Numerous drugs and endogenous ligands bind to cell surface receptors leading to modulation of downstream signaling cascades and frequently to adaptation of the plasma membrane proteome. In-depth ...analysis of dynamic processes at the cell surface is challenging due to biochemical properties and low abundances of plasma membrane proteins. Here we introduce cell surface thermal proteome profiling for the comprehensive characterization of ligand-induced changes in protein abundances and thermal stabilities at the plasma membrane. We demonstrate drug binding to extracellular receptors and transporters, discover stimulation-dependent remodeling of T cell receptor complexes and describe a competition-based approach to measure target engagement of G-protein-coupled receptor antagonists. Remodeling of the plasma membrane proteome in response to treatment with the TGFB receptor inhibitor SB431542 leads to partial internalization of the monocarboxylate transporters MCT1/3 explaining the antimetastatic effects of the drug.
Tet proteins oxidize 5-methylcytosine (mC) to generate 5-hydroxymethyl (hmC), 5-formyl (fC), and 5-carboxylcytosine (caC). The exact function of these oxidative cytosine bases remains elusive. We ...applied quantitative mass-spectrometry-based proteomics to identify readers for mC and hmC in mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC), and adult mouse brain tissue. Readers for these modifications are only partially overlapping, and some readers, such as Rfx proteins, display strong specificity. Interactions are dynamic during differentiation, as for example evidenced by the mESC-specific binding of Klf4 to mC and the NPC-specific binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation.
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► Identification of readers for 5-(hydroxy)methylcytosine in (differentiated) mESCs ► mC and hmC readers are highly dynamic during development and show limited overlap ► Oxidized derivatives of mC recruit DNA repair proteins in mESCs ► Uhrf2 stimulates the activity of the Tet1 enzyme
Spruijt et al. identified protein binding partners of 5-methylcytosine and its oxidative derivatives. These dynamic and specific readers suggest a role for these bases in active DNA demethylation and a DNA demethylation-independent function.
Posttranslational modifications on core histones can serve as binding scaffolds for chromatin-associated proteins. Proteins that specifically bind to or “read” these modifications were previously ...identified in mass spectrometry-based proteomics screens based on stable isotope-labeling in cell lines. Here we describe a sensitive, label-free histone peptide pull-down technology with extracts of different mouse tissues. Applying this workflow to the classical activating and repressive epigenetic marks on histone H3, H3K4me3, and H3K9me3, we identified known and putative readers in extracts from brain, liver, kidney, and testis. A large class of proteins were specifically repelled by H3K4me3. Our screen reached near-saturation of direct interactors, most of which are ubiquitously expressed. In addition, it revealed a number of specialized readers in tissues such as testis. Apart from defining the chromatin interaction landscape in mouse tissues, our workflow can be used for peptides with different modifications and cell types of any organism.
► Largest catalog of chromatin readers of histone trimethylation marks ► Accurate label-free proteomics interaction technology for tissue material ► CHD5 is a brain-specific NuRD complex subunit binding histone tails directly ► Tissue specificity is generally conveyed by auxiliary chromatin complex members
Mass spectrometry-based secretomics approaches frequently utilize serum-free culture conditions to circumvent serum-induced interference and to increase analytical depth. However, this can negatively ...affect a wide range of cellular functions and cell viability. These effects become particularly apparent when investigating transcriptionally regulated secretion events and feedback-loops in response to perturbations that require 48 h or more to fully manifest. We present an “interval-based” secretomics workflow, which determines protein secretion rates in short serum-free time windows. Relative quantification using tandem mass tags enables precise monitoring of time-dependent changes. We applied this approach to determine temporal profiles of protein secretion in the hepatocyte model cell lines HepG2 and HepaRG after stimulation of the acute-phase response (APR) by the cytokines IL1b and IL6. While the popular hepatocarcinoma cell line HepG2 showed an incomplete APR, secretion patterns derived from differentiated HepaRG cells recapitulated the expected APR more comprehensively. For several APR response proteins, substantial secretion was only observed after 72 h, a time window at which cell fitness is substantially impaired under serum-free cell culture conditions. The interval-based secretomics approach enabled the first comprehensive analysis of time-dependent secretion of liver cell models in response to these proinflammatory cytokines. The extended time range facilitated the observation of distinct chronological phases and cytokine-dependent secretion phenotypes of the APR. IL1b directed the APR toward pathogen defense over three distinct phases—chemotaxis, effector, clearance—while IL6 directed the APR toward regeneration. Protein shedding on the cell surface was pronounced upon IL1b stimulation, and small molecule inhibition of ADAM and matrix metalloproteases identified induced as well as constitutive shedding events. Inhibition of ADAM proteases with TAPI-0 resulted in reduced shedding of the sorting receptor SORT1, and an attenuated cytokine response suggesting a direct link between cell surface shedding and cytokine secretion rates.
