Composition of the myddosome during the innate immune response

Abstract Critical for the innate immune response, the myddosome is a complex of proteins characterized by the presence of myeloid differentiation factor 88 (Myd88). The myddosome acts to transfer signals from the toll-like receptor (TLR) proteins to TNF receptor-associated factor 6 (TRAF6). In addition to Myd88, the myddosome contains multiple copies of the interleukin-1 receptor associated kinases (IRAK) 2 and 4 and is theorized to form following stimulation of the TLR proteins. Using affinity purification – mass spectrometry analysis (AP-MS), we identified the Myd88-associated proteins in mouse immortalized bone marrow-derived macrophages before and after lipopolysaccharide (LPS) treatment. Prior to LPS treatment, we found the stable association of Myd88 to IRAKs 2 and 4 along with the inhibitory factors IRAK3 and Sterile alpha and TIR motif-containing protein 1 (Sarm1). Following LPS treatment, the inhibitory factors were lost and Myd88 associated to the downstream effector proteins, including TRAF6. In contrast to the model, our results suggest that the cellular myddosome is preassembled but only activates upon stimulation. Because of this, we are examining the mechanism(s) that regulate the activity of the myddosome. We are currently using AP-MS followed by phosphopeptide enrichment with MS/MS analysis to identify possible phosphorylation sites. Using these data, we will evaluate the role of phosphorylation on myddosome activity. In addition, we will use SILAC with AP-MS to examine myddosome assembly and protein phosphorylation during a time course. This research was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases.