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Phosphoproteome profiling uncovers a key role for CDKs in TNF signaling

ABSTRACT: Tumor necrosis factor (TNF) is one of the few cytokines successfully targeted by therapies against inflammatory diseases. However, blocking this well studied and pleiotropic ligand can cause dramatic side-effects. We reasoned that a systems-level proteomic analysis of TNF signaling could dissect its diverse functions and offer a base for developing more targeted therapies. Combining phosphoproteomics time course experiments with spatial and kinase inhibitor analysis clusters phosphorylations into functional modules. The majority of regulated phosphorylations could be assigned to an upstream kinase by inhibiting master kinases and spatial proteomics revealed phosphorylation-dependent translocations of hundreds of proteins upon TNF stimulation. Phosphoproteome analysis of TNF-induced apoptosis and necroptosis revealed a key role for transcriptional cyclin-dependent kinase (CDK) activity to promote cytokine production and prevent excessive cell death downstream of the TNF signaling receptor. Our comprehensive interrogation of TNF induced pathways and sites can be explored at http://tnfviewer.biochem.mpg.de/.

INSTRUMENT(S): Q Exactive

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

TISSUE(S): Myeloid Cell Line

SUBMITTER: Mario Oroshi

LAB HEAD: Matthias Mann

PROVIDER: PXD021366 | Pride | 2021-10-11

REPOSITORIES: Pride

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MDP phosphorylation by NAGK is essential for NOD2 activation

Project description:Bacterial cell wall components provide various unique molecular structures that are detected by pattern recognition receptors (PRRs) of the innate immune system as non-self. Most bacterial species form a cell wall that consists of peptidoglycan, a polymeric structure comprising of alternating amino sugars. Muramyl dipeptide (MDP) is the minimal, biologically active molecular structure derived from peptidoglycan, and its immunogenicity is well documented in numerous studies. MDP is sensed by the cytosolic nucleotide-binding and nucleotide oligomerization domain-like receptor 2 (NOD2), which triggers a pro-inflammatory response upon engagement 1,2. Conducting a forward genetic screen to identify factors required for MDP detection, we discovered that N-acetylglucosamine kinase (NAGK) is essential for the immunostimulatory activity of MDP. NAGK, which has previously been identified to contribute to the hexosamine salvage pathway in humans, is broadly expressed and inducible in innate immune cells. Mechanistically, NAGK functions upstream of NOD2 in that it directly phosphorylates the N-acetylmuramic moiety of MDP at the hydroxyl group of its C6 position, yielding 6-O-phospho-MDP. NAGK-phosphorylated MDP constitutes an essential component of NOD2 dependent signal transduction. Altogether, these results unveil a previously unknown interconnection of amino sugar metabolism and innate immunity to bacterial cell walls.

2022-06-13 | PXD022384 | Pride

Multi-omics approaches reveal TNF-mediated inhibition of tissue reparative macrophages is via a gene-selective transcriptional mechanism

Project description:Anti-TNF therapies are a core anti-inflammatory approach for chronic diseases such as rheumatoid arthritis and Crohn’s Disease. Previously, we and others found that TNF blocks the emergence and function of alternatively-activated or M2 macrophages involved in wound healing and tissue-reparative functions. Conceivably, anti-TNF drugs could mediate their protective effects in part by an altered balanced of macrophage activity. To understand the mechanistic basis of how TNF regulates tissue-reparative macrophages we used RNAseq, scRNAseq, ATACseq, time-resolved phospho-proteomics, gene-specific approaches, metabolic analysis and signaling pathway deconvolution. Our findings reveal that TNF controls tissue-reparative macrophage gene expression in a highly gene-specific way dependent on JNK signaling. We uncover principles of the selectively inhibition by TNF via the type 1 TNF receptor on specific populations of alternative activated macrophages.

