Mitochondrial Oxidative Phosphorylation Complex Regulates NLRP3 Inflammasome Activation and Predicts Patient Survival in Nasopharyngeal Carcinoma
2019; Elsevier BV; Volume: 19; Issue: 1 Linguagem: Inglês
10.1074/mcp.ra119.001808
ISSN1535-9484
AutoresI‐Che Chung, Lih‐Chyang Chen, Ngan‐Ming Tsang, Wen‐Yu Chuang, Tzu-Chieh Liao, Sheng-Ning Yuan, Chun‐Nan OuYang, David M. Ojcius, Chih‐Ching Wu, Yu‐Sun Chang,
Tópico(s)Heme Oxygenase-1 and Carbon Monoxide
ResumoWe previously reported that tumor inflammasomes play a key role in tumor control and act as favorable prognostic markers in nasopharyngeal carcinoma (NPC). Activated inflammasomes frequently form distinguishable specks and govern the cellular secretion of IL-1β. However, we know little about the biological and biochemical differences between cells with and without apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) speck formation. In this study, we used proteomic iTRAQ analysis to analyze the proteomes of NPC cells that differ in their ASC speck formation upon cisplatin treatment. We identified proteins that were differentially over-expressed in cells with specks, and found that they fell into two Gene ontology (GO) pathways: mitochondrial oxidative phosphorylation (OxPhos) and ubiquinone metabolism. We observed up-regulation of various components of the OxPhos machinery (including NDUFB3, NDUFB8 and ATP5B), and subsequently found that these changes lead to mitochondrial ROS (mtROS) production, which promotes the formation and activation of NLRP3 inflammasomes and subsequent pyroptosis. In NPC patients, better local recurrence-free survival was significantly associated with high-level expression of NDUFB8 (p = 0.037) and ATP5B (p = 0.029), as examined using immunohistochemistry. However, there were no significant associations between the expression of NDUFB8 and ATP5B with overall survival of NPC patients. Together, our results demonstrate that up-regulated mitochondrial OxPhos components are strongly associated with NLRP3 inflammasome activation in NPC. Our findings further suggest that high-level expression of OxPhos components could be markers for local recurrence and/or promising therapeutic targets in patients with NPC. We previously reported that tumor inflammasomes play a key role in tumor control and act as favorable prognostic markers in nasopharyngeal carcinoma (NPC). Activated inflammasomes frequently form distinguishable specks and govern the cellular secretion of IL-1β. However, we know little about the biological and biochemical differences between cells with and without apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) speck formation. In this study, we used proteomic iTRAQ analysis to analyze the proteomes of NPC cells that differ in their ASC speck formation upon cisplatin treatment. We identified proteins that were differentially over-expressed in cells with specks, and found that they fell into two Gene ontology (GO) pathways: mitochondrial oxidative phosphorylation (OxPhos) and ubiquinone metabolism. We observed up-regulation of various components of the OxPhos machinery (including NDUFB3, NDUFB8 and ATP5B), and subsequently found that these changes lead to mitochondrial ROS (mtROS) production, which promotes the formation and activation of NLRP3 inflammasomes and subsequent pyroptosis. In NPC patients, better local recurrence-free survival was significantly associated with high-level expression of NDUFB8 (p = 0.037) and ATP5B (p = 0.029), as examined using immunohistochemistry. However, there were no significant associations between the expression of NDUFB8 and ATP5B with overall survival of NPC patients. Together, our results demonstrate that up-regulated mitochondrial OxPhos components are strongly associated with NLRP3 inflammasome activation in NPC. Our findings further suggest that high-level expression of OxPhos components could be markers for local recurrence and/or promising therapeutic targets in patients with NPC. Inflammation plays important roles at different stages of tumorigenesis. A key signal involved in driving acute and chronic inflammation is controlled by the inflammasome, which is an inducible cytoplasmic multiprotein complex that serves as a platform for sensing danger signals in the tumor microenvironment (1He Q. Fu Y. Tian D. Yan W. The contrasting roles of inflammasomes in cancer.Am. J. Cancer Res. 2018; 8: 566-583PubMed Google Scholar, 2Moossavi M. Parsamanesh N. Bahrami A. Atkin S.L. Sahebkar A. Role of the NLRP3 inflammasome in cancer.Mol. Cancer. 2018; 17: 158Crossref PubMed Scopus (226) Google Scholar). Various inflammasomes have been identified. Some comprise intracellular pattern recognition receptors, such as nod-like receptors and AIM2-like receptors, which can be activated by pathogen-associated molecule patterns and damage-associated molecule patterns (3Awad F. Assrawi E. Louvrier C. Jumeau C. Georgin-Lavialle S. Grateau G. Amselem S. Giurgea I. Karabina S.A. Inflammasome biology, molecular pathology and therapeutic implications.Pharmacol. Therapeutics. 2018; 187: 133-149Crossref PubMed Scopus (75) Google Scholar). The apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) 1The abbreviations used are:ASCapoptosis-associated speck-like protein containing a caspase-recruitment domainCIDcollision-induced dissociationsEB1end-binding protein 1EBVEpstein-Barr virusETCelectron transport chainFLICAfluorochrome labeled inhibitors of caspase-1GOGene ontologyHCDhigher-energy collision-induced dissociationsIHCimmunohistochemistryIRBInstitutional Review BoardLDHlactate dehydrogenaseLMP1latent membrane protein 1MAVSmitochondrial antiviral signaling proteinMfn2mitofusin 2mtDNAmitochondrial DNAmtΔΨmitochondrial membrane potentialmtROSmitochondrial reactive oxygen speciesNPCnasopharyngeal carcinomaOxPhosoxidative phosphorylationPIpropidium iodidePYCARDPYD and CARD domain containingSDstandard deviationTEABCtriethylammonium bicarbonateTFAtrifluoroacetic acid. 1The abbreviations used are:ASCapoptosis-associated speck-like protein containing a caspase-recruitment domainCIDcollision-induced dissociationsEB1end-binding protein 1EBVEpstein-Barr virusETCelectron transport chainFLICAfluorochrome labeled inhibitors of caspase-1GOGene ontologyHCDhigher-energy collision-induced dissociationsIHCimmunohistochemistryIRBInstitutional Review BoardLDHlactate dehydrogenaseLMP1latent membrane protein 1MAVSmitochondrial antiviral signaling proteinMfn2mitofusin 2mtDNAmitochondrial DNAmtΔΨmitochondrial membrane potentialmtROSmitochondrial reactive oxygen speciesNPCnasopharyngeal carcinomaOxPhosoxidative phosphorylationPIpropidium iodidePYCARDPYD and CARD domain containingSDstandard deviationTEABCtriethylammonium bicarbonateTFAtrifluoroacetic acid. protein, also called PYD and CARD domain containing (PYCARD), is encoded by PYCARD gene in human and is a key component of all known inflammasomes. Upon initiation of inflammasome activation, all inflammasomes recruit the adaptor protein ASC assembles into a large helical fibril called the ASC speck (4Hoss F. Rodriguez-Alcazar J.F. Latz E. Assembly and regulation of ASC specks.Cell. Mol. Life Sci. 2017; 74: 1211-1229Crossref PubMed Scopus (68) Google Scholar). The ASC speck serves as signaling platform for caspase-1, leading to the activation of caspase-1, the maturation of pro-inflammatory cytokines (e.g. IL-1β) and the initiation of a lytic form of programmed cell death known as pyroptosis (5Kovacs S.B. Miao E.A. Gasdermins: effectors of pyroptosis.Trends Cell Biol. 2017; 27: 