{"id":{"repo_id":"zurich","oai_identifier":"oai:www.zora.uzh.ch:20.500.14742/218644"},"canonical_url":"https://search.dev.ndltd.org/etd/zurich/oai:www.zora.uzh.ch:20.500.14742/218644","repository":{"repo_id":"zurich","name":"Universität Zurich","base_url":"https://www.zora.uzh.ch/server/oai/request"},"display":{"title":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Schiefer, Samira Nadine"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-03-25","date_published":"2024-03-25","updated_at":"2026-08-21T16:51:01Z","subjects":["610 Medicine & health","570 Life sciences; biology"],"languages":["eng"],"rights":["info:eu-repo/semantics/openAccess"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":null,"outbound_label":null,"outbound_source":null},"source_record":{"url":"https://www.zora.uzh.ch/server/oai/request?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Awww.zora.uzh.ch%3A20.500.14742%2F218644","prefix":"oai_dc"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Schiefer, Samira Nadine"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2024-03-25"]},{"key":"dc:relation","label":"Dc Relation","values":["https://www.zora.uzh.ch/handle/20.500.14742/218644"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["610 Medicine & health","570 Life sciences; biology"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["info:eu-repo/semantics/openAccess"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["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."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:source","label":"Dc Source","values":["Schiefer, S. N. (2024). Analysis of Human Interferon Signaling and its Regulation. (Dissertation, University of Zurich) https://doi.org/10.5167/uzh-258788"]},{"key":"dc:title","label":"Title","values":["Analysis of Human Interferon Signaling and its Regulation"]}]}],"canonical_facts":{"dc:creator":["Schiefer, Samira Nadine"],"dc:date":["2024-03-25"],"dc:description":["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."],"dc:format":["application/pdf"],"dc:language":["eng"],"dc:relation":["https://www.zora.uzh.ch/handle/20.500.14742/218644"],"dc:rights":["info:eu-repo/semantics/openAccess"],"dc:source":["Schiefer, S. N. (2024). Analysis of Human Interferon Signaling and its Regulation. (Dissertation, University of Zurich) https://doi.org/10.5167/uzh-258788"],"dc:subject":["610 Medicine & health","570 Life sciences; biology"],"dc:title":["Analysis of Human Interferon Signaling and its Regulation"],"dc:type":["Dissertation"]},"updated_at":"2026-08-21T16:51:01Z"}