The role of ANKIB1 in regulating cell death signaling versus geneactivation following TLR3 activation

Biochemical reactions in cells are necessary to fulfill underlying cellular processes and signal transduction from both, the extracellular and intracellular environment. Their dysregulation can lead to an increase of morbidity and mortality in organisms. Because of that, they are strictly regulated to hamper adverse outcomes. One of the most common regulatory mechanisms are post-translational modifications (PTMs) of proteins, including by ubiquitination. Different pairs of E2 ubiquitin conjugating enzymes and E3 ubiquitin ligases can generate different types of ubiquitination that lead to diverse outcomes for the substrate protein. Nonetheless, their implication in the control of the different immune and inflammatory signaling pathways needs to be further studied. In the current thesis, I dissected the role of a new E3 ubiquitin ligase, Ankyrin repeat and IBR domain- containing protein 1 (ANKIB1), in the Toll-like Receptor 3 (TLR3) signaling pathway and in the antiviral response. The Walczak group previously showed that ANKIB1 has an active catalytic domain and interacts with the TLR3 signaling complex (TLR3-SC) upon stimulation. I have demonstrated that ANKIB1, especially its catalytic activity, is required for the activation of TANK-Binding Kinase 1 (TBK1) and, consequently, type I interferon (IFN) production following TLR3 activation. Oppositely, the deletion of ANKIB1 does not have any consequences on Tumor Necrosis Factor (TNF) signaling. I have shown that, upon TLR3 stimulation, ANKIB1 does not only interact with activated TBK1 but also with optineurin (OPTN) and the NF-kappa-B Essential Modulator (NEMO), the primordial protein for TBK1 activation. To further elucidate the regulation of this process, I have performed an unbiased proteomic analysis of the ubiquitinated proteins upon TLR3 stimulation and found that TBK1, Interferon regulatory factor 3 (IRF3), and TNF Induced Protein 3 (A20) contain ubiquitinated sites that are dependent on the presence of ANKIB1. Interestingly, I have also seen the same consequence on Gasdermin E suggesting a potential role of this new E3 ligase in the regulation of cell death. Therefore, I dissected the potential role of ANKIB1 on TLR3-induced cell death in vitro using WT and ANKIB1 KO cells. This analysis revealed that ANKIB1 deficiency in HT29 cells results in higher susceptibility to TLR3-induced cell death. Overall, this study highlights the requirement of ANKIB1, a previously uncharacterized E3 ligase, for TBK1 activation and, consequently, type I IFN production in TLR3 signaling. Several components of this signaling complex are impacted by the absence of ANKIB1 in terms of ubiquitination, implying an important contribution of ANKIB1 to TLR3-induced gene activation and regulation of cell death, primordial to the induction of an efficient immune response against pathogens.