673-684Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). apoptosis-associated speck-like protein containing a caspase-recruitment domain collision-induced dissociations end-binding protein 1 Epstein-Barr virus electron transport chain fluorochrome labeled inhibitors of caspase-1 Gene ontology higher-energy collision-induced dissociations immunohistochemistry Institutional Review Board lactate dehydrogenase latent membrane protein 1 mitochondrial antiviral signaling protein mitofusin 2 mitochondrial DNA mitochondrial membrane potential mitochondrial reactive oxygen species nasopharyngeal carcinoma oxidative phosphorylation propidium iodide PYD and CARD domain containing standard deviation triethylammonium bicarbonate trifluoroacetic acid. apoptosis-associated speck-like protein containing a caspase-recruitment domain collision-induced dissociations end-binding protein 1 Epstein-Barr virus electron transport chain fluorochrome labeled inhibitors of caspase-1 Gene ontology higher-energy collision-induced dissociations immunohistochemistry Institutional Review Board lactate dehydrogenase latent membrane protein 1 mitochondrial antiviral signaling protein mitofusin 2 mitochondrial DNA mitochondrial membrane potential mitochondrial reactive oxygen species nasopharyngeal carcinoma oxidative phosphorylation propidium iodide PYD and CARD domain containing standard deviation triethylammonium bicarbonate trifluoroacetic acid. Nasopharyngeal carcinoma (NPC) is a cancer that is prominent in the Taiwanese population. It is closely associated with Epstein-Barr virus (EBV) infection. In tumor cells, EBV-encoded latent membrane protein 1 (LMP1) mediates pro-IL-1β gene expression through NF-κB signaling (6Chen L.C. Wang L.J. Tsang N.M. Ojcius D.M. Chen C.C. Ouyang C.N. Hsueh C. Liang Y. Chang K.P. Chen C.C. Chang Y.S. Tumour inflammasome-derived IL-1beta recruits neutrophils and improves local recurrence-free survival in EBV-induced nasopharyngeal carcinoma.EMBO Mol. Med. 2012; 4: 1276-1293Crossref PubMed Scopus (120) Google Scholar). Within the NPC tumor mass or tumor microenvironment, the levels of proinflammatory cytokines, including IL-1β, are elevated (7Huang Y.T. Sheen T.S. Chen C.L. Lu J. Chang Y. Chen J.Y. Tsai C.H. Profile of cytokine expression in nasopharyngeal carcinomas: a distinct expression of interleukin 1 in tumor and CD4+ T cells.Cancer Res. 1999; 59: 1599-1605PubMed Google Scholar). Indeed, elevated levels of IL-1β and the NLRP3, AIM2 and RIG-I inflammasomes were reported to be useful as prognostic biomarkers for predicting better recurrence-free survival of NPC patients treated with the current protocols (6Chen L.C. Wang L.J. Tsang N.M. Ojcius D.M. Chen C.C. Ouyang C.N. Hsueh C. Liang Y. Chang K.P. Chen C.C. Chang Y.S. Tumour inflammasome-derived IL-1beta recruits neutrophils and improves local recurrence-free survival in EBV-induced nasopharyngeal carcinoma.EMBO Mol. Med. 2012; 4: 1276-1293Crossref PubMed Scopus (120) Google Scholar). End-binding protein 1 (EB1), which regulates microtubule polymerization, was reported to co-immunoprecipitate with ASC of the NLRP3, AIM2, and RIG-I inflammasomes in NPC cells and was found to be required for speck formation in response to AIM2 inflammasome activation (8Wang L.J. Hsu C.W. Chen C.C. Liang Y. Chen L.C. Ojcius D.M. Tsang N.M. Hsueh C. Wu C.C. Chang Y.S. Interactome-wide analysis identifies end-binding protein 1 as a crucial component for the speck-like particle formation of activated absence in melanoma 2 (AIM2) inflammasomes.Mol. Cell. Proteomics. 