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Landscape of protein-protein interactions in Drosophila immune deficiency signaling during bacterial challenge.

ABSTRACT: The Drosophila defense against pathogens largely relies on the activation of two signaling pathways: immune deficiency (IMD) and Toll. The IMD pathway is triggered mainly by Gram-negative bacteria, whereas the Toll pathway responds predominantly to Gram-positive bacteria and fungi. The activation of these pathways leads to the rapid induction of numerous NF-?B-induced immune response genes, including antimicrobial peptide genes. The IMD pathway shows significant similarities with the TNF receptor pathway. Recent evidence indicates that the IMD pathway is also activated in response to various noninfectious stimuli (i.e., inflammatory-like reactions). To gain a better understanding of the molecular machinery underlying the pleiotropic functions of this pathway, we first performed a comprehensive proteomics analysis to identify the proteins interacting with the 11 canonical members of the pathway initially identified by genetic studies. We identified 369 interacting proteins (corresponding to 291 genes) in heat-killed Escherichia coli-stimulated Drosophila S2 cells, 92% of which have human orthologs. A comparative analysis of gene ontology from fly or human gene annotation databases points to four significant common categories: (i) the NuA4, nucleosome acetyltransferase of H4, histone acetyltransferase complex, (ii) the switching defective/sucrose nonfermenting-type chromatin remodeling complex, (iii) transcription coactivator activity, and (iv) translation factor activity. Here we demonstrate that sumoylation of the I?B kinase homolog immune response-deficient 5 plays an important role in the induction of antimicrobial peptide genes through a highly conserved sumoylation consensus site during bacterial challenge. Taken together, the proteomics data presented here provide a unique avenue for a comparative functional analysis of proteins involved in innate immune reactions in flies and mammals.

SUBMITTER: Fukuyama H

PROVIDER: S-EPMC3696746 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Landscape of protein-protein interactions in Drosophila immune deficiency signaling during bacterial challenge.

Fukuyama Hidehiro H Verdier Yann Y Guan Yongsheng Y Makino-Okamura Chieko C Shilova Victoria V Liu Xi X Maksoud Elie E Matsubayashi Jun J Haddad Iman I Spirohn Kerstin K Ono Kenichiro K Hetru Charles C Rossier Jean J Ideker Trey T Boutros Michael M Vinh Joëlle J Hoffmann Jules A JA

Proceedings of the National Academy of Sciences of the United States of America 20130607 26

The Drosophila defense against pathogens largely relies on the activation of two signaling pathways: immune deficiency (IMD) and Toll. The IMD pathway is triggered mainly by Gram-negative bacteria, whereas the Toll pathway responds predominantly to Gram-positive bacteria and fungi. The activation of these pathways leads to the rapid induction of numerous NF-κB-induced immune response genes, including antimicrobial peptide genes. The IMD pathway shows significant similarities with the TNF recepto ...[more]

PMID: 23749869

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Project description: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).

Project description:Assembly of infectious hepatitis C virus (HCV) particles requires multiple cellular proteins including for instance apolipoprotein E (ApoE). To describe these protein-protein interactions, we performed an affinity purification mass spectrometry screen of HCV-infected cells. We used functional viral constructs with epitope-tagged envelope protein 2 (E2), protein (p) 7, or nonstructural protein 4B (NS4B) as well as cells expressing a tagged variant of ApoE. We also evaluated assembly stage-dependent remodeling of protein complexes by using viral mutants carrying point mutations abrogating particle production at distinct steps of the HCV particle production cascade. Five ApoE binding proteins, 12 p7 binders, 7 primary E2 interactors, and 24 proteins interacting with NS4B were detected. Cell-derived PREB, STT3B, and SPCS2 as well as viral NS2 interacted with both p7 and E2. Only GTF3C3 interacted with E2 and NS4B, highlighting that HCV assembly and replication complexes exhibit largely distinct interactomes. An HCV core protein mutation, preventing core protein decoration of lipid droplets, profoundly altered the E2 interactome. In cells replicating this mutant, E2 interactions with HSPA5, STT3A/B, RAD23A/B, and ZNF860 were significantly enhanced, suggesting that E2 protein interactions partly depend on core protein functions. Bioinformatic and functional studies including STRING network analyses, RNA interference, and ectopic expression support a role of Rad23A and Rad23B in facilitating HCV infectious virus production. Both Rad23A and Rad23B are involved in the endoplasmic reticulum (ER)-associated protein degradation (ERAD). Collectively, our results provide a map of host proteins interacting with HCV assembly proteins, and they give evidence for the involvement of ER protein folding machineries and the ERAD pathway in the late stages of the HCV replication cycle.IMPORTANCEHepatitis C virus (HCV) establishes chronic infections in the majority of exposed individuals. This capacity likely depends on viral immune evasion strategies. One feature likely contributing to persistence is the formation of so-called lipo-viro particles. These peculiar virions consist of viral structural proteins and cellular lipids and lipoproteins, the latter of which aid in viral attachment and cell entry and likely antibody escape. To learn about how lipo-viro particles are coined, here, we provide a comprehensive overview of protein-protein interactions in virus-producing cells. We identify numerous novel and specific HCV E2, p7, and cellular apolipoprotein E-interacting proteins. Pathway analyses of these interactors show that proteins participating in processes such as endoplasmic reticulum (ER) protein folding, ER-associated protein degradation, and glycosylation are heavily engaged in virus production. Moreover, we find that the proteome of HCV replication sites is distinct from the assembly proteome, suggesting that transport process likely shuttles viral RNA to assembly sites.

Project description:Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. Drosophila metamorphosis represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, nothing is known about the mechanisms that coordinate development and immune cell activity in the transition from larva to adult. Here, we reveal that regulation of macrophage-like cells (hemocytes) by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. Although it is generally accepted that steroid hormones impact immunity in mammals, their action on monocytes (e.g. macrophages and neutrophils) is still not well understood. Here in a simpler model system, we used an approach that allows in vivo, cell autonomous analysis of hormonal regulation of innate immune cells, by combining genetic manipulation with flow cytometry, high-resolution time-lapse imaging and tissue-specific transcriptomic analysis. We show that in response to ecdysone, hemocytes rapidly upregulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic and local production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential for hemocyte immune functions and survival after infection. Microarray analysis of hemocytes revealed a large set of genes regulated at metamorphosis by EcR signaling, among which many are known to function in cell motility, cell shape or phagocytosis. This study demonstrates an important role for steroid hormone regulation of immunity in vivo in Drosophila, and paves the way for genetic dissection of the mechanisms at work behind steroid regulation of innate immune cells.

Dataset Information

Landscape of protein-protein interactions in Drosophila immune deficiency signaling during bacterial challenge.

Publications

Landscape of protein-protein interactions in Drosophila immune deficiency signaling during bacterial challenge.

Similar Datasets

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