K27-linked ubiquitination restrains the NOD2-RIP2 signaling pathway

Nucleotide-binding oligomerization domain 2 (NOD2) signaling is a critical arm of host defense and immune homeostasis but requires strict regulation to prevent self-injury. Central to this regulation is the polyubiquitination of the adaptor protein RIP2, yet the precise ubiquitin linkages and their dynamic control remain incompletely understood. Here, we identify K27-linked polyubiquitination of RIP2 by the E3 ligase XIAP as a novel inhibitory signal that attenuates RIP2 complex assembly and downstream NF-{kappa}B activation, thereby fine-tuning NOD2-induced inflammatory signaling. We show that XIAP-mediated K63-linked ubiquitination promotes NOD2-RIP2 activation, whereas K27-linked ubiquitination impairs the recruitment of TAK1 and NEMO to the NOD2-RIP2 complex, dampening downstream signaling and cytokine production. We further demonstrate that this regulation is counterbalanced by the deubiquitinase MYSM1, which selectively removes K27-, K63-. and M1-polyubiquitin chains via its SWIRM-MPN domains to restore balanced signaling. Together, these findings define a dynamic XIAP-RIP2-MYSM1 axis in which opposing ubiquitin linkages fine-tune NOD2 responses, establishing a molecular rheostat for immune signaling with implications for inflammatory disease pathogenesis and opportunities for targeted therapeutic intervention.