydroxylation; Inhibitors of sterol biosynthesis; Cholesterol lowering drug;

Incorporation and remodeling of extracellular phosphatidylcholine with short acyl residues in Saccharomyces cerevisiae by Kunihiko Tanaka; Ryouichi Fukuda; Yusuke Ono; Hiroki Eguchi; Shinya Nagasawa; Yusuke Nakatani; Hidenori Watanabe; Hiroki Nakanishi; Ryo Taguchi; Akinori Ohta (391-399).
The pem1/cho2 pem2/opi3 double mutant of Saccharomyces cerevisiae, which is auxotrophic for choline because of the deficiency in methylation activities of phosphatidylethanolamine, grew in the presence of 0.1 mM dioctanoyl-phosphatidylcholine (diC8PC). Analysis of the metabolism of methyl-13C-labeled diC8PC ((methyl-13C)3-diC8PC) by electrospray ionization tandem mass spectrometry (ESI-MS/MS) revealed that it was rapidly converted to (methyl-13C)3-PCs containing C16 or C18 acyl chains. (Methyl-13C)3-8:0-lyso-PC, (methyl-13C)3-8:0-16:0-PC and (methyl-13C)3-8:0-16:1-PC, which are the probable intermediate molecular species of acyl chain remodeling, appeared immediately after 5 min of pulse-labeling and decreased during the subsequent chase period. These results indicate that diC8PC was taken up by the pem1 pem2 double mutant and that the acyl chains of diC8PC were exchanged with longer yeast fatty acids. The temporary appearance of (methyl-13C)3-8:0-lyso-PC suggests that the remodeling reaction may consist of deacylation and reacylation by phospholipase activities and acyltransferase activities, respectively. The detailed analyses of the structures of (methyl-13C)3-8:0-16:0-PC and (methyl-13C)3-8:0-16:1-PC by MS/MS and MS3 strongly suggest that most (methyl-13C)3-8:0-16:0-PCs have a C16:0 acyl chain at sn-1 position, whereas (methyl-13C)3-8:0-16:1-PCs have a C16:1 acyl chain at either sn-1 or sn-2 position in a similar frequency, implying that the initial C16:0 acyl chain substitution prefers the sn-1 position; however, the C16:1 acyl chain substitution starts at both sn-1 and sn-2 positions. The current study provides a pivotal insight into the acyl chain remodeling of phospholipids in yeast.
Keywords: Saccharomyces cerevisiae; Phosphatidylcholine; Remodeling; Acyl chain;

Polyunsaturated fatty acids can be ω-oxidized to dicarboxylic polyunsaturated fatty acids (DC-PUFA), bioactive compounds which cause vasodilatation and activation of PPARα and γ. DC-PUFA can be shortened by β-oxidation, and to determine whether mitochondria and/or peroxisomes are responsible for this degradation 20-carboxy-[1-14C]-eicosatetraenoic acid (20-COOH-AA) was synthesized and given to hepatocytes from mouse models with peroxisomal dysfunctions. In contrast to wild type cells, hepatocytes from mice with liver-selective elimination of peroxisomes, due to Pex5p deficiency, failed to produce 14CO2 and labeled acid-soluble oxidation products, indicating that peroxisomes are involved in the degradation of 20-COOH-AA. Subsequently, the oxidation of 20-COOH-AA was analyzed in hepatocytes lacking multifunctional protein 1 (MFP1) or MFP2, key enzymes of the peroxisomal β-oxidation. Degradation of 20-COOH-AA was partially impaired in MFP1, but not in MFP2 knockout hepatocytes. Taken together, peroxisomes and not mitochondria are the site of β-oxidation of DC-PUFA, and MFP1 is involved in this process.
Keywords: Arachidonic acid; Multifunctional protein 1; Peroxisomes; Pex5; PPARα; ω-oxidation; Zellweger syndrome;

