Evidence for the importance of OxPAPC interaction with cysteines in regulating endothelial cell function
2012; Elsevier BV; Volume: 53; Issue: 7 Linguagem: Inglês
10.1194/jlr.m025320
ISSN1539-7262
AutoresJames R. Springstead, Bogdan G. Gugiu, Sangderk Lee, Seung Cha, Andrew D. Watson, Judith A. Berliner,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoOxidation 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 and regulate over 1,000 genes in human aortic endothelial cells (HAEC). We previously demonstrated that OxPNB, a biotinylated analog of OxPAPC, covalently binds to a number of proteins in HAEC. The goal of these studies was to gain insight into the binding mechanism and determine whether binding regulates activity. In whole cells, N-acetylcysteine inhibited gene regulation by OxPAPC, and blocking cell cysteines with N-ethylmaleimide strongly inhibited the binding of OxPNB to HAEC proteins. Using MS, we demonstrate that most of the binding of OxPAPC to cysteine is mediated by PEIPC. We also show that OxPNB and PEIPE-NB, the analog of PEIPC, bound to a model protein, H-Ras, at cysteines previously shown to regulate activity in response to 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2). This binding was observed with recombinant protein and in cells overexpressing H-Ras. OxPAPC and PEIPC compete with OxPNB for binding to H-Ras. 15dPGJ2 and OxPAPC increased H-Ras activity at comparable concentrations. Using microarray analysis, we demonstrate a considerable overlap of gene regulation by OxPAPC, PEIPC, and 15dPGJ2 in HAEC, suggesting that some effects attributed to 15dPGJ2 may also be regulated by PEIPC because both molecules accumulate in inflammatory sites. Overall, we provide evidence for the importance of OxPAPC-cysteine interactions in regulating HAEC function. 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 and regulate over 1,000 genes in human aortic endothelial cells (HAEC). We previously demonstrated that OxPNB, a biotinylated analog of OxPAPC, covalently binds to a number of proteins in HAEC. The goal of these studies was to gain insight into the binding mechanism and determine whether binding regulates activity. In whole cells, N-acetylcysteine inhibited gene regulation by OxPAPC, and blocking cell cysteines with N-ethylmaleimide strongly inhibited the binding of OxPNB to HAEC proteins. Using MS, we demonstrate that most of the binding of OxPAPC to cysteine is mediated by PEIPC. We also show that OxPNB and PEIPE-NB, the analog of PEIPC, bound to a model protein, H-Ras, at cysteines previously shown to regulate activity in response to 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2). This binding was observed with recombinant protein and in cells overexpressing H-Ras. OxPAPC and PEIPC compete with OxPNB for binding to H-Ras. 15dPGJ2 and OxPAPC increased H-Ras activity at comparable concentrations. Using microarray analysis, we demonstrate a considerable overlap of gene regulation by OxPAPC, PEIPC, and 15dPGJ2 in HAEC, suggesting that some effects attributed to 15dPGJ2 may also be regulated by PEIPC because both molecules accumulate in inflammatory sites. Overall, we provide evidence for the importance of OxPAPC-cysteine interactions in regulating HAEC function. Minimally modified LDL (mm-LDL) was demonstrated by our group to stimulate endothelial cells, resulting in the recruitment of monocytes to the vascular wall, an important initial event in the early stages of 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, and genetics.Circulation. 1995; 91: 2488-2496Crossref PubMed Scopus (1583) Google Scholar). We have subsequently shown that several biologically active components of mm-LDL are the oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphatidylcholine (PAPC), a naturally occurring phospholipid found in cell membranes and lipoproteins (2.Berliner J.A. Leitinger N. Tsimikas S. The role of oxidized phospholipids in atherosclerosis.J. Lipid Res. 2009; 50: S207-S212Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). OxPAPC was also shown to regulate over 1,000 genes in human aortic endothelial cells (HAEC) (3.Romanoski C.E. Che N. Yin F. Mai N. Pouldar D. Civelek M. Pan C. Lee S. Vakili L. Yang