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Identification of proteins that interact with TRAF6 in ADP-heptose- or IL-1β-stimulated HEK293 cells

ABSTRACT: The bacterial metabolite ADP-heptose activates the mammalian protein kinase ALPK1 (alpha protein kinase 1), which then phosphorylates TIFA (TRAF interacting protein with FHA domain). This leads to the oligomerisation of TIFA and the recruitment and oligomerisation of TRAF6 (TNF receptor-associated factor 6), activating a signalling network that culminates in the production of inflammatory mediators that mount responses to combat microbial infection. To identify other protein(s) that associate with TRAF6 in ADP-heptose-stimulated cells, we re-expressed FLAG-TRAF6 in TRAF6 KO cells, stimulated the cells with ADP-heptose and immunoprecipitated TRAF6 from the cell extracts using a FLAG antibody. Proteins associated with TRAF6 were identified by mass spectrometry and included the components of LUBAC (the linear ubiquitin assembly complex), the components of the TAK1 and IB kinase (IKK) complexes, TRAF2 and c-IAP1, providing insight into the molecular details and mode of operation of the ADP-heptose signalling pathway. In parallel experiments the cells were stimulated with IL-1b, a cytokine that also signals via TRAF6, which served as a control.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Frederic Lamoliatte

LAB HEAD: Professor Sir Philip Cohen

PROVIDER: PXD034964 | Pride | 2022-09-14

REPOSITORIES: Pride

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A study into the ADP-ribosylome of IFN--stimulated THP-1 human macrophage-like cells identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-ribosylation

Project description:ADP-ribosylation is a post-translational modification that, until recently, has remained elusive to study at the cellular level. Previously dependent on radioactive tracers to identify ADP-ribosylation targets, several advances in mass spectrometric workflows now permit global identification of ADP-ribosylated substrates. In this study, we capitalized on two ADP-ribosylation enrichment strategies, and multiple activation methods performed on the Orbitrap Fusion Lumos, to identify IFN--induced ADP-ribosylation substrates in macrophages. The ADP-ribosyl binding protein, Af1521, was used to enrich ADP-ribosylated peptides, and the anti-poly-ADP-ribosyl antibody, 10H, was used to enrich ADP-ribosylated proteins. ADP-ribosyl-specific mass spectra were further enriched by an ADP-ribose product ion triggered EThcD and HCD activation strategy, in combination with multiple acquisitions that segmented the survey scan into smaller ranges. HCD and EThcD resulted in overlapping and unique ADP-ribosyl peptide identifications, with HCD providing more peptide identifications but EThcD providing more reliable ADP-ribosyl acceptor sites. Our acquisition strategies also resulted in the first ever characterization of ADP-ribosyl on three poly-ADP-ribose polymerases, ARTD9/PARP9, ARTD10/PARP10, and ARTD8/PARP14. IFN- increased the ADP-ribosylation status of ARTD9/PARP9, ARTD8/PARP14, and proteins involved in RNA processes. This study therefore summarizes specific molecular pathways at the intersection of IFN- and ADP-ribosylation signaling pathways.

2020-01-27 | PXD011690 | Pride

miRNA expression profile in platelets after ex vivo activation

Project description:We performed microRNA sequencing (miRNA-seq) according to Illumina sequencing protocols. Platelet-rich plasma (PRP) has been obtained by centrifuging whole blood at 160 x g for 15 min at room temperature. Platelets have been activated with 3 different physiologic agonists, namely ADP (20uM), collagen (60ug/mL), or Thrombin Receptor Activating Peptide (TRAP 25uM) under continuous stirring. An aliquot of platelets has been obtained at 120 minutes following ADP, collagen and TRAP and then processed to determine miRNA expression profiles. Time 0, indicating samples before any agonist treatment, was used as baseline. Total RNA was extracted by using mirVana Paris kit (life technologies) and then was subjected to size selection library preparation and retrotranscribed. Obtained cDNA was sequenced with the Illumina HiSeq 1000 sequencer .

2015-04-27 | E-MTAB-2078 | biostudies-arrayexpress

IL-17-induced NF-kB activation via CIKS/Act1: Physiologic significance and signaling mechanisms

Project description:Interleukin-17 (IL-17) is essential in host defense against extracellular bacteria and fungi, especially at mucosal sites, but it also contributes significantly to inflammatory and autoimmune disease pathologies. Binding of IL-17 to its receptor leads to recruitment of the adaptor protein CIKS/Act1 via heterotypic association of their respective SEFIR domains and to activation of the transcription factor NF-kB; it is not known whether CIKS and/or NF-kB are required for all gene induction events. Here we report that CIKS is essential for all IL-17 induced immediate-early genes in primary mouse embryo fibroblasts, while NF-kB is profoundly involved. We also identify a novel sub-domain in the N-terminus of CIKS that is essential for IL-17-mediated NF-kB activation. This domain is both necessary and sufficient for the interaction between CIKS and TRAF6, an adaptor required for NF-kB activation. The ability of decoy peptides to block this interaction may provide a new therapeutic strategy for intervention in IL-17-driven autoimmune and inflammatory diseases. Keywords: strain comparisons strain comparisons

2011-10-18 | E-GEOD-24873 | biostudies-arrayexpress

Gas-phase Fragmentation of ADP-Ribosylated Peptides: Arginine-Specific Side-Chain Losses and their Implication in Database Searches