2012; 11: 1230-1244Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). EB1 has also been shown to mediate the autophagy-dependent secretion of IL-1β, and the EB1-mediated, autophagy-based and inflammasome-induced secretion of IL-1β was found to be regulated by 5′AMP activated protein kinase (9Wang L.J. Huang H.Y. Huang M.P. Liou W. Chang Y.T. Wu C.C. Ojcius D.M. Chang Y.S. The microtubule-associated protein EB1 links AIM2 inflammasomes with autophagy-dependent secretion.J. Biol. Chem. 2014; 289: 29322-29333Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Other studies found that the NLRP3 inflammasome is activated by EBV-LMP1 and the chemotherapeutic agent, cisplatin, in NLRP3-knockdown NPC cells (6Chen L.C. Wang L.J. Tsang N.M. Ojcius D.M. Chen C.C. Ouyang C.N. Hsueh C. Liang Y. Chang K.P. Chen C.C. Chang Y.S. Tumour inflammasome-derived IL-1beta recruits neutrophils and improves local recurrence-free survival in EBV-induced nasopharyngeal carcinoma.EMBO Mol. Med. 2012; 4: 1276-1293Crossref PubMed Scopus (120) Google Scholar, 10Cai T.T. Ye S.B. Liu Y.N. He J. Chen Q.Y. Mai H.Q. Zhang C.X. Cui J. Zhang X.S. Busson P. Zeng Y.X. Li J. LMP1-mediated glycolysis induces myeloid-derived suppressor cell expansion in nasopharyngeal carcinoma.PLoS Pathogens. 2017; 13e1006503Crossref PubMed Scopus (76) Google Scholar). However, we know relatively little about the changes in other cellular components of activated inflammasomes. Emerging evidence indicates that mitochondria not only act as the energy powerhouses of the cell, they also function as signaling organelles that can activate signal pathways to regulate innate and adaptive immunity (11Weinberg S.E. Sena L.A. Chandel N.S. Mitochondria in the regulation of innate and adaptive immunity.Immunity. 2015; 42: 406-417Abstract Full Text Full Text PDF PubMed Scopus (524) Google Scholar). Recent studies have indicated that mitochondrial dysfunctions may crucially modulate NLRP3 inflammasome activation (12Liu Q. Zhang D. Hu D. Zhou X. Zhou Y. The role of mitochondria in NLRP3 inflammasome activation.Mol. Immunol. 2018; 103: 115-124Crossref PubMed Scopus (203) Google Scholar). NLRP3 inflammasome activation involves various parameters that can be altered under mitochondrial dysfunction, such as mitochondrial membrane potential (mtΔΨ) (13Zhong Z. Umemura A. Sanchez-Lopez E. Liang S. Shalapour S. Wong J. He F. Boassa D. Perkins G. Ali S.R. McGeough M.D. Ellisman M.H. Seki E. Gustafsson A.B. Hoffman H.M. Diaz-Meco M.T. Moscat J. Karin M. NF-kappaB restricts inflammasome activation via elimination of damaged mitochondria.Cell. 2016; 164: 896-910Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar), mitochondrial outer membrane permeabilization (14Horng T. Calcium signaling and mitochondrial destabilization in the triggering of the NLRP3 inflammasome.Trends Immunol. 2014; 35: 253-261Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), and the levels of mitochondrion-derived molecules [(e.g. mitochondrial ROS (mtROS) (15Zhou R. Yazdi A.S. Menu P. Tschopp J. A role for mitochondria in NLRP3 inflammasome activation.Nature. 2011; 469: 221-225Crossref PubMed Scopus (3447) Google Scholar), release of mitochondrial DNA (mtDNA) (16Zhong Z. Liang S. Sanchez-Lopez E. He F. Shalapour S. Lin X.J. Wong J. Ding S. Seki E. Schnabl B. Hevener A.L. Greenberg H.B. Kisseleva T. Karin M. New mitochondrial DNA synthesis enables NLRP3 inflammasome activation.Nature. 2018; 560: 198-203Crossref PubMed Scopus (483) Google Scholar), and cardiolipin (17Iyer S.S. He Q. Janczy J.R. Elliott E.I. Zhong Z. Olivier A.K. Sadler J.J. Knepper-Adrian V. Han R. Qiao L. Eisenbarth S.C. Nauseef W.M. Cassel S.L. Sutterwala F.S. Mitochondrial cardiolipin is required for Nlrp3 inflammasome activation.Immunity. 