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Activation of the NF-κB family of transcription factors is related to diverse biological processes, such as development, immune responses, and inflammation. As a result, mutations affecting the NF-κB signaling pathway cause syndromes characterized by immunodeficiency and autoinflammaotory disease. Recently, we identified the C-terminus of NEMO to be a regulator of NF-κB signaling, as a class of NEMO mutations lacking the C-terminus (ΔCT-NEMO) confer gain-of-function properties to the IKK complex due to impaired recruitment of A20 to the TNFR. Here, we investigated details by which A20 recruitment to the TNFR suppresses NF-κB signaling. The C-terminus of NEMO has been previously identified to preferentially recognize K63-linked polyubiquitin that in its unanchored form can mediate NEMO/A20 interaction. We found that the IKK complex containing ΔCT-NEMO forms but not other mutant forms of NEMO exhibit impaired K63 polyubiquitin binding ability. In addition to unanchored K63-lilnked polyubiquitin, we hypothesized that direct polyubiquitination of A20 could enable a functional interaction between A20 and NEMO. Using mass spectroscopy, we identified 4 lysine residues on A20 to be ubiquitination sites. Mutation of these lysines to arginine impaired A20’s ability to suppress TNF induced NF-κB activation. We noted stabilization of NEMO in A20 deficient cells following TNF stimulation. Biochemical studies in Jurkat and 293 cells revealed A20 dependent K48 linked polyubiquitination of NEMO at the TNFR following receptor stimulation. Our data suggest a mechanism of NF-κB regulation in which A20 is directly K63 polyubiquitinated to functionally interacts with NEMO which it targets by K48 linkages proteasomal degradation.

Hypomorphic mutation of the NF-kB essential modulator (NEMO) causes a spectrum of inflammatory disease and immunodeficiency. NEMO is critical in immunity, regulating the activation of transcription factors NF-kB and IRF3 to control inflammation and antiviral type I interferon. In an individual with persistent inflammatory disease, we identified a synonymous mutation in the gene encoding NEMO that enhances alternative splicing and production of a mutant protein (Δ-NEMO). Co-immunoprecipitation studies in patient T cells revealed impaired association of Δ-NEMO with TANK (TRAF family member-associated NF-kB activator) and, consequently, with the IRF3 kinase TANK-binding kinase 1 (TBK1). Concomitantly, patient fibroblasts displayed impaired production of IRF3-dependent type I and III interferon in response to poly(I:C) and permitted increased virus propagation. However, cells containing Δ-NEMO exhibited enhanced IRF3 nuclear translocation and binding to the IFNB1 promoter. Our results suggest that defective interferon production in this patient lies instead in impaired NF-kB activation, as poly(I:C)- and 5’pppRNA induced translocation of NF-kB in patient fibroblasts was reduced. This impairment in NF-kB was specific, as TNF-induced NF-kB activation and gene induction was normal. These data describe a novel mechanism of autoinflammatory disease due to a type I interferon deficiency. Thus, this represents a model of inflammatory diseases responsive to type I IFN therapy.

The RIG-I-like receptors mediate antiviral signaling through NEMO-dependent activation of transcription factors NF-kB and IRF3, which are recognized for their roles in inflammation and the induction of Type I Interferon, respectively. We identified an individual with increased susceptibility to viral infection and persistent inflammatory disease. Sanger sequencing revealed a de novo synonymous mutation in the gene encoding NEMO that results in an in-frame splicing event and exclusive production of a shortened protein. To understand the role of NEMO in regulating the antiviral response, we stimulated patient and healthy control fibroblasts with RIG-I-like receptor ligand poly(I:C), which mimics viral RNA. Induction of IRF3 response genes IFNB1 and CXCL10 was reduced 35-fold and 500-fold (n=2), respectively, compared to healthy control, and patient cells were unable to control viral infection. In contrast, TNF induced the expression of proinflammatory genes TNFSF10 and IL17 7-fold and 4-fold higher in patient cells than in healthy control cells. Increased TNF induced gene expression was accompanied by increased p65 nuclear translocation indicative of increased canonical IKK activation. These results suggest that aberrant expression of an alternately spliced NEMO isoform constitutes a novel autoinflammatory disease characterized by unbalanced NF-kB and IRF3 mediated responses to viral infection.