Deubiquitination of RIPK2 by OTUB2 augments NOD2 signalling and protective effects in intestinal inflammation

Abstract

Background: Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract, but the molecular mechanisms underlying IBD are incompletely understood. In this study, we explored the role and regulating mechanism of otubain 2 (OTUB2), a deubiquitinating enzyme, in IBD.

Methods: To study the function of OTUB2 in IBD, we generated Otub2^–/– mice and treated them with dextran sulfate sodium (DSS) to induce experimental colitis. Bone marrow transplantation was performed to identify the cell populations that were affected by OTUB2 in colitis. The molecular mechanism of OTUB2 in signal transduction was studied by various biochemical methods.

Results: OTUB2 was highly expressed in colon-infiltrating macrophages in both humans with IBD and mice with DSS-induced experimental colitis. Colitis was significantly aggravated in Otub2^–/– mice and bone marrow chimeric mice receiving Otub2^–/– bone marrow. OTUB2-deficiency impaired the production of cytokines and chemokines in macrophages in response to the NOD2 agonist muramyl dipeptide (MDP). Upon MDP stimulation, OTUB2 promoted NOD2 signaling by stabilizing RIPK2. Mechanistically, OTUB2 inhibited the proteasomal degradation of RIPK2 by removing K48-linked polyubiquitination on RIPK2, which was mediated by the active C51 residue in OTUB2. In mice, OTUB2 ablation abolished the protective effects of MDP administration in colitis.

Conclusion: This study identified OTUB2 as a novel regulator of intestinal inflammation.

Keywords

inflammatory bowel disease / OTUB2 / RIPK2 / signal transduction / ubiquitination

References

[1] Roda G, Chien Ng S, Kotze PG, et al. Crohn’s disease. Nat Rev Dis Primers. 2020; 6: 22.

[2] Kobayashi T, Siegmund B, Le Berre C, et al. Ulcerative colitis. Nat Rev Dis Primers. 2020; 6: 74.

[3] Mehandru S, Colombel JF. The intestinal barrier, an arbitrator turned provocateur in IBD. Nat Rev Gastroenterol Hepatol. 2021; 18: 83-84.

[4] Wan T, Wang Y, He K, Zhu S. Microbial sensing in the intestine. Protein Cell. 2023; 14: 824-860.

[5] Noble AJ, Nowak JK, Adams AT, Uhlig HH, Satsangi J. Defining interactions between the genome, epigenome, and the environment in inflammatory bowel disease: progress and prospects. Gastroenterology. 2023; 165: 44-60. e42.

[6] Hegarty LM, Jones GR, Bain CC. Macrophages in intestinal homeostasis and inflammatory bowel disease. Nat Rev Gastroenterol Hepatol. 2023; 20: 538-553.

[7] Dikic I, Schulman BA. An expanded lexicon for the ubiquitin code. Nat Rev Mol Cell Biol. 2023; 24: 273-287.

[8] Ruan J, Schlüter D, Naumann M, Waisman A, Wang Xu. Ubiquitin-modifying enzymes as regulators of colitis. Trends Mol Med. 2022; 28: 304-318.

[9] Jostins L, Ripke S, Weersma RK, et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature. 2012; 491: 119-124.

[10] Liu JZ, Van Sommeren S, Huang H, et al. Association analyses identify 38 susceptibility loci for inflammatory bowel disease and highlight shared genetic risk across populations. Nat Genet. 2015; 47: 979-986.

[11] Lu TT, Onizawa M, Hammer GE, et al. Dimerization and ubiquitin mediated recruitment of A20, a complex deubiquitinating enzyme. Immunity. 2013; 38: 896-905.

[12] Onizawa M, Oshima S, Schulze-Topphoff U, et al. The ubiquitin-modifying enzyme A20 restricts ubiquitination of the kinase RIPK3 and protects cells from necroptosis. Nat Immunol. 2015; 16: 618-627.

[13] Hammer GE, Turer EE, Taylor KE, et al. Expression of A20 by dendritic cells preserves immune homeostasis and prevents colitis and spondyloarthritis. Nat Immunol. 2011; 12: 1184-1193.

