University of the Pacific Scholarly Commons Dugoni School of Dentistry Faculty Articles Arthur A. Dugoni School of Dentistry 8-6-2012 Interactome-wide analysis identifies nd-bindine g protein 1 as a crucial component for the speck-like particle formation of activated absence in melanoma 2 (AIM2) inflammasomes Li-Jie Wang Chang Gung University Chia-Wei Hsu Chang Gung University Chiu-Chin Chen Chang Gung University Ying Liang Chang Gung University Lih-Chyang Chen Chang Gung University FSeoe nelloxtw pa thige fors aaddndition addal aitutionhorsal works at: https://scholarlycommons.pacific.edu/dugoni-facarticles Part of the Biochemistry Commons, Immunity Commons, Immunology of Infectious Disease Commons, and the Medical Immunology Commons Recommended Citation Wang, L., Hsu, C., Chen, C., Liang, Y., Chen, L., Ojcius, D. M., Tsang, N., Hsueh, C., Wu, C., & Chang, Y. (2012). 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. Molecular and Cellular Proteomics, 11, 1230–1244. DOI: 10.1074/mcp.M112.020594 https://scholarlycommons.pacific.edu/dugoni-facarticles/32 This Article is brought to you for free and open access by the Arthur A. Dugoni School of Dentistry at Scholarly Commons. It has been accepted for inclusion in Dugoni School of Dentistry Faculty Articles by an authorized administrator of Scholarly Commons. For more information, please contact [email protected]. Authors Li-Jie Wang, Chia-Wei Hsu, Chiu-Chin Chen, Ying Liang, Lih-Chyang Chen, David M. Ojcius, Ngan-Ming Tsang, Chuen Hsueh, Chih-Ching Wu, and Yu-Sun Chang This article is available at Scholarly Commons: https://scholarlycommons.pacific.edu/dugoni-facarticles/32 Research © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org 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*□S Li-Jie Wang‡, Chia-Wei Hsu‡, Chiu-Chin Chen§, Ying Liang§, Lih-Chyang Chen§, David M. Ojcius¶ʈ, Ngan-Ming Tsang**, Chuen Hsueh§‡‡, Chih-Ching Wu§§§¶¶, and Yu-Sun Chang‡§¶¶ Inflammasomes are cytoplasmic receptors that can rec- related with AIM2 and ASC expression in nasopharyngeal ognize intracellular pathogens or danger signals and are carcinoma tumor cells. In sum, we profiled the interac- critical for interleukin 1␤ production. Although several key tome components of three inflammasomes and show for components of inflammasome activation have been iden- the first time that end binding protein 1 is crucial for the tified, there has not been a systematic analysis of the speck-like particle formation that represents activated protein components found in the stimulated complex. In inflammasomes. Molecular & Cellular Proteomics 11: this study, we used the isobaric tags for relative and 10.1074/mcp.M112.020594, 1230–1244, 2012. absolute quantification approach to systemically analyze the interactomes of the NLRP3, AIM2, and RIG-I inflam- masomes in nasopharyngeal carcinoma cells treated with Nasopharyngeal carcinoma (NPC)1 is a malignancy of the specific stimuli of these interactomes (H2O2, poly (dA:dT), head and the neck that is highly prevalent in Southern China and EBV noncoding RNA, respectively). We identified a and Southeast Asia (1). Both environmental and genetic risk number of proteins that appeared to be involved in the factors are considered to be important for the development of interactomes and also could be precipitated with anti- NPC (2, 3); among them, Epstein-Barr virus (EBV) infection of apoptosis-associated speck-like protein containing cas- the epithelium is the most important known factor (1). In pase activation and recruitment domain antibodies after addition to the EBV-encoded oncoprotein-mediated blockade stimulation. Among them, end binding protein 1 was an interacting component in all three interactomes. Silencing of intracellular mechanisms in EBV-associated tumors (1), of end binding protein 1 expression by small interfering chronic inflammation is considered to be an important onco- RNA inhibited the activation of the three inflammasomes, genic factor in NPC (4). Interleukin 1 beta (IL-1␤), which is an as indicated by reduced levels of interleukin 1␤ secretion. inflammatory cytokine that has oncogenic effects in many We confirmed that end binding protein 1 directly inter- tumors (5), can be detected in NPC tumor tissues (6, 7). IL-1␤ acted with AIM2 and ASC in vitro and in vivo. Most impor- secretion is mediated by cytosolic protein complexes called tantly, fluorescence confocal microscopy showed that inflammasomes, which induce IL-1␤ secretion by activating end binding protein 1 was required for formation of the catalytic caspase 1 (8). However, no previous study has ex- speck-like