Rapid production of type I Interferon is pivotal to initiate cellular antiviral host defense and adaptive immunity. In order to facilitate innate immune processes, a cell harbors pattern recognition receptors (PRRs) which sense distinctive forms of pathogen associated molecular patterns (PAMPs). For example, Toll like receptors (TLRs) and RIG-I like receptors (RLRs) were discovered as PRRs for pathogen derived molecules and the production of type I Interferon (IFN). To induce type I IFN, several transcription factors such as nuclear factor-kappaB (NF-ĸB), interferon regulatory factor 3 (IRF3), interferon regulatory factor 7 (IRF7), and activating protein-1 (AP-1) need to be stimulated through the specific signaling adaptors. Among them, our lab is interesting in the death domain (DD) containing proteins Receptor interacting kinase1 (RIP1) and Fas-associated death domain protein (FADD), which we showed were important for innate signaling processes. RIP1 and FADD were initially identified as Fas and TNFR interacting proteins which were involved in death receptor mediated apoptosis. Aside from apopotic function, recent publications indicate that RIP1 and FADD mediate cell survival, proliferation, and cytokine production through NF-ĸB activation. Here, we show that RIP1 and FADD are essential for efficient TLR-independent signaling. We report that RIP1 and FADD lacking MEF cells are sensitive to viral cytolysis and also exhibit impaired IFN production against dsRNA virus infection. RIP1 acts as a scaffolding protein for death receptor mediated apoptosis and NF-ĸB activation, necrosis, and innate immunity. As mentioned, we demonstrate that cells lacking RIP1 are sensitive to RNA virus infection. To understand the detailed mechanisms of RIP1 function in innate signaling, we first tested whether RIP1 is involved in RIG-I signaling. We found that RIP1 forms a complex with RIG-I in the presence of dsRNA. Additionally, we showed that RIP1 is required for optimal RIG-I and melanoma differentiation-associated protein 5 (MDA-5) activity. We also find that FADD, a RIP1 interaction protein, is implicated in innate immunity. To study the precise mechanisms of FADD in type I IFN signaling, we generated FADD variants and used luciferase reporter assays to indicate that the FADD death effector domain (DED) is crucial for IFN-β signaling. In order to identify interacting partners of FADD, yeast two hybrid assays were performed and indicated that FADD binds to protein inhibitor of activated STAT (PIAS1), part of the SUMO machinery. SUMOylation is a reversible post-translational modification of a protein by SUMO, a 100 amino acid protein. The consequence of SUMOylation alters specific proteins’ function by affecting activity, localization, stability or influencing molecular interactions by interfering with or linking to a target protein. To confirm FADD-PIAS interactions, we conducted in-vitro SUMOylation assays by using Ubc9 conjugated FADD and found possible FADD SUMOylation sites. We also discovered that FADD and SUMO are co-localized in the nucleus. This result reveals that FADD undergoes SUMOylations and its modification might regulate FADD’s function, including role in innate signaling. Furthermore, we report here that HTLV-1 Tax protein interacts with RIP1 and inhibits IFN-β inducing signaling by abrogating RIP1 and IRF7 interaction. This implies that RIP1 is involved in the regulation of IRF7 and is essential for IFN-β production. Collectively, our data demonstrate the significance of RIP1 and FADD in dsRNA recognition pathways in mammalian cells that are essential for the optimal induction of type I IFNs and other innate genes important for host defense.
| Identifer | oai:union.ndltd.org:UMIAMI/oai:scholarlyrepository.miami.edu:oa_dissertations-1573 |
| Date | 10 May 2011 |
| Creators | Hyun, Jinhee |
| Publisher | Scholarly Repository |
| Source Sets | University of Miami |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Open Access Dissertations |
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