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BBA - Molecular and Cell Biology of Lipids (v.1791, #10)

Editorial Board (pp. i).

TLR-4 mediated group IVA phospholipase A₂ activation is phosphatidic acid phosphohydrolase 1 and protein kinase C dependent by Andrej Grkovich; Aaron Armando; Oswald Quehenberger; Edward A. Dennis ⁎ (pp. 975-982).

Group IVA phospholipase A₂ (GIVA PLA₂) catalyzes the release of arachidonic acid (AA) from the sn-2 position of glycerophospholipids. AA is then further metabolized into terminal signaling molecules including numerous prostaglandins. We have now demonstrated the involvement of phosphatidic acid phosphohydrolase 1 (PAP-1) and protein kinase C (PKC) in the Toll-like receptor-4 (TLR-4) activation of GIVA PLA₂. We also studied the effect of PAP-1 and PKC on Ca+² induced and synergy enhanced GIVA PLA₂ activation. We observed that the AA release induced by exposure of RAW 264.7 macrophages to the TLR-4 specific agonist Kdo₂-Lipid A is blocked by the PAP-1 inhibitors bromoenol lactone (BEL) and propranolol as well as the PKC inhibitor Ro 31-8220; however these inhibitors did not reduce AA release stimulated by Ca+² influx induced by the P2X7 purinergic receptor agonist ATP. Additionally, stimulation of cells with diacylglycerol (DAG), the product of PAP-1 mediated hydrolysis, initiated AA release from unstimulated cells as well as restored normal AA release from cells treated with PAP-1 inhibitors. Finally, neither PAP-1 nor PKC inhibition reduced GIVA PLA₂ synergistic activation by stimulation with Kdo₂-Lipid A and ATP.

Keywords: Phosphatidic acid phosphohydrolase 1 (PAP-1); Group IVA phospholipase-2 (GIVA PLA; ₂; ); Protein Kinase C (PKC); Lipopolysaccharide (LPS); Macrophage; Signal transduction; Inflammation; Arachidonic acid (AA); Eicosanoid

Continuous measurement of galactolipid hydrolysis by pancreatic lipolytic enzymes using the pH-stat technique and a medium chain monogalactosyl diglyceride as substrate by Sawsan Amara; Dominique Lafont; Brice Fiorentino; Paul Boullanger; Frédéric Carrière ⁎; Alain De Caro ⁎ (pp. 983-990).

Galactolipids are the main lipids from plants and galactolipases play a major role in their metabolism. These enzymes were however poorly studied so far and only few assays have been developed. A specific and continuous galactolipase assay using synthetic medium chain monogalactosyl diacylglycerol (MGDG) as substrate was developed using the pH-stat technique and recombinant human (rHPLRP2) and guinea pig (rGPLRP2) pancreatic lipase-related protein 2 as model enzymes. PLRP2s are the main enzymes involved in the digestion of galactolipids in the gastrointestinal tract. Monogalactosyl di-octanoylglycerol was mixed with bile salt solutions by sonication to form a micellar substrate before launching the assay. The nature of the bile salt and the bile salt to MGDG ratio were found to significantly affect the rate of MGDG hydrolysis by rHPLRP2 and rGPLRP2. The maximum galactolipase activity of both enzymes was recorded with sodium deoxycholate (NaDC) and at a NaDC to MGDG ratio of 1.33 and at basic pH values (8.0–9.0). The maximum rates of hydrolysis were obtained using a MGDG concentration of 10−² M and calcium chloride was found to be not necessary to obtain the maximum of activity. Under these conditions, the maximum turnovers of rGPLRP2 and rHPLRP2 on mixed NaDC/MGDG micelles were found to be 8000±500 and 2800±60 μmol/min/mg (U/mg), respectively. These activities are in the same order of magnitude as the activities on triglycerides of lipases and they are the highest specific activities ever reported for galactolipases. For the sake of comparison, the hydrolysis of mixed bile salt/MGDG micelles was also tested using other pancreatic lipolytic enzymes and only native and recombinant human carboxyl ester hydrolase were found to display significant but lower activities (240±17 and 432±62 U/mg, respectively) on MGDG.

Keywords: Bile salt; Enzyme assay; Galactolipase; Galactolipid; Mixed micelle; Pancreas; Pancreatic lipase-related protein 2; Monogalactosyl diacylglycerol

Forkhead box transcription factor O1 inhibits cholesterol 7α-hydroxylase in human hepatocytes and in high fat diet-fed mice by Tiangang Li; Huiyan Ma; Young Joo Park; Yoon-Kwang Lee; Stephen Strom; David D. Moore; John Y.L. Chiang ⁎ (pp. 991-996).