W.P. et al.Network for activation of human endothelial cells by oxidized phospholipids: a critical role of heme oxygenase 1.Circ. Res. 2011; 109: e27-e41Crossref PubMed Scopus (92) Google Scholar). Although our group and others have demonstrated that OxPAPC activates several signaling pathways in HAECs, the primary event in OxPAPC signaling has not been determined. The goal of this study was to gain insight into this primary event. In addition to our studies showing strong activity of OxPAPC in regulating gene expression in endothelial cells, we have shown that a biotinylated analog of OxPAPC, OxPAPE-N-biotin (OxPNB), covalently binds to a group of endothelial cell proteins and contains similar oxidation products (4.Gugiu B.G. Mouillesseaux K. Duong V. Herzog T. Hekimian A. Koroniak L. Vondriska T.M. Watson A.D. Protein targets of oxidized phospholipids in endothelial cells.J. Lipid Res. 2008; 49: 510-520Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). We hypothesized that this covalent binding plays a role in some aspects of OxPAPC action. For these studies, we developed methods to synthesize and oxidize PAPE-N-biotin to produce OxPNB. We subsequently demonstrated that OxPNB regulates a number of genes that are regulated by OxPAPC, suggesting that OxPNB is an appropriate analog of OxPAPC. We demonstrated covalent binding of OxPNB to a group of proteins in HAECs (4.Gugiu B.G. Mouillesseaux K. Duong V. Herzog T. Hekimian A. Koroniak L. Vondriska T.M. Watson A.D. Protein targets of oxidized phospholipids in endothelial cells.J. Lipid Res. 2008; 49: 510-520Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). However, we previously had not determined the mechanism of the covalent binding by OxPAPC, including which lipid is responsible for this binding and which amino acid is bound by OxPAPC. We hypothesize that a primary mechanism of this binding is the interaction of electrophilic OxPAPC components, such as 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphatidylcholine (PEIPC), with available cysteines on endothelial proteins, and we address this mechanism in this article. PEIPC is the most bioactive component in OxPAPC with respect to gene regulation in HAECs, and it is active at the lowest concentration of the eight OxPAPC lipids that we have tested in several assays (3.Romanoski C.E. Che N. Yin F. Mai N. Pouldar D. Civelek M. Pan C. Lee S. Vakili L. Yang W.P. et al.Network for activation of human endothelial cells by oxidized phospholipids: a critical role of heme oxygenase 1.Circ. Res. 2011; 109: e27-e41Crossref PubMed Scopus (92) Google Scholar). This lipid is formed by the free radical oxidation and cyclization of the arachidonic acid group of PAPC, resulting in a 5,6-expoxyisoprostane. PEIPC has an electrophilic α,β-unsaturated enone group in the sn-2 position, which is capable of Michael addition with nucleophilic amino acid residues like cysteine and lysine (Fig. 1). We hypothesize that PEIPC interacts with cysteines on proteins, and that this interaction of PEIPC is an important mechanism in the action of OxPAPC on the endothelium. Although the structure of PEIPC suggests that its interaction with cysteines is important, there are several reasons why this may not be true. In a recent publication by Gao et al., it is reported that for two other bioactive, α,β-unsaturated enone-containing oxidized phos-pholipids, KOdiaPC and KDdiaPC, the α,β-unsaturation does not play a major role in the interaction of these phospholipids with CD36 (5.Gao D. Ashraf M.Z. Kar N.S. Lin D. Sayre L.M. Podrez E.A. Structural basis for the recognition of oxidized phospholipids in oxidized low density lipoproteins by class B scavenger receptors CD36 and SR-BI.J. Biol. Chem. 2010; 285: 4447-4454Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Furthermore, we previously demonstrated that the epoxide group of PEIPC bound strongly to a model amine-containing compound (used as a surrogate compound for peptides containing lysine) (6.Jung M.E. Berliner J.A. Koroniak L. Gugiu B.G. Watson A.D. Improved synthesis of the epoxy isoprostane phospholipid PEIPC and its reactivity with amines.Org. Lett. 2008; 10: 4207-4209Crossref PubMed Scopus (35) Google Scholar), and