2013; 39: 311-323Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar)] and mitochondrion-residing molecules [(e.g. mitochondrial antiviral signaling protein (MAVS) (18Subramanian N. Natarajan K. Clatworthy M.R. Wang Z. Germain R.N. The adaptor MAVS promotes NLRP3 mitochondrial localization and inflammasome activation.Cell. 2013; 153: 348-361Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar) and mitofusin 2 (Mfn2) (19Ichinohe T. Yamazaki T. Koshiba T. Yanagi Y. Mitochondrial protein mitofusin 2 is required for NLRP3 inflammasome activation after RNA virus infection.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 17963-17968Crossref PubMed Scopus (173) Google Scholar)]. Notably, MAVS and Mfn2 help redistribute cytosolic NLRP3 to mitochondrial outer membrane and enhance NLRP3 inflammasome activation. Further, mtROS can oxidize mtDNA, leading to further NLRP3 inflammasome activation. The electron transport chain (ETC) is in the mitochondrial inner membrane. It is the major source of oxidative phosphorylation (OxPhos) and is involved in the generation of energy: oxygen acts as an electron acceptor, and electrons from NADH and FADH2 generated by glycolysis and the TCA cycle are used to generate ATP. Through the ETC, protons are transported across the inner membrane of mitochondria to generate mtΔΨ. Mechanistically, the loss of mitochondrial potential has been associated with the production of mtROS (15Zhou R. Yazdi A.S. Menu P. Tschopp J. A role for mitochondria in NLRP3 inflammasome activation.Nature. 2011; 469: 221-225Crossref PubMed Scopus (3447) Google Scholar) and changes in cellular Ca2+ (20Triantafilou K. Hughes T.R. Triantafilou M. Morgan B.P. The complement membrane attack complex triggers intracellular Ca2+ fluxes leading to NLRP3 inflammasome activation.J. Cell Sci. 2013; 126: 2903-2913Crossref PubMed Scopus (256) Google Scholar) in the context of NLRP3 inflammasome activation. Moreover, mitochondria are the major source of cellular ROS (21Mills E.L. Kelly B. O'Neill L.A.J. Mitochondria are the powerhouses of immunity.Nature Immunol. 2017; 18: 488-498Crossref PubMed Scopus (513) Google Scholar). Several lines of evidence support the notion that mtROS activates NLRP3 inflammasome formation and activation. For example, inhibitors of OxPhos trigger mtROS production, which can lead to NLRP3 inflammasome activation (15Zhou R. Yazdi A.S. Menu P. Tschopp J. A role for mitochondria in NLRP3 inflammasome activation.Nature. 2011; 469: 221-225Crossref PubMed Scopus (3447) Google Scholar). Meanwhile, we previously showed that the E3 ubiquitin ligase, Cbl, maintains mitochondrial size and reduces mtROS production by suppressing NLRP3 inflammasome activation (22Chung I.C. Yuan S.N. OuYang C.N. Lin H.C. Huang K.Y. Chen Y.J. Chung A.K. Chu C.L. Ojcius D.M. Chang Y.S. Chen L.C. Src-family kinase-Cbl axis negatively regulates NLRP3 inflammasome activation.Cell Death Dis. 2018; 91109Crossref PubMed Scopus (21) Google Scholar). Together, the previous results reveal that mtROS can be an upstream regulator for NLRP3 inflammasome activation. Here, we report that mitochondrial OxPhos components are enriched in ASC speck-positive NPC cells and are likely to participate in NLRP3 inflammasome activation and cell pyroptosis. Further, the components of OxPhos are highly expressed in tumor cells and significantly correlated with better post-treatment survival of NPC patients. Our findings further clarify the molecular mechanisms of mitochondria-mediated NLRP3 inflammasome activation and suggest that overexpressed OxPhos proteins could prove useful as favorable markers for local recurrence in NPC patients. EBV-positive NPC cell line, NPC-HK1-EBV, and an EBV-negative NPC cell line, NPC-HK1, were kindly provided by Dr. S. W. Tsao (Hong Kong University, SAR, China) and cultured as previously described (6Chen L.C. Wang L.J. Tsang N.M. Ojcius D.M. Chen C.C. Ouyang C.N. Hsueh C. Liang Y. Chang K.P. Chen C.C. Chang Y.S. Tumour inflammasome-derived IL-1beta recruits neutrophils and improves local recurrence-free survival in EBV-induced nasopharyngeal carcinoma.EMBO Mol. Med. 2012; 4: 1276-1293Crossref PubMed Scopus (120) Google Scholar, 23Lo A.K. Lo K.W. Tsao S.W. Wong H.L. Hui J.W. To K.F. Hayward D.S. Chui Y.L. Lau Y.L. Takada K. Huang D.P. Epstein-Barr virus infection alters cellular signal cascades in human nasopharyngeal epithelial cells.Neoplasia. 