Lipopolysaccharide Upregulates Palmitoylated Enzymes of the Phosphatidylinositol Cycle: An Insight from Proteomic Studies
2017; Elsevier BV; Volume: 17; Issue: 2 Linguagem: Inglês
10.1074/mcp.ra117.000050
ISSN1535-9484
AutoresJustyna Sobocińska, Paula Roszczenko, Monika Zaręba-Kozioł, Aneta Hromada‐Judycka, Orest V. Matveichuk, Gabriela Traczyk, Katarzyna Łukasiuk, Katarzyna Kwiatkowska,
Tópico(s)Diabetes and associated disorders
ResumoLipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria that induces strong proinflammatory reactions of mammals. These processes are triggered upon sequential binding of LPS to CD14, a GPI-linked plasma membrane raft protein, and to the TLR4/MD2 receptor complex. We have found earlier that upon LPS binding, CD14 triggers generation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a lipid controlling subsequent proinflammatory cytokine production. Here we show that stimulation of RAW264 macrophage-like cells with LPS induces global changes of the level of fatty-acylated, most likely palmitoylated, proteins. Among the acylated proteins that were up-regulated in those conditions were several enzymes of the phosphatidylinositol cycle. Global profiling of acylated proteins was performed by metabolic labeling of RAW264 cells with 17ODYA, an analogue of palmitic acid functionalized with an alkyne group, followed by detection and enrichment of labeled proteins using biotin-azide/streptavidin and their identification with mass spectrometry. This proteomic approach revealed that 154 fatty-acylated proteins were up-regulated, 186 downregulated, and 306 not affected in cells stimulated with 100 ng/ml LPS for 60 min. The acylated proteins affected by LPS were involved in diverse biological functions, as found by Ingenuity Pathway Analysis. Detailed studies of 17ODYA-labeled and immunoprecipitated proteins revealed that LPS induces S-palmitoylation, hence activation, of type II phosphatidylinositol 4-kinase (PI4KII) β, which phosphorylates phosphatidylinositol to phosphatidylinositol 4-monophosphate, a PI(4,5)P2 precursor. Silencing of PI4KIIβ and PI4KIIα inhibited LPS-induced expression and production of proinflammatory cytokines, especially in the TRIF-dependent signaling pathway of TLR4. Reciprocally, this LPS-induced signaling pathway was significantly enhanced after overexpression of PI4KIIβ or PI4KIIα; this was dependent on palmitoylation of the kinases. However, the S-palmitoylation of PI4KIIα, hence its activity, was constitutive in RAW264 cells. Taken together the data indicate that LPS triggers S-palmitoylation and activation of PI4KIIβ, which generates PI(4)P involved in signaling pathways controlling production of proinflammatory cytokines. Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria that induces strong proinflammatory reactions of mammals. These processes are triggered upon sequential binding of LPS to CD14, a GPI-linked plasma membrane raft protein, and to the TLR4/MD2 receptor complex. We have found earlier that upon LPS binding, CD14 triggers generation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a lipid controlling subsequent proinflammatory cytokine production. Here we show that stimulation of RAW264 macrophage-like cells with LPS induces global changes of the level of fatty-acylated, most likely palmitoylated, proteins. Among the acylated proteins that were up-regulated in those conditions were several enzymes of the phosphatidylinositol cycle. Global profiling of acylated proteins was performed by metabolic labeling of RAW264 cells with 17ODYA, an analogue of palmitic acid functionalized with an alkyne group, followed by detection and enrichment of labeled proteins using biotin-azide/streptavidin and their identification with mass spectrometry. This proteomic approach revealed that 154 fatty-acylated proteins were up-regulated, 186 downregulated, and 306 not affected in cells stimulated with 100 ng/ml LPS for 60 min. The acylated proteins affected by LPS were involved in diverse biological functions, as found by Ingenuity Pathway Analysis. Detailed studies of 17ODYA-labeled and immunoprecipitated proteins revealed that LPS induces S-palmitoylation, hence activation, of type II phosphatidylinositol 4-kinase (PI4KII) β, which phosphorylates phosphatidylinositol to phosphatidylinositol 4-monophosphate, a PI(4,5)P2 precursor. Silencing of PI4KIIβ and PI4KIIα inhibited LPS-induced expression and production of proinflammatory cytokines, especially in the TRIF-dependent signaling pathway of TLR4. Reciprocally, this LPS-induced signaling pathway was significantly enhanced after overexpression of PI4KIIβ or PI4KIIα; this was dependent on palmitoylation of the kinases. However, the S-palmitoylation of PI4KIIα, hence its activity, was constitutive in RAW264 cells. Taken together the data indicate that LPS triggers S-palmitoylation and activation of PI4KIIβ, which generates PI(4)P involved in signaling pathways controlling production of proinflammatory cytokines. Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. Upon infection, LPS induces strong proinflammatory responses that facilitate eradication of bacteria. These proinflammatory reactions are triggered upon recognition of LPS by Toll-like receptor 4 (TLR4) (1.Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: Mutations in Tlr4 gene.Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6421) Google Scholar), which is expressed in cells of myeloid lineage, such as monocytes, macrophages, and dendritic cells, and certain nonimmune cells like endothelial and epithelial cells. TLR4 belongs to so-called pattern recognizing receptors specialized in identification of evolutionarily conserved constituents of cell wall/membranes of microbes, their RNA, and unmethylated CpG DNA motifs. These receptors trigger innate immune responses constituting the first line of defense against pathogens and initiate appropriate adaptive immune responses (2.Brubaker S.W. Bonham K.S. Zanoni I. Kagan J.C. Innate immune pattern recognition: A cell biological perspective.Annu. Rev. Immunol. 2015; 33: 257-290Crossref PubMed Scopus (878) Google Scholar). The LPS-induced proinflammatory reaction, although initially beneficial, when exaggerated can lead to a potentially fatal systemic inflammation called sepsis, severe sepsis, and septic shock (3.Angus D.C. van der Poll T. Severe sepsis and septic shock.N. Engl. J. Med. 2013; 369: 840-851Crossref PubMed Scopus (1837) Google Scholar). 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We have found earlier that LPS induces accumulation of S-palmitoylated Lyn kinase in the raft-enriched fraction of RAW264 macrophage-like cells, which determines negative regulation of TLR4 signaling by the kinase (39.Borzęcka-Solarz K. Dembińska J. Hromada-Judycka A. Traczyk G. Ciesielska A. Ziemlińska E. Świątkowska A. Kwiatkowska K. Association of Lyn kinase with membrane rafts determines its negative influence on LPS-induced signaling.Mol. Biol. Cell. 2017; 28: 1147-1159Crossref PubMed Google Scholar). Taking into account the redistribution and activation of proteins involved in the MyD88- and TRIF-dependent signaling pathways of TLR4, we assumed that changes of the level of S-palmitoylated proteins could contribute substantially to those complex cascades of events. In order to identify acylated proteins affected by LPS, in this study we metabolically labeled RAW264 cells with a palmitic acid analogue, 17-octadecynoic acid (17ODYA) 1The abbreviations used are: 17ODYA, 17-octadecynoic acid; AP-1, adaptor protein-1; BPA, bromohexadecanoic acid; BSA, bovine serum albumin; CCL5/RANTES, C-C motif chemokine ligand 5/regulated upon activation, normal T cell expressed and secreted; DGKε, diacylglycerol kinase-ε; eIF5A2, eukaryotic translation initiation factor 5A2; FBS, fetal bovine serum; GPI, glycosylphosphatidylinositol; IP3, inositol 1,4,5-trisphosphate; IPA, Ingenuity Pathway Analysis; ISRE, interferon-stimulated response element; LPS, lipopolysaccharide; PBS, phosphate buffered saline; PI, phosphatidylinositol; PI(4)P, phosphatidylinositol 4-monophosphate; PI, phosphatidylinositol; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PI4KII, type II phosphatidylinositol 4-kinase; PIP5KI, type I phosphatidylinositol 4-phosphate 5-kinase; TBTA, Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride; TIR, Toll/interleukin-1 receptor; TLR4, Toll-like receptor 4; VSVG, vesicular stomatitis virus G-protein; zDHHC, zinc finger DHHC; emPAI, exponentially modified protein abundance index; FDR, false discovery rate; IRAK, interleukin-1 receptor-associated kinase; IRF, interferon regulatory factor; TIRAP, TIR domain-containing adaptor protein; TNFα, tumor necrosis factor α; TRAM, TRIF-related adaptor molecule; TRIF, TIR domain-containing adapter-inducing interferon-β; qPCR, quantitative polymerase chain reaction. 1The abbreviations used are: 17ODYA, 17-octadecynoic acid; AP-1, adaptor protein-1; BPA, bromohexadecanoic acid; BSA, bovine serum albumin; CCL5/RANTES, C-C motif chemokine ligand 5/regulated upon activation, normal T cell expressed and secreted; DGKε, diacylglycerol kinase-ε; eIF5A2, eukaryotic translation initiation factor 5A2; FBS, fetal bovine serum; GPI, glycosylphosphatidylinositol; IP3, inositol 1,4,5-trisphosphate; IPA, Ingenuity Pathway Analysis; ISRE, interferon-stimulated response element; LPS, lipopolysaccharide; PBS, phosphate buffered saline; PI, phosphatidylinositol; PI(4)P, phosphatidylinositol 4-monophosphate; PI, phosphatidylinositol; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PI4KII, type