Double stranded RNA (dsRNA), the genomic material of some viruses and a replication intermediate in others, is recognized by multiple signaling receptors that initiate the anti-viral response1. Viruses have developed mechanisms to circumvent the anti-viral response by targeting components of the signaling pathway. An example of one such pathway is the TLR3 signaling pathway, which contains a kinase complex that activates interferon regulatory factor 3 (IRF3), leading to production of type I interferons. The kinase complex consists of a scaffold protein, NAK-associated protein 1 (NAP1), and two kinases, TANK-binding kinase 1 (TBK-1) and IκB kinase epsilon (IKKε). A fourty residue sequence in NAP1 was discovered that mediated its interaction with TBK1 and IKKε, termed the kinase binding domain (KBD)1. However, the function of NAP1 in mediating kinase activation is unknown and understanding this is the long-term goal of this project. The goal of this thesis was to test the dependency of NAP1’s dimeric structure on mediating interactions with the kinases. Biochemical characterization of recombinant targets was completed using size-exclusion chromatography (SEC) and NAP1 KBD WT eluted as a dimeric species. CFP/YFP/Alexa Fluor 546 fusion proteins of the NAP1 KBD and scaffold binding motif (SBM) of the kinases, TBK-1 and IKKε, were generated to assess interactions using fluorescence resonance energy transfer (FRET). NAP1 KBD directly interacts with TBK1 and IKKε, with low micromolar affinity in vitro. Mutagenesis was attempted to identify the residues necessary for NAP1 dimerization and any effect dimerization may have on kinase recognition. This thesis shows data to support that NAP1 KBD forms stable homo-oligomers and directly interacts with a small C-terminal portion of TBK1 and IKKε.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-3501 |
| Date | 27 May 2011 |
| Creators | Call, Richard |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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