[14] Vereecke L, Sze M, Guire CMc, et al. Enterocyte-specific A20 deficiency sensitizes to tumor necrosis factor-induced toxicity and experimental colitis. J Exp Med. 2010; 207: 1513-1523.

[15] Vereecke L, Vieira-Silva S, Billiet T, et al. A20 controls intestinal homeostasis through cell-specific activities. Nat Commun. 2014; 5: 5103.

[16] Pu T, Liu W, Wu Y, Zhao Y. A20 functions as a negative regulator in macrophage for DSS-induced colitis. Int Immunopharmacol. 2021; 97: 107804.

[17] Dufner A, Kisser A, Niendorf S, et al. The ubiquitin-specific protease USP8 is critical for the development and homeostasis of T cells. Nat Immunol. 2015; 16: 950-960.

[18] van Loosdregt J, Fleskens V, Fu J, et al. Stabilization of the transcription factor Foxp3 by the deubiquitinase USP7 increases Treg-cell-suppressive capacity. Immunity. 2013; 39: 259-271.

[19] Wu Bo, Qiang L, Zhang Y, et al. The deubiquitinase OTUD1 inhibits colonic inflammation by suppressing RIPK1-mediated NF-kappaB signaling. Cell Mol Immunol. 2022; 19: 276-289.

[20] Masuta Y, Minaga K, Kurimoto M, et al. Activation of nucleotide-binding oligomerization domain 2 by muramyl dipeptide negatively regulates Toll-like receptor 9-mediated colonic inflammation through the induction of deubiquitinating enzyme A expression. Int Immunol. 2023; 35: 79-94.

[21] Balakirev MY, Tcherniuk SO, Jaquinod M, Chroboczek J. Otubains: a new family of cysteine proteases in the ubiquitin pathway. EMBO Rep. 2003; 4: 517-522.

[22] Chang W, Luo Q, Wu X, et al. OTUB2 exerts tumor-suppressive roles via STAT1-mediated CALML3 activation and increased phosphatidylserine synthesis. Cell Rep. 2022; 41: 111561.

[23] Li J, Cheng D, Zhu M, et al. OTUB2 stabilizes U2AF2 to promote the Warburg effect and tumorigenesis via the AKT/mTOR signaling pathway in non-small cell lung cancer. Theranostics. 2019; 9: 179-195.

[24] Zhang Z, Du J, Wang S, et al. OTUB2 promotes cancer metastasis via hippo-independent activation of YAP and TAZ. Mol Cell. 2019; 73: 7-21. e27.

[25] Ren W, Xu Z, Chang Y, et al. Pharmaceutical targeting of OTUB2 sensitizes tumors to cytotoxic T cells via degradation of PD-L1. Nat Commun. 2024; 15: 9.

[26] Li S, Zheng H, Mao Ai-P, et al. Regulation of virus-triggered signaling by OTUB1-and OTUB2-mediated deubiquitination of TRAF3 and TRAF6. J Biol Chem. 2010; 285: 4291-4297.

[27] Kato K, Nakajima K, Ui A, Muto-Terao Y, Ogiwara H, Nakada S. Fine-tuning of DNA damage-dependent ubiquitination by OTUB2 supports the DNA repair pathway choice. Mol Cell. 2014; 53: 617-630.

[28] Liu Y, et al. Epithelial EZH2 serves as an epigenetic determinant in experimental colitis by inhibiting TNFalpha-mediated inflammation and apoptosis. Proc Natl Acad Sci U S A. 2017; 114: E3796-E3805.

[29] Liu X, Fang Yi, Lv X, et al. Deubiquitinase OTUD6A in macrophages promotes intestinal inflammation and colitis via deubiquitination of NLRP3. Cell Death Differ. 2023; 30: 1457-1471.

[30] Ogura Y, Bonen DK, Inohara N, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature. 2001; 411: 603-606.

[31] Hugot J-P, Chamaillard M, Zouali H, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature. 2001; 411: 599-603.

