Analysis of Human Interferon Signaling and its Regulation

Abstract

Type I interferon (IFN) signaling is a crucial first line in the defense against viral pathogens. After virus recognition by infected cells, type I IFN is produced and signals to neighboring cells. The IFN is bound by receptors on the cell surface, which induces an intracellular phosphorylation-dependent signaling cascade. The active Janus kinases JAK1 and tyrosine kinase 2 (TYK2) phosphorylate signal transducer and activator of transcription 1 (STAT1) and STAT2, which act as a transcription factor together with IFN-regulatory factor 9 (IRF9) to induce antiviral IFN-stimulated genes (ISGs). Unraveling the complex and dynamic processes that control type I IFN signaling is essential for understanding the regulatory mechanisms involved in the host defense against viruses.

In the main part of this thesis, I applied TurboID-based proximity labeling to identify putative interactors of all seven canonical type I IFN signaling members, IFNAR1, IFNAR2, JAK1, TYK2, STAT1, STAT2, and IRF9, at various times post type I IFN stimulation. Using highly stringent selection criteria and label-free quantification (LFQ) this led to the identification of 103 proximal proteins beyond the canonical type I IFN signaling members themselves. Amongst those putative interactors were both previously validated interactors and novel, previously unassociated proteins. After functional screening of 50 candidates using small interfering RNA (siRNA)-mediated depletion, I selected the E3 ubiquitin ligase PJA2 for more in-depth characterization. PJA2 was detected in proximity to TYK2 in the TurboID screen and was further confirmed as a negative regulator of IFN signaling. Depletion of PJA2 increased ISG expression and antiviral activity, while overexpression of PJA2 led to a dose-dependent reduction in ISG expression. Notably, I discovered that the E3 ubiquitin ligase activity of PJA2 was required for its suppression of type I IFN signaling. I further verified that PJA2 interacted with TYK2, as well as JAK1, the other Janus kinase involved in type I IFN signaling. PJA2 induced the non-lysine and non-degradative ubiquitination of TYK2, and likely JAK1, to restrain downstream STAT1 phosphorylation.

In the second part of this thesis, I re-analyzed the data from the TurboID screen using different selection criteria and different protein quantification methods (LFQ and spectral counting, SPC), and then compared these results to the original LFQ-based analysis. This led me to identify a higher number of proximal proteins, which contained previously annotated interactors of type I IFN signaling components as well as novel, previously unassociated proteins. Taken together with the identification of some known regulators only in the SPC-based analysis, this implies that new relevant factors were revealed. Furthermore, the deubiquitinase, ubiquitin-specific peptidase 9X (USP9X), was selected for a more in-depth characterization. USP9X was identified in proximity to both STAT2 and IRF9 in the TurboID screen, and siRNA-based functional studies suggested that it was another promising candidate that modulates the antiviral function of type I IFN. Indeed, I could confirm these interactions and could provide evidence that USP9X negatively regulates type I IFN signaling, but I could not observe a functional dependence on its deubiquitinase activity.

In summary, I identified and characterized two ubiquitin-system enzymes, PJA2 and USP9X, as negative regulators of type I IFN signaling. Furthermore, I uncovered a network of 888 putative interactors of the canonical type I IFN signaling members, which may impact antiviral type I IFN signaling. This knowledge provides a valuable foundation for future characterization of potential IFN signaling regulators.