Innate immunity provides the first line of defense against invading pathogen by recognizing and mounting a response to the pathogenic challenge. Among the cellular mechanisms of the innate immune response, Toll-like receptor 3 (TLR3) recognizes viral dsRNA and signals subsequent production of type-I interferon. The TLR3:interferon signaling cascades contains a kinase complex composed of two kinases and a scaffold protein, NAK-associated protein 1 (NAP1). The role of NAP1 in modulating kinase activation or regulation is unknown. A key inhibitory protein identified in the TLR3:interferon pathway, silencer of inhibitor of κBα kinase ε (SIKE), blocks the activity of this kinase complex through an unknown mechanism. The long term goal of this project is to define how protein:protein interactions modulate signal transduction in this pathway as mediated by host in the context of an immune response, pathogen as it attempts to subvert the host immune system and in disease states such as cancer and obesity. Objectives for this thesis were to produce recombinant SIKE and NAP1 material that could be used to elucidate the self-association pattern and hetero-interactions mediated by these components within the pathway. SIKE full-length, SIKE 72 (residues 72-207), NAP 270 (residues 1-270) and NAP 255 (residues 1-255) expression constructs were completed and recombinant protein produced using either a bacterial or baculovirus/insect cell expression system. Self-association was characterized by size exclusion chromatography and analytical ultracentrifugation. Hetero-interactions were explored via co-precipitation assays of recombinant proteins. SIKE 72 formed a primarily dimeric structure whereas NAP 255 forms a single species that appears to be monomeric at this stage of analysis. Hetero-interactions form between the kinase TBK-1 and NAP 255 and also TBK-1 and SIKE 72. TBK-1 shows a higher preference for binding NAP 255 in the prescence of both NAP 255 and SIKE 72. This work provides methodology to produce recombinant material for two components of the TLR3:interferon pathway and their initial biochemical characterization for both self-association and hetero-interactions.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-1085 |
| Date | 01 January 2010 |
| Creators | Forbes, Jonathan |
| 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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