[32] Yang S, Wang B, Humphries F, et al. Pellino3 ubiquitinates RIP2 and mediates Nod2-induced signaling and protective effects in colitis. Nat Immunol. 2013; 14: 927-936.

[33] Wu Q, Huang Y, Gu L, Chang Z, Li GM. OTUB1 stabilizes mismatch repair protein MSH2 by blocking ubiquitination. J Biol Chem. 2021; 296: 100466.

[34] Zhao Y, Ruan J, Li Z, et al. OTUB1 inhibits breast cancer by non-canonically stabilizing CCN6. Clin Transl Med. 2023; 13: e1385.

[35] Nayar S, Morrison JK, Giri M, et al. A myeloid-stromal niche and gp130 rescue in NOD2-driven Crohn’s disease. Nature. 2021; 593: 275-281.

[36] Watanabe T, et al. Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis. J Clin Invest. 2008; 118: 545-559.

[37] Bertrand MJM, Doiron K, Labbé K, Korneluk RG, Barker PA, Saleh M. Cellular inhibitors of apoptosis cIAP1 and cIAP2 are required for innate immunity signaling by the pattern recognition receptors NOD1 and NOD2. Immunity. 2009; 30: 789-801.

[38] Girardin SE, Boneca IG, Viala J, et al. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J Biol Chem. 2003; 278: 8869-8872.

[39] Inohara N, Ogura Y, Fontalba A, et al. Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn’s disease. J Biol Chem. 2003; 278: 5509-5512.

[40] Hasegawa M, Fujimoto Y, Lucas PC, et al. A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-kappaB activation. EMBO J. 2008; 27: 373-383.

[41] Li Qi, Lee CH, Peters LA, et al. Variants in TRIM22 that affect NOD2 signaling are associated with very-early-onset inflammatory bowel disease. Gastroenterology. 2016; 150: 1196-1207.

[42] Ranjan K, Hedl M, Abraham C. The E3 ubiquitin ligase RNF186 and RNF186 risk variants regulate innate receptor-induced outcomes. Proc Natl Acad Sci U S A. 2021; 118.

[43] Damgaard RB, Nachbur U, Yabal M, et al. The ubiquitin ligase XIAP recruits LUBAC for NOD2 signaling in inflammation and innate immunity. Mol Cell. 2012; 46: 746-758.

[44] Hitotsumatsu O, Ahmad R-C, Tavares R, et al. The ubiquitin-editing enzyme A20 restricts nucleotide-binding oligomerization domain containing 2-triggered signals. Immunity. 2008; 28: 381-390.

[45] Panda S, Gekara NO. The deubiquitinase MYSM1 dampens NOD2-mediated inflammation and tissue damage by inactivating the RIP2 complex. Nat Commun. 2018; 9: 4654.

[46] Lee KH, Biswas A, Liu YJ, Kobayashi KS. Proteasomal degradation of Nod2 protein mediates tolerance to bacterial cell wall components. J Biol Chem. 2012; 287: 39800-39811.

[47] Bist P, Cheong WS, Ng A, et al. E3 Ubiquitin ligase ZNRF4 negatively regulates NOD2 signalling and induces tolerance to MDP. Nat Commun. 2017; 8: 15865.

[48] Sivakumar D, Kumar V, Naumann M, Stein M. Activation and selectivity of OTUB-1 and OTUB-2 deubiquitinylases. J Biol Chem. 2020; 295: 6972-6982.

[49] Mevissen TET, Hospenthal MK, Geurink PP, et al. OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis. Cell. 2013; 154: 169-184.

[50] Wang Xu, Mulas F, Yi W, et al. OTUB1 inhibits CNS autoimmunity by preventing IFN-gamma-induced hyperactivation of astrocytes. EMBO J. 2019; 38.

[51] Mulas F, Wang Xu, Song S, et al. The deubiquitinase OTUB1 augments NF-kappaB-dependent immune responses in dendritic cells in infection and inflammation by stabilizing UBC13. Cell Mol Immunol. 2021; 18: 1512-1527.

RIGHTS & PERMISSIONS

2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.