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2021-05-19 | PXD017013 | Pride

Analysis of CNK1-SAM domain phosphorylation sites

Project description:Scaffold proteins such as CNK1 are hubs for coordinating signalling pathways. Here, we demonstrate that CNK1 is a crucial transducer of growth factor-stimulated and oncogenic signalling. Activation of CNK1 depends on its dimerization executed by the N-terminal sterile motif alpha (SAM) domain. Accordingly, a CNK1 mutant lacking the SAM domain prevents CNK1-driven cell proliferation and matrix metalloproteinase 14 promoter activation. We identified phosphorylation of Ser22 by AKT as trigger for CNK1 dimerisation. Consistently, the mutant CNK1S22D mimicking constitutive phosphorylation stimulates CNK1 signalling, whereas the phosphoylation-dead mutant CNK1S22A does not. Searching the COSMIC database revealed Ser22 as target for oncogenic activation of CNK1. The mutant CNK1S22D and the oncogenic mutant CNK1S22F form clusters in serum-starved cells comparable to clusters of CNK1 in growth factor stimulated cells. Light-activatable CNK1, optoCNK1, based on the light-induced oligomerisation of cryptochrome 2 confirms that dimerization is the trigger for CNK1 activation. CNK1 dimers induced by activating Ser22 mutants or by light enhance cell invasion and ADP ribosylation factors 1 signalling associated with tumour formation. Positive and negative feedback mechanisms regulates CNK1 dimerisation and in this way its activity. Oncogenic mutants of CNK1 support the positive feedback while escape from negative feedback regulation.

2022-02-23 | PXD003797 | Pride

Analysis of CNK1-SAM domain phosphorylation sites

2022-02-23 | PXD003831 | Pride

Proteomic analyses identify ARH3 as a serine mono-ADP-ribosylhydrolase

Project description:ADP-ribosylation is a posttranslational modification that exists in monomeric and polymeric forms. Whereas the writers (e.g. ARTD1/PARP1) and erasers (e.g. PARG, ARH3) of poly-ADP-ribosylation (PARylation) are relatively well described, the enzymes involved in mono-ADP-ribosylation (MARylation) have been less well investigated. While erasers for the MARylation of glutamate/aspartate and arginine have been identified, the respective enzymes with specificity for serine are still missing. Here, we report that in vitro, ARH3 specifically binds and demodifies proteins and peptides that are MARylated. Molecular modeling and site-directed mutagenesis of ARH3 revealed that numerous residues are critical for both the mono- and the poly-ADP-ribosylhydrolase activity of ARH3. Notably, a mass spectrometry approach showed that ARH3-deficient MEFs are characterized by a specific increase in serine-ADP-ribosylation in vivo under untreated conditions as well as following hydrogen-peroxide stress. Together, our results establish ARH3 as a serine mono-ADP-ribosylhydrolase and as an important regulator of the basal and stress induced ADP-ribosylome.

2017-12-14 | PXD008083 | Pride

Engineering Af1521 macro domain increases ADP-ribose binding affinity and leads to the identification of more ADP-ribosylated proteins

Project description:Protein ADP-ribosylation is a covalent, reversible post-translational modification that has important functions in several cellular mechanisms. The identification of modified proteins in cells has proven challenging and, due to the low abundance of protein ADP-ribosylation, has mainly been possible via enrichment methodologies using ADP-ribose binding domains. Here, using random mutagenesis and in vitro selection with an ADP-ribosylated histone peptide, we engineered the archaeal Af1521 macro domain to generate an engineered isoform containing nine mutations that displays significantly increased affinity towards ADP-ribose. Comparison of the wild-type and the engineered Af1521 macro domains using our proteomic ADP-ribosylome enrichment workflow demonstrated an increased identification rate of ADP-ribosylated proteins with the engineered Af1521.

2020-10-20 | PXD016686 | Pride

Proteomic characterization of the heart and skeletal muscle reveals widespread Arginine ADP-ribosylation by the ectopic ADP-ribosyltransferase ARTC1

Project description:ADP-ribosylation has thus far been studied on the proteomic level mainly in cell lines and in combination with genotoxic stress. The clostridium-like ADP-ribosyltransferases (i.e. ARTC) are GPI-anchored or secreted proteins that are expressed in a highly tissue specific manner. Transcriptomic data revealed that ARTC1 is expressed in skeletal muscle and heart tissue. Although ARTC1 is highly active in vitro, identification of ARTC1 targets in vivo and subsequent characterization of ARTC1-regulated cellular processes on the proteome level has been challenging and only a few ADP-ribosylated targets are known. Using a new MS-based workflow, we provide the thus far most extensive identification of endogenous ADP-ribosylomes with hundreds of ARTC1-ADP-ribosylated proteins in C2C12 myotubes and in skeletal muscle and heart tissues from wild type and newly generated ARTC1 knockout mice. These proteins are ADP-ribosylated on arginine residues and mainly located on the cell surface or in the extracellular space. They are associated with signal transduction, transmembrane transport and muscle function. Together we provide the first comprehensive systems-level analysis of endogenous ADP-ribosylation in two different muscle tissues revealing a widespread function of ARTC1 and its targets.

2018-08-17 | PXD008041 | Pride

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

Project description: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.

2020-06-17 | PXD016469 | Pride

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