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•Interval-based secretomics enables extended time course analysis.•Time-resolved acute phase response in liver model systems HepG2 and HepaRG.•IL1b response clusters in three phases.•Cell surface shedding is amplified during acute-phase response.•ADAM inhibition dampens secretion of inflammatory cytokines.
Using short serum-free incubation intervals allows extended time course analysis downstream of perturbations and addresses limitations of classical serum-free secretomics workflows. The time-dependent secretion of liver cell models HepG2 and HepaRG upon stimulation of the acute-phase response with the proinflammatory cytokines IL1b and IL6 was studied and revealed time- and stimulus-dependent phases of protein secretion. Monitoring proteolysis at the cell surface linked ADAM inhibition to inflammatory cytokine secretion.
Monitoring drug-target interactions with methods such as the cellular thermal-shift assay (CETSA) is well established for simple cell systems but remains challenging in vivo. Here we introduce tissue ...thermal proteome profiling (tissue-TPP), which measures binding of small-molecule drugs to proteins in tissue samples from drug-treated animals by detecting changes in protein thermal stability using quantitative mass spectrometry. We report organ-specific, proteome-wide thermal stability maps and derive target profiles of the non-covalent histone deacetylase inhibitor panobinostat in rat liver, lung, kidney and spleen and of the B-Raf inhibitor vemurafenib in mouse testis. In addition, we devised blood-CETSA and blood-TPP and applied it to measure target and off-target engagement of panobinostat and the BET family inhibitor JQ1 directly in whole blood. Blood-TPP analysis of panobinostat confirmed its binding to known targets and also revealed thermal stabilization of the zinc-finger transcription factor ZNF512. These methods will help to elucidate the mechanisms of drug action in vivo.
Trimethyl-lysine (me3) modifications on histones are the most stable epigenetic marks and they control chromatin-mediated regulation of gene expression. Here, we determine proteins that bind these ...marks by high-accuracy, quantitative mass spectrometry. These chromatin “readers” are assigned to complexes by interaction proteomics of full-length BAC-GFP-tagged proteins. ChIP-Seq profiling identifies their genomic binding sites, revealing functional properties. Among the main findings, the human SAGA complex binds to H3K4me3 via a double Tudor-domain in the C terminus of Sgf29, and the PWWP domain is identified as a putative H3K36me3 binding motif. The ORC complex, including LRWD1, binds to the three most prominent transcriptional repressive lysine methylation sites. Our data reveal a highly adapted interplay between chromatin marks and their associated protein complexes. Reading specific trimethyl-lysine sites by specialized complexes appears to be a widespread mechanism to mediate gene expression.
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► MS-based proteomics screen identifies novel histone lysine trimethylation readers ► Readers are assembled into complexes and their genome-wide occupancy is determined ► Sgf29 links the SAGA complex to H3K4me3 via a double tudor domain ► Trimethyl reader complexes determine the biological function of their epigenetic mark
The plasma membrane proteome plays a crucial role in inter- and intracellular signaling, cell survival, and cell identity. As such, it is a prominent target for pharmacological intervention. The ...relatively low abundance of this subproteome in conjunction with challenging extractability and solubility still hampers its comprehensive analysis. Here, we combined a chemical glycoprotein-tagging strategy with mass spectrometry to enable comprehensive analysis of the cell-surface glycoproteome. To benchmark this workflow and to provide guidance for cell line selection for functional experiments, we generated an inventory of the N-linked cell-surface glycoproteomes of 15 standard laboratory human cell lines and three primary lymphocytic cell types. On average, about 900 plasma membrane and secreted proteins were identified per experiment, including more than 300 transporters and ion channels. Primary cells displayed distinct expression of surface markers and transporters underpinning the importance of carefully validating model cell lines selected for the study of cell surface-mediated processes. To monitor dynamic changes of the cell-surface proteome in a highly multiplexed experiment, we employed an isobaric mass tag-based chemical labeling strategy. This enabled the time-resolved analysis of plasma membrane protein presentation during differentiation of the monocytic suspension cell line THP-1 into macrophage-like adherent cells. Time-dependent changes observed in membrane protein presentation reflect functional remodeling during the phenotypic transition in three distinct phases: rapid surface presentation and secretion of proteins from intracellular pools concurrent with rapid internalization of no longer needed proteins and finally delayed presentation of newly synthesized macrophage markers. Perturbation of this process using marketed receptor tyrosine kinase inhibitors revealed dasatinib to severely compromise macrophage differentiation due to an off-target activity. This finding suggests that dynamic processes can be highly vulnerable to drug treatment and should be monitored more rigorously to identify adverse drug effects.