Dietary polyunsaturated fatty acids (C18:2 ω6 and C18:3 ω3) do not suppress hepatic lipogenesis by Whitney Sealls; Monica Gonzalez; M. Julia Brosnan; Paul N. Black; Concetta C. DiRusso (406-414).
Omega 3 polyunsaturated fatty acids are promoted as beneficial in the prevention of metabolic and cardiovascular diseases. In general, dietary omega 3 fatty acids are derived from plant sources as linolenic acid (LNA, C18:3 ω3) the precursor to eicosapentaenoic acid (EPA, C20:5 ω3) and docosahexaenoic acid (DHA, C22:6 ω3). However, it remains unclear if the polyunsaturated fatty acid (PUFA) LNA can provide the same health benefits as the very long chain highly unsaturated fatty acids (HUFA) EPA and DHA generally derived from oily fish. In this study, mice were fed synthetic diets containing lard (low in PUFA and HUFA), canola oil (to supply PUFA), or a mixture of menhaden and arasco (fish and fungal) oils (to supply HUFA) for 8 weeks. The diets were neither high in calories nor fat, which was supplied at 6%. The lard and canola oil diets resulted in high levels of hepatic triglycerides and cholesterol and elevation of lipogenic gene expression. By comparison livers from mice fed the fish/fungal oil diet had low levels of lipid accumulation and more closely resembled livers from mice fed standard laboratory chow. SREBP1c and PPARγ gene and protein expression were high in livers of animals fed diets containing lard or canola oil compared with fish/fungal oil. Hepatic fatty acid analyses indicated that dietary PUFA were efficiently converted to HUFA regardless of source. Therefore, differences in hepatic lipid levels and gene expression between dietary groups were due to exogenous fatty acid supplied rather than endogenous pools. These results have important implications for understanding the regulation of hepatic lipogenesis by dietary fatty acids.
Keywords: Fatty liver; PUFA; HUFA; Canola oil; Fish oil; Lard; Dietary fat; SREBP1c; PPARγ;

A negative regulator of delayed prostaglandin D2 production in mouse mast cells by Noriko Ueno; Yoshitaka Taketomi; Kumiko Koga; Yohei Atsumi; Rei Kikuchi-Yanoshita; Ichiro Kudo; Makoto Murakami (415-421).
We have previously shown that maturation of mouse bone marrow-derived mast cells (BMMCs) into connective tissue mast cells (CTMCs) upon coculture with fibroblasts in the presence of stem cell factor (kit ligand) is accompanied by marked induction of a panel of genes, one of which was identified as NLRP3. Here we report that NLRP3 acts as a novel negative regulator of delayed prostaglandin (PG) D2 production in BMMCs. We found that, apart from its cell maturation-associated induction, NLRP3 expression was markedly induced in BMMCs several hours after FcɛRI crosslinking or cytokine stimulation. Ectopic expression of NLRP3 in BMMCs resulted in marked attenuation of cyclooxygenase (COX)-2-dependent delayed PGD2 generation, whereas it had no effects on other effector functions, including degranulation, COX-1-dependent immediate PGD2 generation and cytokine/chemokine expression. The suppression of delayed PGD2 generation by NLRP3 was preceded by a transient decrease of NF-κB activation and a marked reduction in the expression of COX-2, but not that of cytosolic phospholipase A2 α (cPLA2α), COX-1 and hematopoietic PGD2 synthase. Moreover, in CTMC-like differentiated cells in which endogenous NLRP3 expression was induced, cytokine-stimulated induction of COX-2 and attendant delayed PGD2 generation were markedly reduced. Our results suggest that, in mouse mast cells, NLRP3 counter-regulates COX-2-dependent sustained production of PGD2, a prostanoid that exhibits both pro- and anti-allergic effects, thereby potentially influencing the duration of allergic and other mast cell-associated inflammatory diseases.
Keywords: Allergy; Cyclooxygenase; Mast cell; Prostaglandin D2;