Hydrolysis by phospholipase D of phospholipids in solution state or adsorbed on a silica matrix

Phospholipases D (PLD) catalyse hydrolysis and transphosphatidylation reactions in phospholipids. In the present study, the hydrolytic activity for cabbage PLD was investigated with five different substrates (dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylcholine (DPPC), didecanoylphosphatidylcholine (DDPC), 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine and lyso-phosphatidylcholine (lyso-PC)) in solution or adsorbed on a silica matrix. In the specific buffer solutions, where the substrates were proved to form large multilamellar polydisperse aggregates, PLD showed preference for DPPC > DPPE > DDPC > 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine > lyso-PC. When the substrates were adsorbed on the silica matrix, PLD hydrolysed 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine and lyso-PC, DDPC, but not DPPC or DPPE. Theoretical studies of the simplest possible adducts between the phospholipids and the silica matrix were performed. Examination of local geometries of DPPC showed a significant blocking of the P-O-X bond-prone to hydrolysis, which could possibly block the access of PLD. Immobilization of phospholipids could be applied for improving the yield of reactions catalysed by PLD as well as for performing a targeted production of short-chain length phosphatidic acid analogs.     

Incorporation and remodeling of phosphatidylethanolamine containing short acyl residues in yeast

Phosphatidylethanolamine (PE) is one of the essential phospholipids in the yeast Saccharomyces cerevisiae. We have previously shown that a yeast strain, the endogenous PE synthesis of which was controllable, grew in the presence of PE containing decanoyl residues (diC10PE) when PE synthesis was repressed. In this study, we investigated the fate of diC10PE, its uptake and remodeling in yeast. Deletion of the genes encoding Lem3p/Ros3p or P-type ATPases, Dnf1p and Dnf2p, impaired the growth of the mutants in the medium containing diC10PE, suggesting the involvement of these proteins in the uptake of diC10PE. Analysis of the metabolism of deuterium-labeled diC10PE by electrospray ionization tandem mass spectrometry revealed that it was rapidly converted to deuterium-labeled PEs containing C16 or C18 acyl residues. The probable intermediate PEs that contained decanoic acid and C16 or C18 fatty acids as acyl residues were also detected. In addition, a substantial amount of decanoic acid was released into the culture medium during growth in the presence of diC10PE. These results imply that diC10PE was remodeled to PEs with longer acyl residues and used as membrane components. Defects in the remodeling of diC10PE in the deletion mutants of ALE1 and SLC1, products of which were capable of acyl-transfer to the sn− 2 position of lyso-phospholipids, suggested their involvement in the introduction of acyl residues to the sn− 2 position of lyso-phosphatidylethanolamine in the remodeling reaction of diC10PE. Our results also suggest the presence of a mechanism to maintain the physiological length of PE acyl residues in yeast.     

A ménage à trois made in heaven: G-protein-coupled receptors, lipids and TRP channels

Recent technical and conceptual advances in lipid analysis have given us a glimpse into the true versatility of the lipidome and the complexity of lipid signaling species. Progress alike in protein chemistry and genetics has presented us with new signal pathways and molecular mechanisms for the lipid actions. G-protein-coupled receptors (GPCR) appear to play a central role in the regulation of many lipid signals and are also themselves targets for some of these. TRP channels have recently been acknowledged as one of the most important GPCR effectors; in many cases the signals from GPCRs to TRPs are mediated via lipid signals. This review aims at presenting a view into the complex lipid signaling networks, their possible regulation by GPCRs and the signals transmitted to the TRP channels. Critical views and possible shortcomings in the composition of the studies are also presented.     

Identification of membrane O-acyltransferase family motifs

Cellular membranes contain several classes of glycerophospholipids, which have numerous structural and functional roles in cells. Membrane diversity and asymmetry are important for membrane fluidity, curvature, and storage of lipid mediator precursors. Using acyl-CoAs, glycerophospholipids are first formed in the de novo pathway (Kennedy pathway), and then modified in the remodeling pathway (Lands’ cycle) to generate mature membrane. Recently, several lysophospholipid acyltransferases (LPLATs) from two families, the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family and the membrane bound O-acyltransferase (MBOAT) family, were shown to function in the remodeling pathway. The MBOAT family possesses either LPLAT activity or protein O-acyltransferase activity. While the motifs of the AGPAT family have been well characterized, the MBOAT motifs remain unclear. In this study, we identified four MBOAT motifs essential for LPLAT activities by extensive site-directed mutagenesis. These findings further our understanding of the enzyme reaction mechanisms and will contribute to structure predictions for the MBOAT family enzymes.