II phosphatidylinositol 4-kinase; PIP5KI, type I phosphatidylinositol 4-phosphate 5-kinase; TBTA, Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride; TIR, Toll/interleukin-1 receptor; TLR4, Toll-like receptor 4; VSVG, vesicular stomatitis virus G-protein; zDHHC, zinc finger DHHC; emPAI, exponentially modified protein abundance index; FDR, false discovery rate; IRAK, interleukin-1 receptor-associated kinase; IRF, interferon regulatory factor; TIRAP, TIR domain-containing adaptor protein; TNFα, tumor necrosis factor α; TRAM, TRIF-related adaptor molecule; TRIF, TIR domain-containing adapter-inducing interferon-β; qPCR, quantitative polymerase chain reaction. and then tagged the 17ODYA-labeled proteins with biotin-azide in a click chemistry reaction and analyzed them by mass spectrometry. Proteomic analysis has recently been used to identify global protein acylation in diverse cells, including RAW264 macrophage-like cells and the dendritic cell line DC2.4 (31.Thinon E. Percher A. Hang H.C. Bioorthogonal chemical reporters for monitoring unsaturated fatty-acylated proteins.ChemBioChem. 2016; 17: 1800-1803Crossref PubMed Scopus (17) Google Scholar, 40.Merrick B.A. Dhungana S. Williams J.G. Aloor J.J. Peddada S. Tomer K.B. Fessler M.B. Proteomic profiling of S-acylated macrophage proteins identifies a role for palmitoylation in mitochondrial targeting of phospholipid scramblase 3.Mol. Cell. Proteomics. 2011; 10Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 41.Chesarino N.M. Hach J.C. Chen J.L. Zaro B.W. Rajaram M.V. Turner J. Schlesinger L.S. Pratt M.R. Hang H.C. Yount J.S. Chemoproteomics reveals Toll-like receptor fatty acylation.BMC Biol. 2014; 12: 91Crossref PubMed Scopus (45) Google Scholar). It should be emphasized, however, that none of the earlier studies examined the influence of cells stimulation with LPS on protein palmitoylation, and thus the contribution of palmitoylated proteins to LPS-induced signaling remains largely unknown. We identified 154 fatty-acylated proteins up-regulated and 186 downregulated ones in cells stimulated with 100 ng/ml LPS for 60 min, which is a time window where the signaling events related to both MyD88- and TRIF-dependent pathways of TLR4 occur. Ingenuity Pathway Analysis (IPA) revealed several functional networks involving the fatty-acylated proteins affected by LPS. By adding an immunoprecipitation step to the above procedure, we confirmed the proteomic data pointing to LPS-induced S-palmitoylation, hence activation, of type II phosphatidylinositol 4-kinase (PI4KII) β, which phosphorylates phosphatidylinositol to PI(4)P. S-palmitoylation of the related isoform PI4KIIα was constitutive in RAW264 cells. Our data indicate that LPS induces production of PI(4,5)P2 and its PI(4)P precursor controlling downstream proinflammatory signaling and that S-palmitoylation of PI4KIIβ is a crucial step in this cascade of events. RAW264.7 and J774A.1 macrophage-like cells, and HEK293 cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 2 mm l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Metabolic labeling of RAW264 cells with 17ODYA (Sigma-Aldrich, Poznan, Poland) was performed in DMEM containing 2% charcoal-stripped FBS (ThermoFisher Scientific, Waltham, MA, USA), l-glutamine, and antibiotics as above and 30 mm Hepes, pH 7.4. For stimulation, cells were exposed to 100 ng/ml LPS (ultrapure smooth LPS from Escherichia. coli O111:B4, List Biological Laboratories, Campbell, CA, USA). In some experiments, prior to labeling with 17ODYA, cells were incubated with 125–250 μm bromohexadecanoic acid (BPA) for 1 h (37 °C) in DMEM/2% charcoal-stripped FBS, and the drug was present during subsequent labeling of cells with 17ODYA, stimulation with LPS, and cytokine production. BPA was precomplexed to fatty-acid-free bovine serum albumin (BSA; Sigma-Aldrich) at a 4:1 molar ratio, essentially as described earlier (42.Kwiatkowska K. Frey J. Sobota A. Phosphorylation of FcγRIIA is required for the receptor-induced actin rearrangement and capping: the role of membrane rafts.J. Cell Sci. 2003; 116: 537-550Crossref PubMed Scopus (90) Google Scholar). When indicated, cells cultured overnight in DMEM/2% FBS were incubated with 150–500 μm palmitic acid, prepared according to (43.Jin J. Zhang X. Lu Z. Perry D.M. Li Y. Russo S.B. Cowart L.A. Hannun Y.A. Huang Y. Acid sphingomyelinase plays a key role in palmitic acid-amplified inflammatory signaling triggered by lipopolysaccharide at low concentrations in macrophages.Am. J. Physiol. Endocrinol. Metab. 2013; 305: E853-E867Crossref PubMed Scopus (61) Google Scholar), for 30 min (37 °C), and labeled with 17ODYA in its presence. pCMV5-Myc plasmid encoding rat PI4KIIα or its deletion mutant lacking the
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