Dataset Information

Protein ADP-ribosylation, phosphorylation, and interactome dynamics in macrophage activation.

ABSTRACT: The innate immune response relies on efficient, robust and fast protein signaling networks to relay information related to pathogen or viral detection. This communication is mediated primarily through protein-protein interactions and post-translational modifications (PTMs), events which are best characterized by mass spectrometry (MS)-based proteomics. This in-depth study uses MS to identify changes in protein signaling networks of Lipopolysaccharide (LPS)-stimulated human and mouse macrophages, at the level of single PTMs (via phosphorylation and ADP-ribosylation site ID) and protein complexes (via size exclusion chromatography and immunoprecipitation). The result is a curated meta-database of 6,475 proteins including 2,311 ADP-ribosylated proteins and 2,284 phosphoproteins present in LPS-stimulated macrophages. Follow up studies characterized the ASK protein complex – which appeared to dissociate upon LPS stimulation – and a complex which formed upon LPS stimulation and contained the poly(ADP-ribosyl) transferase PARP9 protein.

INSTRUMENT(S): Q Exactive HF, Velos Plus

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Bone Marrow, Blood, Macrophage

SUBMITTER: Casey Daniels

LAB HEAD: Aleksandra Nita-Lazar

PROVIDER: PXD016469 | Pride | 2020-06-17

REPOSITORIES: Pride

ACCESS DATA

Publications

Dynamic ADP-Ribosylome, Phosphoproteome, and Interactome in LPS-Activated Macrophages.

Daniels Casey M CM Kaplan Pauline R PR Bishof Isaac I Bradfield Clinton C Tucholski Trisha T Nuccio Arthur G AG Manes Nathan P NP Katz Samuel S Fraser Iain D C IDC Nita-Lazar Aleksandra A

Journal of proteome research 20200701 9

We have used mass spectrometry (MS) to characterize protein signaling in lipopolysaccharide (LPS)-stimulated macrophages from human blood, human THP1 cells, mouse bone marrow, and mouse Raw264.7 cells. Protein ADP-ribosylation was truncated down to phosphoribose, allowing for enrichment and identification of the resulting phosphoribosylated peptides alongside phosphopeptides. Size exclusion chromatography-MS (SEC-MS) was used to separate proteoforms by size; protein complexes were then identifie ...[more]

PMID: 32529831

Similar Datasets

Project description:Dendritic cells (DCs) are a special class of leukocytes able to activate both innate and adaptive immune responses. They interact with microbes through germline-encoded pattern-recognition receptors (PRRs), which recognize molecular patterns expressed by various microorganisms. Upon antigen binding, PRRs instruct DCs for the appropriate priming of natural killer cells, followed by specific T-cell responses. Once completed the effector phase, DCs reach the terminal differentiation stage and eventually die by apoptosis. We have observed that following lipopolysaccharide (LPS)-stimulation the initiation of the apoptotic pathway in DCs is due the activation of NFAT proteins. Indeed, LPS induces Src-family kinase and phospholipase C (PLC)Î³2 activation, influx of extracellular Ca2+ and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. According with this observation CD14-deficient DCs do not die following LPS stimulation. Nevertheless, CD14-deficient DC death following LPS activation can be restored by co-stimulating DCs with LPS and thapsigargin. Thapsigargin empties the intracellular calcium stores by blocking calcium pumping into the sarcoplasmic and endoplasmic reticulum and thereby activates plasma membrane calcium channels. This, in turn, allows an influx of calcium into the cytosol and NFAT activation. To identify the NFAT controlled apoptosis genes in LPS activated DCs we have performed a kinetic microarray analysis (0, 48 and 60 h) in conditions allowing or inhibiting NFAT activation. Four genes have been selected: Nur77, Gadd45g, Ddit3/Gadd153/Chop-10 and Tia1. To identify apoptosis genes selectively modulated by NFAT, a comparative kinetic (time points 0, 48 and 60 h) microarray analysis was performed in the following conditions: 1) wild type bone marrow derived DCs (wtBMDCs) stimulated with LPS; 2) CD14-deficient BMDCs stimulated with LPS; 3) wtBMDCs stimulated with LPS in presence of thapsigargin; 4) CD14-deficient BMDCs stimulated with LPS in presence of thapsigargin.

Project description:Macrophages and dendritic cells (DCs) differently contribute to the generation of coordinated immune system responses against infectious agents. They interact with microbes through germline-encoded pattern-recognition receptors (PRRs), which recognize molecular patterns expressed by various microorganisms. Upon antigen binding, PRRs instruct DCs for the appropriate priming of natural killer cells, followed by specific T-cell responses. Once completed the effector phase, DCs reach the terminal differentiation stage and eventually die by apoptosis. By contrast, following antigen recognition, macrophages initiate first the inflammatory process and then switch to an anti-inflammatory phenotype for the restoration of tissue homeostasis. Following lipopolysaccharide (LPS)-stimulation the initiation of the apoptotic pathway in DCs is due the activation of NFAT proteins. DC stimulation with lipopolysaccharide (LPS) induces Src-family kinase and phospholipase C (PLC)Î³2 activation, influx of extracellular Ca2+ and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. We asked whether macrophage survival after LPS encounter was due to their inability to activate the Ca2+ pathway. As matter of fact, bone marrow-derived macrophages were unable to mobilize Ca2+ and to translocate NFAT to the. To further investigate whether the Ca2+-NFAT pathway played any role in LPS-stimulated macrophages, we performed a comparative kinetic microarray analysis in conditions allowing or inhibiting NFAT activation. We show here that no modulation of gene expression can be attributed to NFAT. Gene expression analyses were performed using Affymetrix GeneChips in the following groups of murine macrophaghes: 1) CD14-deficient macrophages stimulated with LPS; 2) wt macrophages stimulated with LPS in presence of EGTA; 3) wt macrophages stimulated with LPS. The following kinetic time points were examined: 0, 6 and 24 hours following LPS activation. This experimental setting allowed us to select for effects due to Ca2+ fluxes and exclude the effects due to other causes, particularly the block of TRIF recruitment in CD14-deficient cells and the EGTA effects unrelated to Ca2+ chelation.

Dataset Information

Protein ADP-ribosylation, phosphorylation, and interactome dynamics in macrophage activation.

Publications

Dynamic ADP-Ribosylome, Phosphoproteome, and Interactome in LPS-Activated Macrophages.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure