Evidence for the importance of OxPAPC interaction with cysteines in regulating endothelial cell function

• DOI: 10.1194/jlr.m025320
• Publication Date: 2012-05-02T02:45:23Z
• Authors: James R. Springstead; B.Gabriel Gugiu; Sangderk Lee; Seung Cha; Andrew D. Watson; Judith A. Berliner
• Keywords: Arteriogenesis

Oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphatidylcholine (PAPC), referred to as OxPAPC, and an active component, 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphatidylcholine (PEIPC), accumulate in atherosclerotic lesions regulate over 1,000 genes human aortic endothelial cells (HAEC). We previously demonstrated that OxPNB, a biotinylated analog covalently binds number proteins HAEC. The goal these studies was gain insight into the binding mechanism determine whether regulates activity. In whole cells, N-acetylcysteine inhibited gene regulation by blocking cell cysteines with N-ethylmaleimide strongly OxPNB HAEC proteins. Using MS, we demonstrate most OxPAPC cysteine is mediated PEIPC. also show PEIPE-NB, PEIPC, bound model protein, H-Ras, at shown activity response 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2). This observed recombinant protein overexpressing H-Ras. PEIPC compete for 15dPGJ2 increased H-Ras comparable concentrations. microarray analysis, considerable overlap HAEC, suggesting some effects attributed may be regulated because both molecules inflammatory sites. Overall, provide evidence importance OxPAPC-cysteine interactions regulating function. Minimally modified LDL (mm-LDL) our group stimulate resulting recruitment monocytes vascular wall, important initial event early stages atherogenesis (1.Berliner J.A. Navab M. Fogelman A.M. Frank J.S. Demer L.L. Edwards P.A. Watson A.D. Lusis A.J. Atherosclerosis: basic mechanisms. oxidation, inflammation, genetics.Circulation. 1995; 91: 2488-2496Crossref PubMed Scopus (1583) Google Scholar). have subsequently several biologically components mm-LDL are oxidation naturally occurring phospholipid found membranes lipoproteins (2.Berliner Leitinger N. Tsimikas S. role oxidized phospholipids atherosclerosis.J. Lipid Res. 2009; 50: S207-S212Abstract Full Text PDF (173) (HAEC) (3.Romanoski C.E. Che Yin F. Mai Pouldar D. Civelek Pan C. Lee Vakili L. Yang W.P. et al.Network activation phospholipids: critical heme oxygenase 1.Circ. 2011; 109: e27-e41Crossref (92) Although others activates signaling pathways HAECs, primary has not been determined. this study event. addition showing strong expression OxPAPE-N-biotin (OxPNB), contains similar (4.Gugiu B.G. Mouillesseaux K. Duong V. Herzog T. Hekimian A. Koroniak Vondriska T.M. Protein targets cells.J. 2008; 49: 510-520Abstract (33) hypothesized covalent plays aspects action. For studies, developed methods synthesize oxidize PAPE-N-biotin produce OxPNB. appropriate OxPAPC. HAECs However, had determined including which lipid responsible amino acid hypothesize interaction electrophilic components, such available on proteins, address article. bioactive component respect it lowest concentration eight lipids tested assays formed free radical cyclization arachidonic PAPC, 5,6-expoxyisoprostane. α,β-unsaturated enone sn-2 position, capable Michael nucleophilic residues like lysine (Fig. 1). interacts action endothelium. structure suggests its important, there reasons why true. recent publication Gao al., reported two other bioactive, enone-containing phos-pholipids, KOdiaPC KDdiaPC, α,β-unsaturation does play major CD36 (5.Gao Ashraf M.Z. Kar N.S. Lin Sayre L.M. Podrez E.A. Structural basis recognition low density class B scavenger receptors SR-BI.J. Biol. Chem. 2010; 285: 4447-4454Abstract (48) Furthermore, epoxide amine-containing compound (used surrogate peptides containing lysine) (6.Jung M.E. Berliner Gugiu Improved synthesis epoxy isoprostane reactivity amines.Org. Lett. 10: 4207-4209Crossref (35) Scholar), confirmed cysteine. To possibility playing action, employed examined constituents total Ras central pathways, MAPK/ERK Akt induced (7.Zimman K.P. Le Gharavi N.M. Ryvkin Graeber T.G. Chen T.T. Vascular growth factor receptor 2 phospholipids.Arterioscler. Thromb. Vasc. 2007; 27: 332-338Crossref (41) Scholar, 8.Birukov K.G. Bochkov V.N. Garcia J.G. Signal transduction activated pulmonary lipoproteins.Microvasc. 2004; 67: 18-28Crossref (56) (9.Cole A.L. Subbanagounder G. Mukhopadhyay Vora D.K. Oxidized phospholipid-induced cell/monocyte cAMP-dependent R-Ras/PI3-kinase pathway.Arterioscler. 2003; 23: 1384-1390Crossref (121) 10.Fu P. Birukov control inflammation barrier.Transl. 153: 166-176Abstract (62) Scholar–11.Leitinger triggers atherosclerosis.Mol. Nutr. Food 2005; 1063-1071Crossref (63) balance between R-Ras part pathway leads deposition CS-1 fibronectin surface. Monocyte α4β1 then fibronectin. serves good played specific (12.Oliva J.L. Perez-Sala Castrillo Martinez Canada F.J. Bosca Rojas J.M. cyclopentenone 15-deoxy-delta 12,14-prostaglandin H-Ras.Proc. Natl. Acad. Sci. USA. 100: 4772-4777Crossref (118) 13.Roy Plowman Rotblat B. Prior I.A. Muncke Grainger Parton R.G. Henis Y.I. Kloog Y. Hancock J.F. Individual palmitoyl serve distinct roles H-ras trafficking, microlocalization, signaling.Mol. Cell. 25: 6722-6733Crossref (174) 14.Renedo Gayarre J. Garcia-Dominguez C.A. Perez-Rodriguez Prieto Modification prostanoids different site-selective.Biochemistry. 46: 6607-6616Crossref Scholar–15.Oeste C.L. Diez-Dacal Bray de Lacoba la Torre Andreu Ruiz-Sanchez Perez-Inestrosa E. al.The C-terminus target reactive compounds modulating Ras-dependent pathways.PLoS ONE. 6: e15866Crossref (28) current examines developing identify sites protein. striking similarities 15dPGJ2, prompting speculation chemical use elucidate acts compares 15dPGJ2. Finally, article explores using analysis. Ox-PAPC, expression. (PAPC) 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (PAPE) were purchased from Avanti Lipids. Recombinant EMD Biosciences or Abgent, kit Pierce Biotechnologies, anti-HA resin monoclonal antibody Roche, polyclonal Santa Cruz, strepavidin-HRP RD Biosciences, biotin, dimethylaminopyridine (DMAP), dicyclohexylcarbodiimide (DCC) Sigma-Aldrich. PAPE described PEIPE-NB isolated semipreparative, normal Phase LC-MS isocratic mobile phase 77:15:8 acteonitrile:water:methanol, published isolation (16.Watson Welsbie D.S. Faull K.F. Jung identification novel pro-inflammatory epoxyisoprostane mildly lipoprotein.J. 1999; 274: 24787-24798Abstract (185) (17.Berliner Territo M.C. Sevanian Ramin Kim Bamshad Esterson lipoprotein stimulates monocyte interactions.J. Clin. Invest. 1990; 85: 1260-1266Crossref (767) 18.Navab Imes S.S. Hama S.Y. Hough G.P. Ross L.A. Bork R.W. Valente Drinkwater D.C. Laks H. al.Monocyte transmigration modification cocultures wall due induction chemotactic 1 abolished high 1991; 88: 2039-2046Crossref (645) cultured VEC complete media (VEC Technologies), changed 10% FBS (Thermo Scientific) M199 (Mediatech) overnight before experiments. Human (hr-H-Ras) (1 μg) treated 50 μg/ml (52 μM) 12 μM 37°C 30 min. experiments, hr-H-Ras pretreated (NEM; mM) (158 Additional competition performed similarity 100 ng incubated 10 15 min, without pre- cotreatment 60 After incubation, analyzed Western blotting streptavidin-HRP (RD Systems). blots imaged Bio-Rad Versadoc™ 4000 system. Densitometry Quantity One software (Bio-Rad). 3 mM (NAC) 1% h. (64 added media, cotreated NAC (3 additional 4 Cells washed PBS lysed, RNA extracted qPCR analysis performed, measuring GAPDH, IL-8, ATF-3, HO-1 mRNA levels. GAPDH levels showed minimal change. normalized primer sequences used GAPDH: Forward: 5′-CCT CAA GAT CAT CAG TGC CTC CT-3′, Reverse: 5′-GGT GAG TCC TTC CAC ACC AA-3′ HO-1: 5′-ATA GTG GTA GGA GGC CA-3′, 5′-GGC AGA GAA TGA GTT GA-3′ IL-8: 5′-ACC ACA CTG CGC AAT-3′, 5′-TCC AGC TCT CTT CCA TCA ATF-3: 5′-TTG TAA GCA GTC GTA-3′, 5′-ATG TGG TCT-3′. μg total, μg/ml) Analytical LC/MS 1.0 × 150 mm Zorbax 300SB-C18 column (Agilent) flow rate μl/min gradient 5% acetonitrile 100% formic additive positive ion mode 75 held PC-containing species identified MS/MS ABI Sciex Qtrap instrument, searching ions full mass spectra daughter fragment 184 Da representing phosphatidylcholine headgroup. Hr-H-Ras molar ratio 1:10 (hr-H-Ras:OxPNB) 20 each denatured DTT, followed alkylation iodoacetamide. acetone-precipitated, pelleted centrifugation, resuspended pH 7.8 NH4HCO3. Trypsin according manufacturer specifications digestion. Samples desalted C18 ZipTip (Millipore) MS digested During tryptic digests, scans zoom four abundant scan. Database web-based Global Proteome Machine (GPM) Organization match data known peptides, modifications 57 account carbamidomethyl iodoacetimide treatment default settings. X! Tandem algorithm cutoff log(e) value less than −2 options GPM pEGFP-C3 vector (h-H-Ras) ORF kind gift Dr. Junji Yamauchi (Tokyo, Japan). plasmid encoded GFP-HA-H-Ras fusion GFP, 1xHA, sequentially. C181S C184S point mutations, long-range PCR amplification procedure employing primers mutations. GFP sequence removed, tag bind PEIPE-NB. integrity plasmids UCLA Sequencing Core Facility. Overexpression HEK293 plasmid. Our removing performing aforementioned site-directed mutagenesis selected Transfection done Lipofectamine 2000 specifications. grown 85–95% confluence transfection harvested day after transfection. lab achieves 90–100% conditions. transfected (with Ca Mg) NEM min 37°C. Then added, presence scraped ml cold PBS, centrifuged, centrifuged again. lysed RIPA buffer (Sigma) PMSF, phosphate inhibitors (Sigma), protease (Sigma). Lysate ml), one plate μl (Roche) 4°C, rotating overnight, immunoprecipitate beads three times boiled 5 45 Laemmeli sample β-mercaptoethanol elute detecting HA biotin. Active Pull Down detection (Pierce) 700 directions followed. Duplicate wells 64 h, extracted. prepared hybridization Illumina arrays, 45,000 probes, standard protocol Data Genome Studio package. background subtraction, quantile normalization, content descriptor file "HumanHT-12_V4_0_R1_15002873_B.bgx" files. filtered results either P 0.001 PEIPC/15dPGJ2/OxPAPC measurement measurement. fold changes probes calculated average probe level divided converted log values analyze regulation. Up/downregulated those least 1.25-fold lipids. Gene functional annotation NIH DAVID Bioinformatics Resources 6.7 website platform. IDs up/downregulated sent server, enrichment ontology (GO) categories medium stringency setting. 1e−6 lower significant GO category enrichment. Ingenuity database search common 0.05 generated list change 1.5 higher. Array Expression Omnibus accession GSE35709 supplementary tables. first hypothesis Treatment h HO-1, hub functions Pre- genes, measured 2A). next serum-free 2.5 (2.6 2B, lane 1) N-ethylmaleimide, simple cysteine-binding compound. run gel, probed (SA-HRP) detect biotinylated, inhibits 2). large band halfway down blot corresponds intrinsic nonspecific biotin present bands appear clearly diminished about 45, 90, 110, 200, 220 kDa. These marked arrows left figure. screened confirm accounts amount binding. experiment, μg/ml), cysteine-containing compound, reverse-phase LC-MS/MS, screening A sharp peak 40.96 subsequent peaks, corresponding unbound 3A). contained molecule MW 991 NAC. many lipids, adducts undetectable minimally detectable experiment demonstrates N-acetylcysteine. On observation, ability (PEIPE-NB) 3C). Next, analogs experiments expressed cells. lipid, SA-HRP but unoxidized (PNB), modifies commercially available, purified 4A). Covalent examined. HA-tagged PNB immunoprecipitated beads, anti-H-Ras SA-HRP. Similar hr-H-Ras, endogenous 4B). Previous headgroup can further examining (100 ng) incubated. samples 4C). overexpressed 70% inhibition (data shown). differences effect NEM. 0.5 5A). competed inhibit wild-type (10.4 resin, 5B). set modify blocks binding, interaction. (20 10:1 O