2022-01-07 | PXD028667 | Pride

Phosphoproteomics reveals the GSK3-PDX1 axis as a key pathogenic signaling node in diabetic islets

Project description:Progressive decline of pancreatic beta cell function is central to the pathogenesis of type 2 diabetes. Protein phosphorylation regulates glucose-stimulated insulin secretion from beta cells, but how signaling networks are remodeled in diabetic islets in vivo remains unknown. Using high-sensitivity mass spectrometry-based proteomics we quantified 6,500 proteins and 13,000 phosphopeptides in islets of obese diabetic mice and matched controls, revealing drastic remodeling of key kinase hubs and signaling pathways. Integration with a literature-derived signaling network implicated GSK3 kinase in the control of the beta cell-specific transcription factor PDX1. Deep phosphoproteomic analysis of human islets chronically treated with high glucose demonstrated a conserved glucotoxicity-dependent role of GSK3 kinase in regulating insulin secretion. Remarkably, the ability of beta cells to secrete insulin in response to glucose was rescued almost completely by pharmacological inhibition of GSK3. Thus, our resource enables investigation of mechanisms and drug targets in type 2 diabetes.

2019-03-15 | PXD012671 | Pride

Spectral library based analysis of arginine phosphorylations in Staphylococcus aureus

Project description:Reversible protein phosphorylation is one of the major mechanisms in the regulation of protein expression and protein activity, controlling physiological functions of the important human pathogen Staphylococcus aureus. Phosphorylations at serine, threonine and tyrosine are known to influence for example protein activity in central metabolic pathways and the more energy-rich phosphorylations at histidine, aspartate or cysteine can be found as part of two component system sensor domains or mediating bacterial virulence. In addition to these well-known phosphorylations, the phosphorylation at arginine residues plays an essential role. Hence, the deletion mutant S. aureus COL ΔptpB (protein tyrosine phosphatase B) was studied since the protein PtpB is assumed to be an arginine phosphatase. A gel-free approach was applied to analyse the changes in the phosphoproteome of the deletion mutant ΔptpB and the wild type in growing cells, thereby focusing on the occurrence of phosphorylation on arginine residues. In order to enhance the reliability of identified phosphorylation sites at arginine residues, a subset of arginine phosphorylated peptides was chemically synthesised. Combined spectral libraries based on phosphoenriched samples, synthetic arginine phosphorylated peptides and classical proteome samples provide a sophisticated tool for the analysis of arginine phosphorylations. This way, 212 proteins phosphorylated on serine, threonine, tyrosine or arginine residues were identified within the mutant ∆ptpB and 102 in wild type samples. Among them, 207 arginine phosphosites were identified exclusively within the mutant ∆ptpB, widely distributed along the whole bacterial metabolism. This identification of putative targets of PtpB allows further investigation of the physiological relevance of arginine phosphorylations and provides the basis for reliable quantification of arginine phosphorylations in bacteria.

2017-12-06 | PXD007167 | Pride

Glucose-regulated and drug perturbed beta-cell phosphoproteome reveals molecular mechanisms controlling insulin secretion

Project description:Insulin-secreting beta cells play an essential role in maintaining physiological blood glucose levels, and their dysfunction leads to the development of diabetes. To elucidate the signaling events regulating insulin secretion, we applied a recently developed proteomics and phosphoproteomics workflow. We quantified the time-resolved phosphoproteome of beta cells following their exposure to glucose and in combination with small molecule compounds that promote insulin secretion. The quantitative phosphoproteome of 30,000 sites clustered into three main groups in concordance with the modulation of three key signaling pathways. In addition to their differential modulation of key cellular kinases, all compounds affected pathways regulating the cell cycle. A high-resolution time course revealed key novel regulatory sites in early and late insulin secretion, and unexpected connections to epigenetic regulation of gene expression. Our comprehensive and multi-parametric phosphoproteomics study reveals that control of insulin secretion is embedded in an unexpectedly broad and complex range of cellular functions.

2016-12-09 | PXD003850 | Pride

Quantitative Phospho-proteomic Analysis of TNFα/NFκB Signaling Reveals a Role for RIPK1 Phosphorylation in Suppressing Necrotic Cell Death

Project description:TNFα is a potent inducer of inflammation due to its ability to promote gene expression, inpart via the NFκB pathway. Moreover, in some contexts, TNFα promotes Caspase-dependent apoptosis or RIPK1/RIPK3/MLKL-dependent necrosis. Engagement of the TNF Receptor Signaling Complex (TNF-RSC), which contains multiple kinase activities, promotes phosphorylation of several downstream components, including TAK1, IKKα/IKKβ, IκBα and NFκB. However, immediate downstream phosphorylation events occurring in response to TNFα signaling are poorly understood at a proteome-wide level. Here we use Tandem mass tagging-based proteomics to quantitatively characterize acute TNFα-mediated alterations in the proteome and phosphoproteome with or without inhibition of the cIAP-dependent survival arm of the pathway with a SMAC mimetic. We identify and quantify over 8,000 phosphorylated peptides, among which are numerous known sites in the TNF-RSC, NFκB, and MAP kinase signaling systems, as well as numerous previously unrecognized phosphorylation events. Functional analysis of S320 phosphorylation in RIPK1 demonstrates a role for this event in suppressing its kinase activity, association with CASPASE-8 and FADD proteins, and subsequent necrotic cell death during inflammatory TNFα stimulation. This study provides a resource for further elucidation of TNFα-dependent signaling pathways.

2017-05-30 | PXD006055 | Pride

Phosphoproteomic Characterization of Signaling Networks Resulting from Activation of Chemokine Receptor CCR2

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2020-04-08 | PXD015845 | Pride

RNAseq analysis of the human neutrophil transcriptome, with and without in vitro cytokine stimulation

Project description:We report cytokine specific changes in gene expression in the human neutrophil transcriptome using TNF-alpha and GM-CSF stimulation of healthy neutrophils Healthy human neutrophils were stimulated with TNF-alpha or GM-CSF for 1h in vitro. RNA was analysed by SOLiD and Illumina sequencing. RNA from one biological donor was sequenced on both platforms, and two different biological donors were sequenced by Illumina.

2013-03-06 | E-GEOD-40548 | biostudies-arrayexpress

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Project description:Sustained weight loss is the preferred intervention in a wide range of metabolic conditions, but the effects on an individual´s health state remain ill-defined and controversial. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone eight weeks of weight loss (average of 12%) followed by a year of weight maintenance. Using plasma proteome profiling, a mass spectrometry-based approach, we measured 1294 plasma samples to examine dynamic changes of hundreds of human plasma proteins, including the entire apolipoprotein family. Longitudinal monitoring of the cohort revealed individual-specific protein levels and functional connected proteins with correlated regulation over time. The wide-ranging effect of losing weight on the plasma proteome was reflected in 93 significantly affected proteins. The adipocyte secreted SERPINF1 and the apolipoprotein APOF1 were most significantly down and up regulated (p<10**13), although their fold-change was modest (-16% and +37%), respectively. Overall, markers of inflammation went down in nearly all study participants (39 of 42), however, there were diverse patterns of inflammation marker responses, allowing individual-specific interpretation of the effects of weight loss. Plasma proteome profiling can be used broadly to evaluate and monitor intervention in metabolic diseases.

2016-12-24 | PXD004242 | Pride

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Project description:Obesity-related diseases now affect half of the global population, and bariatric surgery is one of the few interventions with long lasting weight loss and cardio-metabolic effects. Here, we investigated the effect of Roux-en-Y gastric bypass surgery on the plasma proteome, hypothesizing that specific proteins or protein patterns may serve as key mediators and markers of the metabolic response. We performed MS-based proteomics on two longitudinal studies encompassing 47 morbidly obese patients, generating quantitative information on more than 1700 proteins. A global correlation matrix incorporating about 200,000 relationships revealed functional connections between proteins and assigned them to physiological processes. The main classes of significantly altered proteins were markers of systemic inflammation and those involved in lipid metabolism, and our data highlight robust correlative and anti-correlative behavior to each other and to clinical parameters. A group of inflammation-related proteins showed distinct inverse relationships to proteins consistently associated with insulin sensitivity.

2018-12-07 | PXD009348 | Pride

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