we had not previously confirmed the binding of PEIPC to cysteine. To address the possibility of OxPAPC-cysteine interactions playing a role in OxPAPC action, we employed OxPNB. We examined the binding of OxPNB and its constituents to total HAEC protein and to a model protein, H-Ras. Ras is a central protein in cell signaling in inflammatory pathways, including the activation of MAPK/ERK and Akt pathways, which have both been reported by our group and others to be induced by OxPAPC (7.Zimman A. Mouillesseaux K.P. Le T. Gharavi N.M. Ryvkin A. Graeber T.G. Chen T.T. Watson A.D. Berliner J.A. Vascular endothelial growth factor receptor 2 plays a role in the activation of aortic endothelial cells by oxidized phospholipids.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 332-338Crossref PubMed Scopus (41) Google Scholar, 8.Birukov K.G. Leitinger N. Bochkov V.N. Garcia J.G. Signal transduction pathways activated in human pulmonary endothelial cells by OxPAPC, a bioactive component of oxidized lipoproteins.Microvasc. Res. 2004; 67: 18-28Crossref PubMed Scopus (56) Google Scholar). Furthermore, our group has reported that H-Ras has a role in the recruitment of monocytes to endothelial cells, an important initial event in atherogenesis (9.Cole A.L. Subbanagounder G. Mukhopadhyay S. Berliner J.A. Vora D.K. Oxidized phospholipid-induced endothelial cell/monocyte interaction is mediated by a cAMP-dependent R-Ras/PI3-kinase pathway.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1384-1390Crossref PubMed Scopus (121) Google Scholar, 10.Fu P. Birukov K.G. Oxidized phospholipids in control of inflammation and endothelial barrier.Transl. Res. 2009; 153: 166-176Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar–11.Leitinger N. Oxidized phospholipids as triggers of inflammation in atherosclerosis.Mol. Nutr. Food Res. 2005; 49: 1063-1071Crossref PubMed Scopus (63) Google Scholar). We demonstrated that the balance between H-Ras and R-Ras is part of a signaling pathway that leads to deposition of CS-1 containing fibronectin on the endothelial cell surface. Monocyte α4β1 then binds to the CS-1 fibronectin. H-Ras also serves as a good model because there has been considerable study of the role played by specific cysteines in Ras activation (12.Oliva J.L. Perez-Sala D. Castrillo A. Martinez N. Canada F.J. Bosca L. Rojas J.M. The cyclopentenone 15-deoxy-delta 12,14-prostaglandin J2 binds to and activates H-Ras.Proc. Natl. Acad. Sci. USA. 2003; 100: 4772-4777Crossref PubMed Scopus (118) Google Scholar, 13.Roy S. Plowman S. Rotblat B. Prior I.A. Muncke C. Grainger S. Parton R.G. Henis Y.I. Kloog Y. Hancock J.F. Individual palmitoyl residues serve distinct roles in H-ras trafficking, microlocalization, and signaling.Mol. Cell. Biol. 2005; 25: 6722-6733Crossref PubMed Scopus (174) Google Scholar, 14.Renedo M. Gayarre J. Garcia-Dominguez C.A. Perez-Rodriguez A. Prieto A. Canada F.J. Rojas J.M. Perez-Sala D. Modification and activation of Ras proteins by electrophilic prostanoids with different structure are site-selective.Biochemistry. 2007; 46: 6607-6616Crossref PubMed Scopus (62) Google Scholar–15.Oeste C.L. Diez-Dacal B. Bray F. Garcia de Lacoba M. de la Torre B.G. Andreu D. Ruiz-Sanchez A.J. Perez-Inestrosa E. Garcia-Dominguez C.A. Rojas J.M. et al.The C-terminus of H-Ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways.PLoS ONE. 2011; 6: e15866Crossref PubMed Scopus (28) Google Scholar). The current study examines the mechanism by which OxPNB interacts with H-Ras. We demonstrate that this interaction is specific by developing methods to identify OxPNB binding sites on the protein. We also demonstrate that OxPAPC activates H-Ras. PEIPC has striking similarities in structure to 15dPGJ2, including an electrophilic enone group and a cyclopentenone component, prompting our speculation of similar chemical activity (Fig. 1). For these studies we use 15dPGJ2 as a model compound to elucidate the mechanism by which OxPAPC acts on HAEC. The current study compares the binding of 15dPGJ2, PEIPC, and OxPNB to H-Ras, and it examines H-Ras activation by OxPAPC and 15dPGJ2. Finally, this article explores similarities in gene regulation by OxPAPC, PEIPC, and 15dPGJ2 using microarray analysis. Overall, we demonstrate the importance of cysteines in OxPNB, Ox-PAPC, and PEIPC binding to endothelial cells and regulation of gene expression. 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphatidylcholine (PAPC) and 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (PAPE) were purchased from Avanti Lipids. Recombinant H-Ras was purchased from EMD Biosciences or Abgent, the H-Ras activation kit from Pierce Biotechnologies, anti-HA resin and monoclonal anti-HA antibody from Roche, polyclonal H-Ras antibody from Santa Cruz, strepavidin-HRP from RD Biosciences, and biotin, dimethylaminopyridine (DMAP), and dicyclohexylcarbodiimide (DCC) from Sigma-Aldrich. PAPE was biotinylated and oxidized as described previously (4.Gugiu B.G. Mouillesseaux K. Duong V. Herzog T. Hekimian A. Koroniak L. Vondriska T.M. Watson A.D. Protein targets of oxidized phospholipids in endothelial cells.J. Lipid Res. 2008; 49: 510-520Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). PEIPE-NB was isolated from OxPNB with semipreparative, normal Phase LC-MS using an isocratic mobile phase of 77:15:8 acteonitrile:water:methanol, as previously published for PEIPC isolation (16.Watson A.D. Subbanagounder G. Welsbie D.S. Faull K.F. Navab M. Jung M.E. Fogelman A.M. Berliner J.A. Structural identification of a novel pro-inflammatory epoxyisoprostane phospholipid in mildly oxidized low density lipoprotein.J. Biol. Chem. 1999; 274: 24787-24798Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). HAECs were isolated as described previously (17.Berliner J.A. Territo M.C. Sevanian A. Ramin S. Kim J.A. Bamshad B. Esterson M. Fogelman A.M. Minimally modified low density lipoprotein stimulates monocyte endothelial interactions.J. Clin. Invest. 1990; 85: 1260-1266Crossref PubMed Scopus (767) Google Scholar, 18.Navab M. Imes S.S. Hama S.Y. Hough G.P. Ross L.A. Bork R.W. Valente A.J. Berliner J.A. Drinkwater D.C. Laks H. et al.Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein.J. Clin. Invest. 1991; 88: 2039-2046Crossref PubMed Scopus (645) Google Scholar). HAECs were cultured in VEC complete media (VEC Technologies), and media was changed to 10% FBS (Thermo Scientific) in M199 media (Mediatech) overnight before use in experiments. Human recombinant H-Ras (hr-H-Ras) (1 μg) was treated with 50 μg/ml (52 μM) OxPNB or 12 μM PEIPE-NB at 37°C for 30 min. In some experiments, hr-H-Ras was pretreated with N-ethylmaleimide (NEM; 1 mM) or 50 μg/ml (158 μM) 15dPGJ2 for 30 min. Additional competition studies were performed to determine similarity in binding of OxPAPC, PEIPC, and OxPNB. In these studies, 100 ng H-Ras was incubated with 10 μM OxPNB for 15 min, with or without pre- and cotreatment with 100 μM OxPAPC or 10 μM PEIPC for 60 min. After lipid incubation, protein was analyzed with Western blotting using streptavidin-HRP (RD Systems). Western blots were imaged on a Bio-Rad Versadoc™ 4000 system. Densitometry was performed using Quantity One software (Bio-Rad). HAECs were cultured and pretreated in 3 mM N-acetylcysteine (NAC) in M199 media containing 1% FBS for 1 h. 50 μg/ml (64 μM) OxPAPC was then added to the media, and the cells were cotreated with NAC (3 mM) and OxPAPC for an additional 4 h. Cells were washed with PBS and lysed, and then RNA was extracted and qPCR analysis was performed, measuring GAPDH, IL-8, ATF-3, and HO-1 mRNA levels. GAPDH levels showed minimal change. IL-8, ATF-3, and HO-1 levels were normalized to GAPDH levels. The primer sequences used for qPCR were GAPDH: Forward: 5′-CCT CAA GAT CAT CAG CAA TGC CTC CT-3′, Reverse: 5′-GGT CAT GAG TCC TTC CAC GAT ACC AA-3′ HO-1: Forward: 5′-ATA GAT GTG GTA CAG GGA GGC CAT CA-3′, Reverse: 5′-GGC AGA GAA TGC TGA GTT CAT GAG GA-3′ IL-8: Forward: 5′-ACC ACA CTG CGC CAA CAC AGA AAT-3′, Reverse: 5′-TCC AGA CAG AGC TCT CTT CCA TCA GA-3′ ATF-3: Forward: 5′-TTG CAG AGC TAA GCA GTC GTG GTA-3′, Reverse: 5′-ATG GTT CTC TGC TGC TGG GAT TCT-3′. N-acetylcysteine (1 μg total, 50 μg/ml) was incubated with 50 μg/ml (64 μM) OxPAPC in PBS at 37°C for 4 h. Analytical LC/MS was then performed on a 1.0 × 150 mm Zorbax 300SB-C18 column (Agilent) using a flow rate of 50 μl/min and a gradient of 5% acetonitrile to 