Fig 2: MYSM1 inactivates RIP2 complex via the SWIRM and MPN domains. a Schematic representation of MYSM1 domain mutant constructs. b Immunofluorescence staining of full-length MYSM1 (MYSM1 FL) or SWIRM+MPN in complemented Mysm1-/- BMDMs 1 h post L18-MDP stimulation. Scale bar (10 µm). c, d Mysm1-/- BMDMs complemented with indicated MYSM1 constructs were stimulated with L18-MDP. Cytoplasmic fractions were immunoprecipitated with anti-RIP2. Pulldowns (c) and the corresponding cytoplasmic or nuclear fractions (d) were analysed for indicated molecules. e–f Mysm1-/- BMDMs or Mysm1-/- BMDMs complemented with indicated constructs were stimulated with L18-MDP for 12 h and analysed for TNF-a and IL-6 secretion by ELISA. Results in e–f are from three independent experiments. Data are shown as mean ± s.e.m. (n = 3). ***p < 0.001 determined by one-way ANOVA followed by Bonferroni’s post-test depicts statistical significance relative to Ctrl. See also Supplementary Figure 6 and Supplementary Figure 7

Fig 3: MYSM1 suppresses NOD2-mediated systemic inflammation and liver injury. a Representative flow cytometry contour plots showing the recruitment of neutrophils (CD11b+, RB6-8c5+) into peritoneal cavities (PECs) of WT (n = 6) and Mysm1−/− (n = 6) mice 6 h post administration of MDP (25 mg/ kg body weight) or control buffer (Ctrl). b–c Corresponding bar graphs showing the mean percentage of neutrophils in PECs (b) and spleen (c). d–e WT (n = 12), Mysm1−/− (n = 12) and Rip2−/− (n = 12) mice were inoculated with GalN (1 g/kg body weight, via intraperitoneal (i.p) route 4 h prior to challenge with MDP (25 mg/ kg body weight, i.p) analysed 6 h later for TNF-α, IL-6 (d) and liver alanine aminotransferase (ALT) in the sera (e). f–g TNF-α, IL-6 (f) and ALT (g) in sera of Mysm1fl/fl (n = 8) or Mysm1fl/fl LysM-Cre (n = 8) mice challenged with GalN and MDP as in d–e. Each data point represents (b-g) an individual mouse. Data are shown as mean ± s.d.. ****p < 0.0001 determined by one-way analysis of variance (ANOVA) with Bonferroni’s multiple- comparison test. Data in b, c and f, g are from two independent experiments, (d, e), from three independent experiments. See Supplementary Figure 10 for gating strategy for the FACs data in panels (a–c)

Fig 4: MYSM1 disrupts the NOD2:RIP2 complex by removing K63, K27, M1 but not K48 polyubiquitins. a Color codes of ubiquitin linkage specificity of DUBs used in the UbiCRest analysis (b, e). b TUBE pulldowns from BMDMs stimulated with L18-MDP (1 h) were subjected to UbiCRest then immunoblotted for RIP2, K63, K48, K27, and M1 polyubiquitin chains. The red dashed line is an arbitrary line highlighting a relative shift from high to lower MW intermediate /complete digestion products of Ub-RIP2. c Native and denatured cytoplasmic fractions from WT and Mysm1-/- BMDMs stimulated with L18-MDP for indicated duration were immunoprecipitated with anti-RIP2 and pulldowns were immunoblotted for indicated molecules. d Ubiquitin linkage specificity of MYSM1. Di-ubiquitins of indicated linkages incubated with recombinant MYSM1 were separated by SDS-PAGE gel and visualized by silver-staining. e TUBE pulldowns from WT BMDMs stimulated with L18-MDP for 1 h were incubated with higher concentration of the indicated DUBs for 3 h. Samples were then immunoblotted for RIP2, K63, and K48 polubiquitin chains. The dotted red lines (b, e) are arbitrary lines indicating a relative shift from high to lower MW Ub-RIP2 forms after digestion. Note that for maximum Ub restriction, in e, a higher concentration of DUBs was used compared to b. f Schematic of proposed model for removal of polyubiquitins from RIP2 by MYSM1. Data are representative of two to six independent experiments. See also Supplementary Figure 5

Fig 5: MYSM1 suppresses proximal NOD2 signaling events and cytokine response. a, b WT, Mysm1-/-, and Rip2-/- BMDMs, either unstimulated (Ctrl) or stimulated with L18-MDP (200 ng/ml), were analyzed by qRT-PCR for Tnfa and Il6 transcript 6 h post stimulation (a) or for secreted TNF-a and IL-6 12 h post stimulation (b). c, d WT, Mysm1-/-, and Rip2-/- BMDMs, either unstimulated (Ctrl) or stimulated with C12-iE DAP (200 ng/ml), were analyzed by qRT-PCR for Tnfa (c) and Il6 (d) transcript 6 h post stimulation. Results in (a–d) are from three independent experiments. Data are shown as mean ± s.e.m. (n = 3). ***p < 0.001 determined by one-way ANOVA followed by Bonferroni’s post-test depicts statistical significance. e WT, Mysm1-/- and Rip2-/- BMDMs, either unstimulated (Ctrl) or stimulated with L18-MDP for indicated duration, were analysed by immunoblotting for MYSM1, p-RIP2, RIP2, p-TAK1, TAK1, p-IKKa/ß, IKKa/ß, p-p38 MAPK, and p38 MAPK. a- tubulin was used as loading control. Data in (e) are representative of three independent experiments