Determinants of pH profile and acyl chain selectivity in lysosomal phospholipase A2 [S]
作者:Vania Hinkovska-Galcheva、Robert Kelly、Kelly A. Manthei、Renee Bouley、Wenmin Yuan、Anna Schwendeman、JohnJ.G. Tesmer、James A. Shayman
DOI:10.1194/jlr.m084012
日期:——
Lysosomal phospholipaseA2 (LPLA2) is characterized by broad substrate recognition, peak activity at acidic pH, and the transacylation of lipophilic alcohols, especially N-acetyl-sphingosine. Prior structural analysis of LPLA2 revealed the presence of an atypical acidic residue, Asp13, in the otherwise hydrophobic activesite cleft. We hypothesized that Asp13 contributed to the pH profile and/or substrate
Positional specificity of lysosomal phospholipase A2
作者:Akira Abe、Miki Hiraoka、James A. Shayman
DOI:10.1194/jlr.m600183-jlr200
日期:2006.10
5-fold higher than that of 1-O-palmitoyl-NAS. Thus, Lpla2 can act on acylgroups at both sn-1 and sn-2 positions of POPC and OPPC. When 1-palmitoyl-2-unsaturated acyl-sn-glycero-3-phosphocholines were used as acyl donors, the transacylation of the acylgroupfrom the sn-2 position to NAS was preferred to that of the palmitoyl groupfrom the sn-1 position. An exception was observed for 1-palmitoyl-2-ar
Metabolomics annotates ABHD3 as a physiologic regulator of medium-chain phospholipids
作者:Jonathan Z Long、Justin S Cisar、David Milliken、Sherry Niessen、Chu Wang、Sunia A Trauger、Gary Siuzdak、Benjamin F Cravatt
DOI:10.1038/nchembio.659
日期:2011.11
An untargeted metabolomics approach identifies C14 phosphatidylcholines (PCs) as specific cellular medium-chain substrates of the lipase ABHD3, as well as C5âC8 short-chain PCs including oxidized pro-atherosclerotic and pro-apoptotic PC phospholipids. All organisms, including humans, possess a huge number of uncharacterized enzymes. Here we describe a general cell-based screen for enzyme substrate discovery by untargeted metabolomics and its application to identify the protein α/β-hydrolase domainâcontaining 3 (ABHD3) as a lipase that selectively cleaves medium-chain and oxidatively truncated phospholipids. Abhd3â/â mice possess elevated myristoyl (C14)-phospholipids, including the bioactive lipid C14-lysophosphatidylcholine, confirming the physiological relevance of our substrate assignments.
The Highly Selective Production of 2-Arachidonoyl Lysophosphatidylcholine Catalyzed by Purified Calcium-independent Phospholipase A2γ
作者:Wei Yan、Christopher M. Jenkins、Xianlin Han、David J. Mancuso、Harold F. Sims、Kui Yang、Richard W. Gross
DOI:10.1074/jbc.m502358200
日期:2005.7
Herein, we report the heterologous expression of the human peroxisomal 63-kDa calcium-independent phospholipase A(2)gamma (iPLA(2)gamma) isoform in Sf9 cells, purification of the N-terminal His-tagged enzyme by affinity chromatography, and the identification of its remarkable substrate selectivity that results in the highly selective generation of 2-arachidonoyl lysophosphatidylcholine. Mass spectrometric analyses demonstrated that purified iPLA(2)gamma hydrolyzed saturated or monounsaturated aliphatic groups readily from either the sn-1 or sn-2 positions of phospholipids. In addition, purified iPLA(2)gamma effectively liberated arachidonic acid from the sn-2 position of plasmenylcholine substrates. In contrast, incubation of iPLA(2)gamma with 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine resulted in the rapid release of palmitic acid and the selective accumulation of 2-arachidonoyl lysophosphatidylcholine (LPC), which was not metabolized further by iPLA(2)gamma. The putative regiospecificity of the 2-arachidonoyl LPC product was authenticated by its diagnostic fragmentation pattern during tandem mass spectrometric analysis. To identify the physiological relevance of iPLA(2)gamma-mediated 2-arachidonoyl LPC production utilizing naturally occurring membranes, we incubated purified rat hepatic peroxisomes with iPLA(2)gamma and similarly identified the selective accumulation of 2-arachidonoyl LPC. Furthermore, tandem mass spectrometric analysis demonstrated that 2-arachidonoyl LPC is a natural product in human myocardium, a tissue in which iPLA(2)gamma expression is robust. Because 2-arachidonoyl LPC represents a key branch point intermediate that can potentially lead to a variety of bioactive molecules in eicosanoid signaling (e.g. arachidonic acid, 2-arachidonoylglycerol), these results have uncovered a novel eicosanoid selective pathway through iPLA(2)gamma-mediated 2-arachidonoyl LPC production to amplify and diversify the repertoire of biologic lipid second messengers in response to cellular stimulation.
Synthesis and Evaluation of Polyunsaturated Fatty Acid-Phenol Conjugates as Anti-Carbonyl-Stress Lipophenols
apoptosis, we designed and synthesized a series of O-alkylated resorcinol derivatives featuring enhanced anti-carbonyl-stress properties. Additionally, these phenolic structures are linked to a polyunsaturatedfatty acid such as docosahexaenoic acid (C22:6 n-3; DHA) or to a lysophosphatidylcholine–DHA conjugate, in order to specifically increase their bioavailability, and thus, to target the retina. Selective