100% acetonitrile with 1 mM formic acid as an additive in positive ion mode over 75 min, and then held at 100% acetonitrile for an additional 30 min. PC-containing species were identified using MS/MS on an ABI Sciex 4000 Qtrap instrument, searching for ions in the full mass spectra with a daughter ion fragment of 184 Da representing the phosphatidylcholine headgroup. Hr-H-Ras was incubated with or without OxPNB at a molar ratio of 1:10 (hr-H-Ras:OxPNB) at 37°C for 30 min. 20 μg H-Ras was used for each MS/MS analysis. Hr-H-Ras was denatured with 1 mM DTT, followed by alkylation with 100 mM iodoacetamide. The protein was acetone-precipitated, pelleted by centrifugation, and resuspended in pH 7.8 50 mM NH4HCO3. Trypsin was added according to manufacturer specifications and incubated at 37°C overnight for complete digestion. Samples were desalted with C18 ZipTip (Millipore) for MS analysis. Analytical LC/MS of digested H-Ras peptides was performed on a 1.0 × 150 mm Zorbax 300SB-C18 column (Agilent) using a flow rate of 50 μl/min and a gradient of 5% acetonitrile to 100% acetonitrile with 1 mM formic acid as an additive in positive ion mode over 75 min, and then held at 100% acetonitrile for an additional 30 min. During analysis of tryptic digests, full MS scans were performed followed by zoom scans and MS/MS scans of the four most abundant ions in the full scan. Database searching for peptides was performed using the web-based Global Proteome Machine (GPM) Organization to match MS/MS data to known peptides, using modifications of 57 Da at cysteines to account for carbamidomethyl modifications from iodoacetimide treatment and other default settings. The X! Tandem algorithm and a cutoff of peptides with a log(e) value of less than −2 were used as options in the GPM analysis. The pEGFP-C3 vector containing human H-Ras (h-H-Ras) ORF was a kind gift from Dr. Junji Yamauchi (Tokyo, Japan). The plasmid encoded GFP-HA-H-Ras fusion protein, which contains GFP, 1xHA, and H-Ras, sequentially. To produce C181S and C184S point mutations, we used a long-range PCR amplification procedure employing appropriate primers with point mutations. Finally, the GFP sequence was removed, as the GFP tag contains cysteines and may bind OxPNB and PEIPE-NB. The integrity of plasmids was confirmed at the UCLA Sequencing Core Facility. Overexpression of H-Ras in HEK293 cells was performed using a modified plasmid. Our group modified the plasmid by removing the GFP tag and performing the aforementioned site-directed mutagenesis on selected cysteine sites. Transfection of HEK293 cells was done with Lipofectamine 2000 according to manufacturer specifications. Cells were grown to 85–95% confluence before transfection and harvested the day after transfection. Our lab achieves 90–100% transfection for HEK293 cells using these conditions. To study binding of OxPNB to transfected HEK293 cells, cells were pretreated in PBS (with Ca and Mg) with or without NEM (1 mM) for 60 min or 50 μg/ml (158 μM) 15dPGJ2 for 30 min at 37°C. Then 50 μg/ml (52 μM) OxPNB was added, and cells were incubated for an additional 15 min in the presence of NEM or 30 min in the presence of 15dPGJ2. Cells were scraped into 3 ml cold PBS, centrifuged, washed with an additional 10 ml PBS, and centrifuged again. Cells were then lysed in RIPA buffer (Sigma) containing PMSF, phosphate inhibitors (Sigma), and protease inhibitors (Sigma). Lysate (1 ml), from one 100 mm plate of transfected HEK293 cells was incubated with 60 μl anti-HA resin (Roche) at 4°C, rotating overnight, to immunoprecipitate H-Ras. We centrifuged and washed the beads three times in cold PBS, and then boiled for 5 min in 45 μl Laemmeli sample buffer with 5% β-mercaptoethanol to elute H-Ras. Western blotting was then performed, detecting HA and biotin. H-Ras activity was tested using an Active Ras Pull Down and detection kit (Pierce) according to manufacturer specifications. HAECs were cultured and washed with PBS and treated with 50 μg/ml (64 μM) OxPAPC or 20 μg/ml (64 μM) 15dPGJ2 in PBS and incubated for 60 min. After incubation, cells were scraped into PBS, centrifuged, washed with PBS, and lysed with 700 μl RIPA buffer (Sigma). The kit directions were then