| Invasions by microorganisms are initially countered by innate defense mechanisms that preexist in all individuals. A proper inflammatory response to an infection with a pathogen is the result of balanced controls at many levels including, in the signal transduction pathway, in the control of gene expression in the nucleus, and in the interactions between different cells of the immune system. Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract where this regulation is perturbed. Mutations in the gene encoding an innate immune recognition molecule, NOD2 have been linked to susceptibility to Crohn's Disease. Mutations in a different part of this gene cause another inflammatory disease, Blau Syndrome (BS). Also known as CARD15, NOD2 is an intracellular protein expressed mainly in macrophages and dendritic cells and it confers responsiveness of these innate immune cells to muramyl-dipeptide (MDP), a long-known adjuvant and a substructure of bacterial cell wall peptidoglycan. Upon stimulation of the cells by MDP, the Nuclear-Factor Kappa-B (NF-kappaB) is activated and several proinflammatory cytokines are produced. To understand how NOD2 functions and how its mutations predispose to development of inflammatory diseases, I aimed to identify signaling components of the NOD2 pathway.;In this thesis, I have described the use of the tandem affinity purification (TAP) technique to identify UBR1, an E3-ubiquitin ligase, as a protein that associates with the wild-type NOD2 protein and the Blau Syndrome mutant NOD2 protein. The association of UBR1 with NOD2 was confirmed by co-immunoprecipitation using an antibody against NOD2. The association of NOD2 with UBR1 is consitutive and independent of MDP stimulation, while the association of a Crohn's disease susceptibility allele from of NOD2 and UBR1 appears to be weaker. In addition, RIP2, an essential adaptor kinase for the NOD2 signaling was downregulated in HEK293Y cells overexpressing the wild-type NOD2 protein and in THP-1 monocytic cell lines stimulated with MDP. Moreover, RIP2 downregulation was not observed in mouse embryonic fibroblasts that lack the UBR1-mediated N-end rule pathway of protein degradation. These results suggest that the function of UBR1-associated with NOD2 is to downregulate signaling by promoting the degradation of RIP2. |