The conversion of cholesterol to bile acids is the major pathway for cholesterol catabolism. Bile acids are metabolic regulators of triglycerides and glucose metabolism in the liver. This study investigated the roles of FoxO1 in the regulation of cholesterol 7α-hydroxylase ( CYP7A1) gene expression in primary human hepatocytes. Adenovirus-mediated expression of a phosphorylation defective and constitutively active form of FoxO1 (FoxO1-ADA) inhibited CYP7A1 mRNA expression and bile acid synthesis, while siRNA knockdown of FoxO1 resulted in a ∼6-fold induction of CYP7A1 mRNA in human hepatocytes. Insulin caused rapid exclusion of FoxO1 from the nucleus and resulted in the induction of CYP7A1 mRNA expression, which was blocked by FoxO1-ADA. In high fat diet-fed mice, CYP7A1 mRNA expression was repressed and inversely correlated to increase hepatic FoxO1 mRNA expression and FoxO1 nuclear retention. In conclusion, our current study provides direct evidence that FoxO1 is a strong repressor of CYP7A1 gene expression and bile acid synthesis. Impaired regulation of FoxO1 may cause down-regulation of CYP7A1 gene expression and contribute to dyslipidemia in insulin resistance.

Keywords: Bile acid synthesis; Insulin; Gene expression; Nuclear receptor; Metabolic disease

Bid binding to negatively charged phospholipids may not be required for its pro-apoptotic activity in vivo by Anna Manara; Jennefer Lindsay; Marta Marchioretto; Alessandra Astegno; Andrew P. Gilmore; Mauro Degli Esposti; Massimo Crimi ⁎ (pp. 997-1010).

Bid is a ubiquitous pro-apoptotic member of the Bcl-2 family that has been involved in a variety of pathways of cell death. Unique among pro-apoptotic proteins, Bid is activated after cleavage by the apical caspases of the extrinsic pathway; subsequently it moves to mitochondria, where it promotes the release of apoptogenic proteins in concert with other Bcl-2 family proteins like Bak. Diverse factors appear to modulate the pro-apoptotic action of Bid, from its avid binding to mitochondrial lipids (in particular, cardiolipin) to multiple phosphorylations at sites that can modulate its caspase cleavage. This work addresses the question of how the lipid interactions of Bid that are evident in vitro actually impact on its pro-apoptotic action within cells. Using site-directed mutagenesis, we identified mutations that reduced mouse Bid lipid binding in vitro. Mutation of the conserved residue Lys157 specifically decreased the binding to negatively charged lipids related to cardiolipin and additionally affected the rate of caspase cleavage. However, this lipid-binding mutant had no discernable effect on Bid pro-apoptotic function in vivo. The results are interpreted in relation to an underlying interaction of Bid with lysophosphatidylcholine, which is not disrupted in any mutant retaining pro-apoptotic function both in vitro and in vivo.

Keywords: Abbreviations; ADIFAB; AcryloDated Intestinal Fatty Acid Binding Protein; CL; cardiolipin; FL Bid; full length Bid; LPC; lysophosphatidylcholine; LPC-C16; 1-palmitoyl-LPC; LPC-C12; 1-dodecyl-LPC; LPG; lysophosphatidylglycerol; LPG-C18:1; 1-oleyl-LPG; MCL; monolysocardiolipin; tBid; truncated (caspase-8-cleaved) Bid; OMM; outer mitochondrial membrane; MOMP; mitochondrial outer membrane permeabilizationApoptosis; Bid; Mitochondria; Phospholipids; Caspase 8

Subcellular localization and lysophospholipase/transacylation activities of human group IVC phospholipase A2 (cPLA2γ) by Atsushi Yamashita; Ken Tanaka; Ryo Kamata; Tsukasa Kumazawa; Naotaka Suzuki; Hiroki Koga; Keizo Waku; Takayuki Sugiura (pp. 1011-1022).

cPLA2γ was identified as an ortholog of cPLA2α, which is a key enzyme in eicosanoid production. cPLA2γ was reported to be located in endoplasmic reticulum (ER) and mitochondria and to have lysophospholipase activity beside phospholipase A2 (PLA2) activity. However, subcellular localization, mechanism of membrane binding, regulation and physiological function have not been fully established. In the present study, we examined the subcellular localization and enzymatic properties of cPLA2γ with C-terminal FLAG-tag. We found that cPLA2γ was located not only in ER but also mitochondria even in the absence of the prenylation. Purified recombinant cPLA2γ catalyzed an acyltransferase reaction from one molecule of lysophosphatidylcholine (LPC) to another, forming phosphatidylcholine (PC). LPC or lysophosphatidylethanolamine acted as acyl donor and acceptor, but lysophosphatidylserine, lysophosphatidylinositol and lysophosphatidic acid (LPA) did not. PC and phosphatidylethanolamine (PE) also acted as weak acyl donors. Reaction conditions changed the balance of lysophospholipase and transacylation activities, with addition of LPA/PA, pH>8, and elevated temperature markedly increasing transacylation activity; this suggests that lysophospholipase/transacylation activities of cPLA2γ may be regulated by various factors. As lysophospholipids are known to accumulate in ischemia heart and to induce arryhthmia, the cPLA2γ that is abundant in heart may have a protective role through clearance of lysophospholipids by its transacylation activity.