followed. Duplicate wells of HAECs were treated with 1% M199 media or media containing 64 μM OxPAPC, 5 μM PEIPC, or 5 μM 15dPGJ2 for 4 h, and then RNA was extracted. RNA was prepared for hybridization to Illumina arrays, measuring 45,000 probes, using a standard protocol described previously (3.Romanoski C.E. Che N. Yin F. Mai N. Pouldar D. Civelek M. Pan C. Lee S. Vakili L. Yang W.P. et al.Network for activation of human endothelial cells by oxidized phospholipids: a critical role of heme oxygenase 1.Circ. Res. 2011; 109: e27-e41Crossref PubMed Scopus (92) Google Scholar). Data were analyzed using the Genome Studio software package. Data were analyzed using background subtraction, quantile normalization, and the content descriptor file "HumanHT-12_V4_0_R1_15002873_B.bgx" in the Genome Studio files. The data were filtered for results with either the detection P value less than 0.001 for the PEIPC/15dPGJ2/OxPAPC RNA measurement or for the control measurement. The fold changes in probes were calculated as the average probe level for each treatment divided by the average probe level for control treatment and converted to log 2 values to analyze for gene regulation. Up/downregulated genes were identified as those which changed at least 1.25-fold from control values in the presence of lipids. Gene functional annotation analysis on regulated genes was performed using the NIH DAVID Bioinformatics Resources 6.7 website platform. The Illumina IDs of probes that were identified as up/downregulated from the initial Genome Studio analysis were sent to the DAVID server, and the genes were analyzed for enrichment of gene ontology (GO) annotation categories using the medium stringency setting. We used a P value cutoff of 1e−6 or lower for significant GO category enrichment. Furthermore, we used the Ingenuity software database to search for common pathways regulated by OxPAPC and 15dPGJ2. For this analysis, we used a detection P value cutoff of 0.05 and generated an Illumina probe list of those with a fold change of 1.5 or higher. Array data are available in Gene Expression Omnibus accession GSE35709 and in the supplementary tables. We first tested the hypothesis that cysteines play an important role in OxPAPC action. Treatment of HAECs with 50 μg/ml (64 μM) OxPAPC for 4 h increased the expression of IL-8, HO-1, and ATF-3, hub genes representing three major functions regulated by OxPAPC. Pre- and cotreatment with 3 mM NAC strongly inhibited OxPAPC induction of these genes, as measured by qPCR (Fig. 2A). We next examined the role of cysteine in covalent binding of OxPAPC to HAEC proteins using the biotinylated analog OxPNB. HAECs were cultured in VEC media and then incubated in serum-free M199 media for 15 min with 2.5 μg/ml (2.6 μM) OxPNB (Fig. 2B, lane 1) with or without pre- and cotreatment with 1 mM N-ethylmaleimide, a simple cysteine-binding compound. The cells were then lysed, run on a gel, and probed by Western blotting with streptavidin-HRP (SA-HRP) to detect biotinylated, modified protein. Treatment of HAECs with NEM strongly inhibits binding of OxPNB to several HAEC proteins (Fig. 2B, lane 2). The large band halfway down the blot corresponds to intrinsic nonspecific biotin binding protein, present in HAECs (4.Gugiu B.G. Mouillesseaux K. Duong V. Herzog T. Hekimian A. Koroniak L. Vondriska T.M. Watson A.D. Protein targets of oxidized phospholipids in endothelial cells.J. Lipid Res. 2008; 49: 510-520Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The bands that appear most clearly diminished are at about 45, 90, 110, 200, and 220 kDa. These bands are marked by arrows on the left of the figure. These studies demonstrate an important role for cysteine in OxPAPC action and covalent binding to HAEC proteins. We next screened for OxPAPC lipids that covalently bind cysteines to confirm our hypothesis that PEIPC accounts for a significant amount of this binding. In this experiment, we incubated NAC (1 μg total, 50 μg/ml), a simple cysteine-containing compound, with 50 μg/ml (64 μM) OxPAPC for 4 h at 37°C and analyzed the sample with reverse-phase LC-MS/MS, screening for compounds with a daughter fragment of 184 Da representing the phosphatidylcholine headgroup. A