Mediator of IRF3 activation (MITA), also known as stimulator of interferon genes (STING), plays a vital role in the innate immune responses to cytosolic dsDNA. The trafficking of MITA from the ER to perinuclear vesicles is necessary for its activation of the downstream molecules, which lead to the production of interferons and pro-inflammatory cytokines. However, the exact mechanism of MITA activation remains elusive. Here, we report that transmembrane emp24 protein transport domain containing 2 (TMED2) potentiates DNA virus-induced MITA signaling. The suppression or deletion of TMED2 markedly impairs the production of type I IFNs upon HSV-1 infection. TMED2-deficient cells harbor greater HSV-1 load than the control cells. Mechanistically, TMED2 associates with MITA only upon viral stimulation, and this process potentiates MITA activation by reinforcing its dimerization and facilitating its trafficking. These findings suggest an essential role of TMED2 in cellular IFN responses to DNA viruses.

The transcription factor NF-κB plays critical roles in many biological processes, especially immunity. The signaling to NF-κB activation is subtly regulated to avoid harmful immune effects. In this report, we identified ubiquitin-specific protease 2 isoform a (USP2a) as a novel negative regulator in Toll-like receptors/IL-1β- and Sendai virus (SeV)-induced NF-κB activation. Overexpression of USP2a inhibited IL-1β- and SeV-induced NF-κB activation and transcription of inflammatory cytokines, whereas the knockdown or knockout of USP2a had opposite effects. USP2a-deficient cells exhibited potentiated ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6) upon stimulation by IL-1β and SeV. Furthermore, USP2a was constitutively associated with TRAF6, and removed K63-linked polyubiquitin chains of TRAF6 induced by IL-1β and SeV stimulation. The residues of USP2a important for their role were also identified. Because of the importance of TRAF6 in multiple pathways leading to NF-κB activation, these findings provide a general regulatory mechanism for NF-κB activation triggered by different stimuli.

Ag receptor engagement triggers lymphocyte activation and proliferation by activating several transcription factors including NF‐κB. Caspase recruitment domain (CARD) containing membrane‐associated guanylate kinase (MAGUK) protein 1 (CARMA1) is an essential adaptor protein that links Ag receptors to NF‐κB activation. Here, we identify stress‐induced‐phosphoprotein 1 homology and U‐box containing protein 1 (STUB1) as a CARMA1‐associated protein. STUB1 constitutively interacted with CARMA1, and the interaction was intensified by TCR stimulation. Downregulation of STUB1 expression by RNAi markedly diminished TCR‐induced canonical NF‐κB activation and IL‐2 production. Furthermore, overexpression of STUB1 enhanced the ubiquitination of CARMA1, whereas knockdown of STUB1 abolished the endogenous ubiquitination of CARMA1 induced by TCR stimulation. Subsequently, the ubiquitination of CARMA1 catalyzed by STUB1 was identified as Lys‐27 linked, which is important for CARMA1‐mediated NF‐κB activation. These data provide the first evidence that ubiquitination of CARMA1 by STUB1 promotes TCR‐induced NF‐κB signaling.

The paracaspase MALT1 is essential for the activation of NF-κB in response to T cell receptor (TCR) stimulation. It recruits downstream TRAF6 and activates the E3 ligase activity of TRAF6 to polyubiquitinate several targets, which ultimately leads to NF-κB activation. Here we identified ubiquitin-specific protease 2a (USP2a) as a MALT1-associated protein by biochemical affinity purification. Endogenous USP2a constitutively interacted with TRAF6, but dynamically interacted with MALT1 and CARMA1 in a stimulation-dependent manner. RNA interference (RNAi)-mediated silencing of USP2a attenuated TCR-induced NF-κB activation and production of interleukin-2 (IL-2). In addition, the ubiquitination of MALT1 and TRAF6 were both suppressed by USP2a knockdown. By knockdown and reconstitution assays, we found that USP2a mediated the interaction between MALT1 and TRAF6 in a catalytic activity-dependent manner. Furthermore, USP2a deSUMOylated TRAF6. Our findings implicate that USP2a plays an important role in TCR signaling by deSUMOylating TRAF6 and mediating TRAF6-MALT1 interaction.