Keywords: Abbreviations; CoA; coenzyme A; GPC; sn; -glycero-3-phosphocholine; GPE; sn; -glycero-3-phosphoethanolamine; LPC; lysophosphatidylcholine; LPE; lysophosphatidylethanolamine; LPL; lysophospholipid; PC; phosphatidylcholine; PE; phosphatidylethanolamineCAAX box motif; Lysophospholipid; Phospholipase A2; cPLA2γ; Lysophospholipase; Transacylation

Subcellular localization of ceramide kinase and ceramide kinase-like protein requires interplay of their Pleckstrin Homology domain-containing N-terminal regions together with C-terminal domains by Philipp Rovina; Andrea Schanzer; Christine Graf; Diana Mechtcheriakova; Markus Jaritz; Frédéric Bornancin ⁎ (pp. 1023-1030).

Ceramide kinase (CERK) and the ceramide kinase-like protein (CERKL), two related members of the diacylglycerol kinase family, are ill-defined at the molecular level. In particular, what determines their distinctive subcellular localization is not well understood. Here we show that the Pleckstrin Homology (PH) domain of CERK, which is required for Golgi complex localization, can substitute for the N-terminal region of CERKL and allow for wild-type CERKL localization, which is typified by nucleolar accumulation. This demonstrates that determinants for localization of these two enzymes do not lie solely in their PH domain-containing N-terminal regions. Moreover, we present evidence for a previously unrecognized participation of CERK distal sequences in structural stability, localization and activity of the full-length protein. Progressive deletion of CERK and CERKL from the C-terminus revealed similar sequential organization in both proteins, with nuclear import signals in their N-terminal part, and nuclear export signals in their C-terminal part. Furthermore, mutagenesis of individual cysteine residues of a CERK-specific C XXXC XXC motif severely compromised both exportation of CERK from the nucleus and its association with the Golgi complex. Altogether, this work identifies conserved domains in CERK and CERKL as well as new determinants for their subcellular localization. It further suggests a nucleocytoplasmic shuttling mechanism for both proteins that may be defective in CERKL mutant proteins responsible for retinal degenerative diseases.

Keywords: Ceramide; Kinase; Nucleocytoplasmic; Nucleolar; Pleckstrin homology; Retinal degeneration

Novel roles of hepatic lipase and phospholipid transfer protein in VLDL as well as HDL metabolism by Rien van Haperen; Hannelore Samyn; Teus van Gent; Adri J. Zonneveld; Matthijs Moerland; Frank Grosveld; Hans Jansen; Geesje M. Dallinga-Thie; Arie van Tol; Rini de Crom (pp. 1031-1036).

Elevated plasma phospholipid transfer protein (PLTP) expression may increase atherosclerosis in mice by reducing plasma HDL and increasing hepatic VLDL secretion. Hepatic lipase (HL) is a lipolytic enzyme involved in several aspects of the same pathways of lipoprotein metabolism. We investigated whether the effects of elevated PLTP activity are compromised by HL deficiency.HL deficient mice were crossbred with PLTP transgenic (PLTPtg) mice and studied in the fasted state. Plasma triglycerides were decreased in HL deficiency, explained by reduced hepatic triglyceride secretion. In PLTPtg mice, a redistribution of HL activity between plasma and tissue was evident and plasma triglycerides were also decreased. HL deficiency mitigated or even abolished the stimulatory effect of elevated PLTP activity on hepatic triglyceride secretion. HL deficiency had a modest incremental effect on plasma HDL, which remained present in PLTP transgenic/HL^−/− mice, thereby partially compensating the decrease in HDL caused by elevation of PLTP activity. HDL decay experiments showed that the fractional turnover rate of HDL cholesteryl esters was delayed in HL deficient mice, increased in PLTPtg mice and intermediate in PLTPtg mice in an HL^−/− background.HL affects hepatic VLDL. Elevated PLTP activity lowers plasma HDL-cholesterol by stimulating the plasma turnover and hepatic uptake of HDL cholesteryl esters. HL is not required for the increase in hepatic triglyceride secretion or for the lowering of HDL-cholesterol induced by PLTP overexpression.

Keywords: PLTP; HL; Triglyceride; Cholesteryl ester

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BBA - Molecular and Cell Biology of Lipids (v.1791, #10)

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BBA - Molecular and Cell Biology of Lipids (v.1791, #10)