2006; 8: 173-180Crossref PubMed Scopus (161) Google Scholar). The NPC-BM1 cell line was established from a bone marrow biopsy of a female Taiwanese patient with NPC and cultured as previously described (24Liao S.K. Perng Y.P. Shen Y.C. Chung P.J. Chang Y.S. Wang C.H. Chromosomal abnormalities of a new nasopharyngeal carcinoma cell line (NPC-BM1) derived from a bone marrow metastatic lesion.Cancer Genet. Cytogenet. 1998; 103: 52-58Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). For generation of NPC-HK1-ASC-GFP and NPC-BM1-ASC-GFP cells, the NPC-HK1 and NPC-BM1 cells were transduced with lentiviral particles containing an ASC-GFP plasmid, using a commercial lentivirus transduction protocol (Sigma-Aldrich, St. Louis, MO). For inflammasome activation, the cells were treated with 40 μm cisplatin (cat# P4393; Sigma-Aldrich), 10 μm nigericin (cat# N7143; Sigma-Aldrich), 5 mm ATP (cat# A7699; Sigma-Aldrich), 200 μm monosodium urate (MSU) crystals (cat# tlrl-msu; InvivoGen, San Diego, CA) or 200 μm Alum (cat# tlrl-alk; InvivoGen). For mtROS scavenger treatment, the cells were preincubated with MitoTEMPO (cat# SML0737; Sigma-Aldrich) for 1 h. NPC-HK1-ASC-GFP and NPC-BM1-ASC-GFP cells were treated with 40 μm cisplatin for 24 h (NPC-HK1-ASC-GFP) or 30 h (NPC-BM1-ASC-GFP), and ASC speck(+) and speck(−) cells were isolated by flow cytometry as previously described (22Chung I.C. Yuan S.N. OuYang C.N. Lin H.C. Huang K.Y. Chen Y.J. Chung A.K. Chu C.L. Ojcius D.M. Chang Y.S. Chen L.C. Src-family kinase-Cbl axis negatively regulates NLRP3 inflammasome activation.Cell Death Dis. 2018; 91109Crossref PubMed Scopus (21) Google Scholar, 25Sester D.P. Thygesen S.J. Sagulenko V. Vajjhala P.R. Cridland J.A. Vitak N. Chen K.W. Osborne G.W. Schroder K. Stacey K.J. A novel flow cytometric method to assess inflammasome formation.J. Immunol. 2015; 194: 455-462Crossref PubMed Scopus (62) Google Scholar). Briefly, the transit of ASC-GFP protein into the speck is detected by a decreased pulse width (W) of emitted fluorescence after the inflammasome activation, and the total amount of ASC-GFP stained per cell (pulse area, A) was higher in cells with ASC speck. Therefore, low and high pulse width to pulse area profile (ASC-W:ASC-A profile) were taken as indicating ASC speck(+) cells and ASC speck(−) cells, respectively). We then sorted for the presence or absence of ASC-GFP specks by flow cytometric method. NPC-HK1-ASC-GFP and NPC-BM1-ASC-GFP cells were isolated by flow cytometry and lysed in buffer containing 100 mm triethylammonium bicarbonate (TEABC; Sigma-Aldrich) and 0.1% RapiGestTM SF (Waters Corporation, Milford, MA) on ice for 15 min. The cell lysate was collected, sonicated on ice and centrifuged at 10,000 × g for 25 min at 4 °C. The resulting supernatant was used as the cell extract. Protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific, San Jose, CA). For tryptic in-solution digestion, 20 μg protein of each sample was reduced with 4.8 mm Tris-(2-carboxyethyl)-phosphine (Sigma-Aldrich) and 260 mm TEABC at 60 °C for 1 h and then alkylated with 9.6 mm methyl methanethiosulfonate (Sigma-Aldrich) at room temperature for 30 min. The proteins were digested at 37 °C overnight with modified sequencing-grade porcine trypsin (Promega, Madison, WI) using a trypsin-to-protein ratio of 1:1 (w/w). Trypsin-digested peptides were labeled with iTRAQ reagents (AB Sciex, Foster