Stimulator of interferon genes (STING) is a receptor in the endoplasmic reticulum that propagates innate immune sensing of cytosolic pathogen-derived and self DNA
. The development of compounds that ...modulate STING has recently been the focus of intense research for the treatment of cancer and infectious diseases and as vaccine adjuvants
. To our knowledge, current efforts are focused on the development of modified cyclic dinucleotides that mimic the endogenous STING ligand cGAMP; these have progressed into clinical trials in patients with solid accessible tumours amenable to intratumoral delivery
. Here we report the discovery of a small molecule STING agonist that is not a cyclic dinucleotide and is systemically efficacious for treating tumours in mice. We developed a linking strategy to synergize the effect of two symmetry-related amidobenzimidazole (ABZI)-based compounds to create linked ABZIs (diABZIs) with enhanced binding to STING and cellular function. Intravenous administration of a diABZI STING agonist to immunocompetent mice with established syngeneic colon tumours elicited strong anti-tumour activity, with complete and lasting regression of tumours. Our findings represent a milestone in the rapidly growing field of immune-modifying cancer therapies.
The active release of proteins into the extracellular space and the proteolytic cleavage of cell surface proteins are key processes that coordinate and fine-tune a multitude of physiological ...functions. The entirety of proteins that fulfill these extracellular tasks are referred to as the secretome and are of special interest for the investigation of biomarkers of disease states and physiological processes related to cell-cell communication. LC-MS–based proteomics approaches are a valuable tool for the comprehensive and unbiased characterization of this important subproteome. This review discusses procedures, opportunities, and limitations of mass spectrometry–based secretomics to better understand and navigate the complex analytical landscape for studying protein secretion in biomedical science.
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•Overview of challenges, approaches, and opportunities of LC-MS–based secretomics.•Guidance for unbiased analysis of secreted proteins from cultured cells.•Strength and weaknesses of different secretomics approaches.
The secretome encompasses a biologically highly relevant and understudied class of proteins that are either actively released by the cell or reach the extracellular milieu via tissue leakage. Due to its comprehensive and unbiased nature, LC-MS–based proteomics is an indispensable tool for secretome characterization but requires specific and tailored workflows. This review illustrates the analytical challenges associated with LC-MS–based secretomics, provides a broad survey of established and emerging concepts of secretome analysis, and gives examples of biomedical applications.
Modification of proteins with polyubiquitin chains is a key regulatory mechanism to control cellular behavior and alterations in the ubiquitin system are linked to many diseases. Linear (M1-linked) ...polyubiquitin chains play pivotal roles in several cellular signaling pathways mediating immune and inflammatory responses and apoptotic cell death. These chains are formed by the linear ubiquitin chain assembly complex (LUBAC), a multiprotein E3 ligase that consists of 3 subunits, HOIP, HOIL-1L, and SHARPIN. Herein, we describe the discovery of inhibitors targeting the active site cysteine of the catalytic subunit HOIP using fragment-based covalent ligand screening. We report the synthesis of a diverse library of electrophilic fragments and demonstrate an integrated use of protein LC–MS, biochemical ubiquitination assays, chemical synthesis, and protein crystallography to enable the first structure-based development of covalent inhibitors for an RBR E3 ligase. Furthermore, using cell-based assays and chemoproteomics, we demonstrate that these compounds effectively penetrate mammalian cells to label and inhibit HOIP and NF-κB activation, making them suitable hits for the development of selective probes to study LUBAC biology. Our results illustrate the power of fragment-based covalent ligand screening to discover lead compounds for challenging targets, which holds promise to be a general approach for the development of cell-permeable inhibitors of thioester-forming E3 ubiquitin ligases.
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