Because IRF8 and several other IRF members are modified by both ubiquitin and SUMO, we asked whether these modifications occur in other proteins during innate immune responses. Macrophages were stimulated with IFNand a toll like receptor (TLR) ligand, CpG which activates the TLR pathway and triggers transcription of many genes important for macrophage function. Some of these genes are responsible for resistance against pathogens and other genes are involved in inflammatory responses in the host. To detect global ubiquitin or SUMO modifications of nuclear proteins in stimulated macrophages, we performed immunoblot analysis using antibody for ubiquitin, SUMO1 or SUMO2/3. To this end we tested nuclear fractions obtained from stimulated RAW264.7 macrophage cells. While there is only one ubiquitin protein, there are three SUMO molecules. SUMO1 is distinct from SUMO2 and SUMO3 that are more than 90% identical in amino acid sequence, and currently there is no antibody that distinguishes SUMO2 and SUMO3. We found that ubiquitin linked proteins were dramatically increased following IFNCpG stimulation. IFNtreatment alone resulted in significant increase in ubiquitin-liked proteins. However, additional stimulation by CpG further increased the amount of ubiquitin bound proteins. Not only CpG, but other TLR ligands tested including LPS and poly IC also caused increased ubiquitination, provided that macrophages were prestimulated with IFNSimilarly, IFN/TLR stimulation caused a marked increase in proteins conjugated to SUMO1 or SUMO2/3. Our data demonstrate that ubiquitin and SUMO modifications are integral part of innate immune responses, likely coupled with large changes in transcription and other nuclear activities, which helps accommodate a rapid response to pathogen stimuli. To begin cataloguing individual proteins modified by ubiquitin, we have performed proteomic analysis of ubiquitin-bound proteins in stimulated macrophages. RAW macrophages stably expressing Flag-tagged ubiquitin were stimulated with IFNCpG. Proteins coprecipitated with anti-Flag antibody were analyzed by MSMS massspectrometry. These proteins included factors in the ubiquitin pathways, those involved in DNA metabolism, chromatin and transcriptional regulation, and mRNA processing. The largest group of proteins found in our list was ribosomal proteins important for protein translation. Other proteins found here were heat shock proteins and stress-response factors, suggesting a link between macrophage activation and stress response. In conclusion, upon macrophage activation, a large number of nuclear proteins become associated with ubiquitin modification, presumably leading to a global shift in the genome activity, important for proper execution of innate immune responses.

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