City, CA) according to the manufacturer's protocol. The peptides from ASC speck(−) and speck(+) cells were labeled with iTRAQ 114 and 115 tags, respectively. For the biological replicates, the iTRAQ 116 and 117 tags were incubated with peptide samples obtained from different batches of ASC speck(−) and speck(+) cells. After incubation for 1 h at room temperature, the four labeled samples were pooled, frozen and lyophilized to dryness using a Proteomic CentriVap Concentrator System (Labconco, Kansas City, MO). The peptides were desalted using a ziptip (Merck Millipore, Billerica, MA) packed with Resin-C18 (GE Healthcare, UK). In brief, a sufficient volume of Resin-C18 (1 μl for 10 μg of proteins) was packed into a ziptip. To activate the Resin-C18, the ziptip was sequentially loaded with 200 μl of acetonitrile (Mallinckrodt Baker, Ireland) at concentrations of 75%, 40 and 3% in 1% trifluoroacetic acid (TFA), and then centrifuged at 300 rpm for 30 s. This step was repeated three times to ensure complete activation. Dried iTRAQ-labeled peptide samples were reconstituted with 0.1% TFA to a final protein concentration of 1 μg/μl. The reconstituted peptides were loaded into the ziptip, centrifuged at 300 rpm for 30 s and washed with 3% acetonitrile in 1% TFA. Finally, the iTRAQ-labeled peptide samples were eluted from the ziptip with 30%, 50%, and 70% acetonitrile in 1% TFA and dried by vacuum centrifugation. The iTRAQ-labeled peptides were separated using an on-line 2D-HPLC system (Dionex Ultimate 3000, Thermo Fisher Scientific) and analyzed as previously described (26Lin M.H. Li C.C. Shu J.C. Chu H.W. Liu C.C. Wu C.C. Exoproteome profiling reveals the involvement of the foldase PrsA in the cell surface properties and pathogenesis of Staphylococcus aureus.Proteomics. 2018; 18e1700195Crossref PubMed Scopus (18) Google Scholar). Briefly, peptides (20 μg) were resuspended in 0.1% formic acid (20 μl) and desalted using a ziptip home-packed with C18 resin (5–20 μm, LiChroprep RP-18; Merck, Taipei, Taiwan). The resultant samples were vacuum-dried, reconstituted in 50 μl of buffer A (0.1% formic acid and 30% acetonitrile), and loaded onto the homemade column (Luna SCX, 5 μm, 0.5 × 180 mm) at a flow rate of 5 μl/min for 30 min. The peptides were then eluted with a 0–100% gradient of buffer B (0.5 m ammonium chloride, 30% acetonitrile and 0.1% formic acid). The resulting 44 peptide fractions were diluted in-line prior to being trapped onto the Zorbax 300SB-C18 column (0.3 × 5 mm; Agilent Technologies, Wilmington, DE). Each fraction was then separated on a homemade column (HydroRP 2.5 μm, 75 μm inner diameter and 20 cm length) with a 15-μm tip using buffer C (acetonitrile containing 0.1% formic acid). A linear gradient of buffer C (3–28% for 37 min, 28–50% for 12 min, 50–95% for 2 min, 95% for 5 min and 3% for 9 min) was applied at a flow rate of 0.3 μl/min. The LC equipment was connected to the LTQ-Orbitrap ELITE mass spectrometer (Thermo Fisher Scientific), which was operated using the Xcalibur software (Version 2.2, Thermo Fisher Scientific). Full-scan MS was performed in the Orbitrap over a range of 400 to 2000 Da and a resolution of 60,000 at m/z 400. The ion signals of (Si(CH3)2O)6H+ at m/z 445.120025, 462.146574 and 536.165365 were used for lock masses and internal calibration. Each 12 data-dependent MS/MS scan events of six collision-induced dissociations (CID) and six higher-energy collision-induced dissociations (HCD) were followed by one MS scan for the six most abundant ions in the preview MS scan. The m/z values selected for MS2 were excluded dynamically for 40 s with a relative mass window of 1.5 Da. The electrospray voltage was set to 1.8 kV and the temperature of the capillary was set to 220 °C. Automatic gain