sharp peak was shown to elute at 40.96 min followed by subsequent peaks, corresponding to unbound OxPAPC lipid components (Fig. 3A). This peak contained a molecule of MW 991 corresponding to PEIPC bound to NAC. Although OxPAPC contains many lipids, NAC adducts with other OxPAPC components were either undetectable or minimally detectable over several experiments. This experiment demonstrates that PEIPC is the primary OxPAPC component that covalently binds N-acetylcysteine. On the basis of this observation, we tested the ability of biotinylated PEIPC (PEIPE-NB) to bind to HAEC proteins. We demonstrated that PEIPE-NB also has strong binding to many HAEC proteins (Fig. 3C). Next, we tested the hypothesis that OxPAPC and PEIPC bind to cysteines in H-Ras by using the biotinylated analogs OxPNB and PEIPE-NB. We performed experiments to determine whether OxPNB and PEIPE-NB bind hr-H-Ras and H-Ras expressed in cells. H-Ras was treated with lipid, run on a gel, and probed with Western blotting with SA-HRP to detect modified protein. We demonstrated that oxidized PAPE-N-biotin (OxPNB), but not unoxidized PAPE-N-biotin (PNB), covalently modifies commercially available, purified hr-H-Ras (Fig. 4A). Covalent binding of H-Ras in cells by OxPNB was also examined. HEK293 cells were transfected with HA-tagged H-Ras. Cells were treated with 50 μg/ml (52 μM) OxPNB or 50 μg/ml (52 μM) PNB for 30 min, lysed, and then H-Ras was immunoprecipitated with anti-HA beads, followed by Western blotting with anti-H-Ras and SA-HRP. Similar to results with hr-H-Ras, endogenous H-Ras in transfected HEK293 cells was covalently modified by OxPNB but not by PNB (Fig. 4B). Previous studies have shown that headgroup can play an important role in the interaction of oxidized phospholipids with cells (5.Gao D. Ashraf M.Z. Kar N.S. Lin D. Sayre L.M. Podrez E.A. Structural basis for the recognition of oxidized phospholipids in oxidized low density lipoproteins by class B scavenger receptors CD36 and SR-BI.J. Biol. Chem. 2010; 285: 4447-4454Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). To provide further evidence that OxPNB is a good surrogate for OxPAPC and its component PEIPC, we performed competition studies, examining the ability of these molecules to compete with OxPNB for H-Ras binding. Hr-H-Ras (100 ng) was incubated in PBS with or without 100 μM OxPAPC or 10 μM PEIPC. Then 10 μM OxPNB was added, and the sample was further incubated. The samples were then examined with Western blotting using SA-HRP to detect modified H-Ras (Fig. 4C). These results show strong competition of OxPAPC and PEIPC for OxPNB binding to H-Ras. We also tested the ability of OxPAPC to compete with OxPNB for binding to H-Ras overexpressed in HEK293 cells. A 70% inhibition was observed (data not shown). These studies demonstrate that headgroup differences have a minimal effect on binding to H-Ras. We next examined the effect of NEM on covalent binding of OxPNB and PEIPE-NB to human recombinant H-Ras. Hr-H-Ras (1 μg) was incubated in PBS with or without 1 mM NEM. Then 1 μg OxPNB or 0.5 μg PEIPE-NB was added, and the samples were further incubated and examined with Western blotting for H-Ras modification (Fig. 5A). We demonstrate that OxPNB and PEIPE-NB binding to hr-H-Ras was strongly competed by NEM. We also tested the ability of NEM to inhibit the binding of OxPNB to H-Ras that is overexpressed in cells. HEK293 cells overexpressing HA-tagged wild-type H-Ras were pretreated with or without 1 mM NEM for 60 min and then cotreated with 10 μg/ml (10.4 μM) OxPNB for 15 min. The cells were lysed, H-Ras was immunoprecipitated with anti-HA resin, and samples were analyzed for modified H-Ras with Western blotting (Fig. 5B). The results show that in cells the binding of OxPNB to H-Ras was inhibited by pre- and cotreatment with NEM. This set of experiments demonstrated that both OxPNB and PEIPE-NB covalently modify H-Ras and that NEM blocks this binding, showing that cysteines are important in this interaction. To determine which cysteines bound H-Ras, we employed both recombinant and overexpressed H-Ras. Hr-H-Ras (20 μg) was incubated with or without OxPNB at 10:1 O
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