particles that represent activation of the AIM2 amined inflammasome components in NPC tumor cells or the inflammasome. In nasopharyngeal carcinoma tissues, im- mechanisms of inflammasome regulation in NPC. munohistochemical staining showed that end binding Inflammasomes are cytoplasmic receptors that act in innate protein 1 expression was elevated and significantly cor- immunity to recognize intracellular pathogen-associated mo- lecular pattern (PAMP) or danger signal-associated molecular From the ‡Graduate Institute of Biomedical Sciences, College of Medicine, §Chang Gung Molecular Medicine Research Center, ¶Cen- 1 The abbreviations used are: NPC, nasopharyngeal carcinoma; ter for Molecular and Clinical Immunology and §§Department of Med- EBV, Epstein-Barr virus; IL-1␤, interleukin 1 beta; PAMP, pathogen- ical Biotechnology and Laboratory Science, College of Medicine, associated molecular pattern; DAMP, danger signal-associated mo- Chang Gung University, Tao-Yuan 333, Taiwan; Departments of **Ra- lecular pattern; NLR, NOD-like receptors; ROS, reactive oxygen spe- diation Oncology and ‡‡Pathology, Chang Gung Memorial Hospital at cies; AIM2, absence in melanoma 2; RIG-I, retinoic acid-inducible Lin-Kou, Tao-Yuan 333, Taiwan; ʈHealth Sciences Research Institute gene I; ASC, apoptosis-associated speck-like protein containing and School of Natural Sciences, University of California, Merced, caspase activation and recruitment domain; HSP90, heat shock pro- California 95343, USA tein 90; EBER, EBV noncoding RNA; EB1, end binding protein 1; APC, Received May 16, 2012, and in revised form, July 3, 2012 adenomatous polyposis coli; ϩTIP, plus-end tracking protein; CT, Published, MCP Papers in Press, August 6, 2012, DOI 10.1074/ C-terminal; CH, calponin homology; iTRAQ, isobaric tags for relative mcp.M112.020594 and absolute quantification. 1230 Molecular & Cellular Proteomics 11.11 EB1 is the Novel Component of Activated Inflammasome pattern (DAMP). A number of inflammasomes have been iden- plex to the plus end of microtubules (26). The interaction of tified in recent years (9), and they can be classified into EB1 and the ϩTIP complex depends on the C-terminal (CT) different subgroups according to their recognizing PAMP. domain of EB1, whereas the calponin homology (CH) domain These groups include the NOD-like receptors (NLR), which of EB1 binds to the microtubule (26). Many studies have sense intracellular pathogens (e.g. bacteria, fungi, and para- shown that EB1 participates in different biological processes, sites) and activate pro-caspase 1 with or without an adaptive including mitosis, migration and signal transduction (27–29), protein called apoptosis-associated speck-like protein con- and also that it plays an oncogenic role in cancer by affecting taining caspase activation and recruitment domain (ASC) (10). cell growth or migration (30, 31). However, although EB1 is Activated caspase 1 then induces IL-1␤ secretion through known to be a cytoskeletal component that is regulated by the direct cleavage of pro-IL-1␤ (8). Among the NLR family mem- small GTPase, RhoA (28), its role in inflammasome activation bers, the NLRP3 inflammasome recognizes both pathogens has not yet been explored. and danger signals such as ATP or reactive oxygen species Here, we used the isobaric tags for relative and absolute (ROS) generation (11, 12). Members of the two other sub- quantification (iTRAQ) approach to systemically analyze the groups, absence in melanoma 2 (AIM2) and retinoic acid- interactomes of the NLRP3, AIM2, and RIG-I inflammasomes inducible gene I (RIG-I), sense cytoplasmic double-strand in NPC cell lines treated with their specific stimuli, H2O2, poly DNA and 5Ј-triphoshphate RNA, respectively, and then recruit (dA:dT), and EBER, respectively. We characterized the inter- ASC to activate pro-caspase 1 (13, 14). Although inflam- actomes of the NLRP3, AIM2, and RIG-I inflammasomes in masomes are important for pathogen defense in immune NPC cells by proteomic analysis, and report for the first time cells, recent studies have shown that inflammasomes also that EB1 can directly bind to the AIM2 inflammasome and is participate in tumorigenesis in colon cancer and melanoma essential for speck-like particle formation in NPC cells. Finally, (15–17). A previous report showed that EBV noncoding we suggest some possible mechanisms for EB1-associated RNAs (EBERs) are recognized by RIG-I and activate signal- AIM2 inflammasome activation via microtubule polymeriza- ing to induce type I IFN in EBV-infected B lymphocytes (18). tion and RhoA activity. This report is consistent with our