control was applied to preclude over-filling of the ion trap, and 1000 ms/2 × 106 ions, 150 ms/5 × 103 ions and 300 ms/3 × 104 ions were set as the maximum accumulated time/ions for the full scan, CID and HCD, respectively. Protein database searching was performed using the Proteome Discoverer software (version 1.4; Thermo Fisher Scientific) with the reporter ion quantifier node for iTRAQ data analysis. The MS/MS spectra were searched against the Swiss-Prot human sequence database (released 2018/03, selected for Homo sapiens; 20,198 entries) using the Mascot search engine (version 2.2.0; Matrix Science, London, UK). For peptide identification, one missing tryptic cleavage was allowed. Mass tolerances of 10 ppm, 0.5 Da and 0.05 Da were permitted for intact peptide masses, CID fragment ions and HCD fragment ions, respectively. Oxidized methionine (+16 Da) was considered a potential variable modification, and iTRAQ (N-terminal, +144 Da), iTRAQ (K, +144 Da) and methyl methanethiosulfonate (C, +46 Da) were considered fixed modifications. The search results (peptide-spectrum matches) were filtered for a highly confident peptide identification in Proteome Discoverer to ensure that the overall false discovery rate was less than 0.01. Identification of epithelial keratins was excluded. Proteins with single peptide hits were removed. Each quantified protein contained at least two quantifiable spectra. The quantitative data were exported from Proteome Discoverer and manually normalized such that the log2 of protein ratios displayed a median value of zero for all peptides in each protein. This was performed across an entire labeling experiment to correct for any variation in protein abundance. The cutoff value for determining whether a protein was considered dysregulated was selected according to a comparison of protein levels between the same ASC speck(−) samples in two different batches. Proteins with a log2 ratio greater than the mean ratio plus one standard deviation (S.D.) and those with a log2 ratio less than the mean ratio minus one S.D. were overexpressed and underexpressed, respectively. NDUFB3 (sense 5′-UGGCU UUGCA AAGAG UGUUU C-3′, 5′- CCGCA AUGAA GCUUG GAGAU A-3′), NDUFB8 (sense 5′-CCAAAG CAGUA UCCUU ACAAU-3′, 5′-CCUGG CUUUC AUGAU AUUCA U-3′), NDUFB5 (sense 5′- GUCAA GCUGA ACUAG CAGAA A-3′, 5′-CGAAA GCAAC UCCUG ACAAU U-3′), MT-CO2 (sense 5′-CCAUC AUCCU AGUCC UCAU-3′, 5′-GAUCC CUCCC UUACC AUCAA A-3′, 5′-GCAAU UCCCG GACGU CUAA-3′), ATP5B (sense 5′-GCACA GUAAG GACUA UUGCU A-3′, 5′-GCGUU UCUUG UCUCA GCCAU U-3′), ATP5H (sense 5′- CCCGU GCCAG AGGAU AAAUA U-3′, 5′-CCAUU GCUAG UUCCC UGAAA U-3′) and ATP5J (sense 5′- GAUCC UAUAC AGAAA CUCUU U-3′, 5′-GAGGA CCUGU UGAUG CUAGU U-3′) siRNAs were purchased from MDBio (Taipei, Taiwan). Negative-control siRNA were purchased from Invitrogen (Carlsbad, CA). Cells were transfected with 50 nm dsRNA duplexes using jetPRIME (Polyplus Transfection, New York, NY) according to the manufacturer's instructions. After 4 h of incubation, the dsRNA complexes were removed, and the cells were re-plated in 5 ml of fresh culture medium. Western blot analysis was performed as described previously (22Chung I.C. Yuan S.N. OuYang C.N. Lin H.C. Huang K.Y. Chen Y.J. Chung A.K. Chu C.L. Ojcius D.M. Chang Y.S. Chen L.C. Src-family kinase-Cbl axis negatively regulates NLRP3 inflammasome activation.Cell Death Dis. 2018; 91109Crossref PubMed Scopus (21) Google Scholar). Briefly, cells were lysed in RIPA buffer containing a protease inhibitor mixture (cat# 04693116001; Roche Applied Science, Indianapolis, IN) on ice for 30 min, followed by centrifugation (12,000 rpm for 10 min at 4 °C). The protein lysates were further resolved by SDS-PAGE and transferred to nitr
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