DOI
https://doi.org/10.25772/C0T2-F683
Defense Date
2013
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Biochemistry
First Advisor
Jessica K. Bell
Abstract
To defend against pathogen challenge, multi-cellular organisms mount an immune response that recognizes, sequesters and eradicates invading infectious agents. Critical to this safeguard is the receptor-mediated detection of pathogens. Pathogen recognition then initiates a variety of signaling cascades that lead to the modulation of genes orchestrating an immune response. Toll-like receptor 3 (TLR3), a transmembrane receptor found in endosomes, is vital to the innate immune response against viruses. Double-stranded RNA (dsRNA) stimulation of TLR3 initiates a signaling cascade that leads to the production of type-I interferons and proinflammatory cytokines necessary to trigger the protective defenses of the immune system. Critical to this pathway is the activation of a kinase, TANK binding kinase 1 (TBK1), which phosphorylates the downstream transcription factors, IRF3 and IRF7, and leads to the production of IFN-beta. Interestingly, TBK1 function has been implicated in a number of other signaling cascades ranging from the insulin response and vesicle transport to xenophagy and anti-viral immunity. Increasingly, however, TBK1 dysregulation has been linked to autoimmune disorders and cancers, heightening the need to understand regulatory controls of TBK1. As a result, this dissertation investigates three components of the TLR3 signaling cascade in an attempt to further advance our understanding of the innate immune response. First, investigations into the adjuvant potential of dsRNA reveal that a 139bp dsRNA molecule is a viable candidate for vaccine adjuvant studies. Next, structural and functional studies of TLR3 with neutralizing antibodies provide evidence for a new TLR-signaling model in which dsRNA:TLR3 signaling units laterally cluster to achieve efficient signaling. Finally, cell-based assays, biophysical experiments and kinetic investigations into the mechanism by which an endogenous regulator of the TLR3 response, SIKE, functions, reveal that SIKE not only inhibits TBK1-mediated IRF3 phosphorylation, but is also a high affinity substrate. Findings from this study further suggest that SIKE regulates a critical catalytic hub not by direct repression of activity, but by redirection of catalysis through substrate affinity. Taken together, the results presented in this dissertation establish a foundation for building long-term studies on the function, regulation and viral subversion of the innate immune response.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
May 2013