ABSTRACT: This project aims to define, by using a functional proteomic approach, the molecular partners associated with the canonical coupound of the IMD pathway. The Drosophila defense against pathogens largely relies on the activation of two major pathways, IMD and TOLL. The IMD pathway is activated mainly by Gram-negative bacteria, whereas the TOLL pathway responds predominantly to Gram-positive bacteria and fungi. The activation of this pathway leads to the rapid induction via NF-kB of numerous genes of the immune system, including various antimicrobial peptide genes. Recent evidence indicates that the IMD pathway is also involved in various other reactions in the absence of infection ("inflammatory-like" reactions). To gain a better understanding of the molecular machineries underlying the pleiotropic functions of this pathway, we have developed a proteomics analysis. Our first aim was to identify the proteins interacting with the 11 canonical members (PGRP-LC, PGRP-LE, IMD, BG4 (dFADD), DREDD, TAK1, TAB2, IAP2, IRD5 (DmIKKbeta), KEY (DmIKKgamma), and RELISH) previously shown to be involved in the activation of the IMD pathway. Each protein was fused N-terminally or C-terminally with a biotin tag and was stably expressed in S2 cells previously subjected to stable integration of the bacterial enzyme BirA to allow for biotinylation of the tags. In total, we established 22 stable transformant cell lines (11 genes x 2 tag locations), which were individually stimulated by heat-killed E.coli before harvest at 4 different time points (t=0 min, 10 min, 2 hour, and 8 hour). This procedure was followed by streptavidin-mediated affinity purification of 96 bait-protein complexes (22 transformants x 4 time points, plus 8 unstimulated controls without baits), on-bead trypsin digestions of the protein complexes and six LC-MS/MS analyses. We have identified 371 interactanting proteins in heat-killed E.coli stimulated Drosophila S2 cells, 90% of which have human orthologues. Comparative analysis of Gene Ontology (GO) annotation datasets from Human and Flies point to three significant common categories: (1) NuA4 Histone acetyltransferase complex, (2) SWI/SNF-type Chromatin remodeling complex, and (3) Transcription cofactor binding. Our Drosophila data point in particular to SUMOylation of the IkB-kinase homologue Ird5 in Drosophila, a level of regulation not described to date. Among numerous directions opened by our proteomics analysis, we have decided to first investigate this facet of the IMD pathway. We show nthat a highly conserved SUMOylation consensus site binds Drosophila SUMO (SMT3) in a challenge-dependent manner and that this process confers an increased level of activation of the antimicrobial peptide genes during bacterial challenge both in vitro and in flies. Our results demonstrate that SUMOylation of IKKbeta plays an important role in the induction of antimicrobial peptide genes. The MS analysis was performed on a FT ICR mass spectrometer (LTQ-FT Ultra, ThermoFisher Scientific, San Jose, CA) with the top 7 acquisition method: MS resolution 60,000, mass range 500-2000 Th, followed by 7 MS/MS (LTQ) on the 7 most intense peaks, with a dynamic exclusion for 90 s. The raw data were processed using Xcalibur 2.0.7 software. Each sample was first analyzed in triplicate then an exclusion list was added for three next runs. The database search was done on merged data using Mascot search engine (Matrix Science Mascot 2.3) on the 17D melanogaster databank (16535 sequences). Proteome Discoverer 1.3 (ThermoFisher Scientific) and Mascot were used to search the data and filter the results. The following parameters were used: up to 2 miss cleavages; MS tolerance 10 ppm; MSMS tolerance 1 Da; full tryptic peptides; partial modifications carbamidomethyla- tion (C), oxydation (M, H, W), Phosphorylation (Y).
Yann Verdier 
