MyD88 is the critical adaptor protein for the entire set of mammalian Toll like receptors (TLRs) mediated pathways except TLRS. Our long-term goal is to characterize the signaling network of innate immune responses mediated by TLRs as a necessary prerequisite to the development of potential drug targets to alter the consequences of innate immune activation through TLRs, an area that has been listed as an NIAID prioritized research area. The central hypothesis of this proposal is that MyD88 may interact with different proteins in order to trigger different pathways following TLR2 and TLR9 activation. We base this hypothesis on the following observations: (1) A subset of TLRs, TLR7, TLRS and TLR9, induces antiviral responses by producing interferon-alpha. Activation of TLR2, on the other hand, utilizes MyD88 as adaptor yet does not lead to the production of IFN-?. (2) TLR9 signaling leading to the production of IFN-? is dependent on MyD88-IRF-7 interaction that only occurs in endosome vesicles. (3) MyD88 is capable of binding proteins that are only involved in a specific TLR pathway. Typical approaches to understand assembly of an immune signaling complex include such approaches as the yeast two-hybrid system, protein homologue search by computational analysis, coimmunoprecipitation, and site directed mutagenesis. However, the above approaches are relatively inefficient in direct identification of components of signaling complexes from actual immune cells. We have previously developed a quantitative mass spectrometry-based approach that allowed ultra sensitive detection of MyD88 interacting proteins directly from murine macrophages following stimulation with TLR4 agonist. In this proposal, we plan to further improve this technology so that MyD88 signaling complex following activation of TLR2 and TLR9 can be detected and characterized. Thus we have two goals: First, to further improve the quantitative proteomic approach for immune signaling study; second, to define the signaling specificity of different TLR pathways mediated by MyD88 by directly identifying interacting proteins formed around MyD88 when different TLR pathways are triggered. Through these studies, knowledge of will be gained. A high throughput technology will also be developed for biologists who are interested in signaling study. ? ? ? ?
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