ABSTRACT
Poly(ADP-ribos)ylation (PARylation) is a reversible posttranslational modification found in higher eukaryotes. However, little is known about PARylation acceptor proteins. Here, we describe a sensitive proteomics approach based on high-accuracy quantitative mass spectrometry for the identification of PARylated proteins induced under different cellular stress conditions. While confirming the majority of known PARylated substrates, our screen identifies numerous additional PARylation targets. In vivo and in vitro validation of acceptor proteins confirms that our methodology targets covalent PARylation. Nuclear proteins encompassing nucleic acid binding properties are prominently PARylated upon genotoxic stress, consistent with the nuclear localization of ARTD1/PARP1 and ARTD2/PARP2. Distinct differences in proteins becoming PARylated upon various genotoxic insults are observed, exemplified by the PARylation of RNA-processing factors THRAP3 and TAF15 under oxidative stress. High-content imaging reveals that PARylation affects the nuclear relocalization of THRAP3 and TAF15, demonstrating the potential of our approach to uncover hitherto unappreciated processes being controlled by specific genotoxic-stress-induced PARylation.
Subject(s)
DNA Damage , Poly Adenosine Diphosphate Ribose/metabolism , Proteome/metabolism , Proteomics/methods , Antineoplastic Agents, Alkylating/pharmacology , Cell Line, Tumor , Cell Nucleus/genetics , Cell Nucleus/metabolism , Chromatography, Liquid , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Electrophoresis, Polyacrylamide Gel , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Hydrogen Peroxide/pharmacology , Methyl Methanesulfonate/pharmacology , Microscopy, Confocal , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oxidants/pharmacology , Protein Processing, Post-Translational/drug effects , Protein Processing, Post-Translational/radiation effects , Proteome/genetics , RNA Interference , Radiation, Ionizing , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/radiation effects , TATA-Binding Protein Associated Factors/genetics , TATA-Binding Protein Associated Factors/metabolism , Tandem Mass Spectrometry , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The covalent attachment of methyl groups to the side-chain of arginine residues is known to play essential roles in regulation of transcription, protein function, and RNA metabolism. The specific N-methylation of arginine residues is catalyzed by a small family of gene products known as protein arginine methyltransferases; however, very little is known about which arginine residues become methylated on target substrates. Here we describe a proteomics methodology that combines single-step immunoenrichment of methylated peptides with high-resolution mass spectrometry to identify endogenous arginine mono-methylation (MMA) sites. We thereby identify 1027 site-specific MMA sites on 494 human proteins, discovering numerous novel mono-methylation targets and confirming the majority of currently known MMA substrates. Nuclear RNA-binding proteins involved in RNA processing, RNA localization, transcription, and chromatin remodeling are predominantly found modified with MMA. Despite this, MMA sites prominently are located outside RNA-binding domains as compared with the proteome-wide distribution of arginine residues. Quantification of arginine methylation in cells treated with Actinomycin D uncovers strong site-specific regulation of MMA sites during transcriptional arrest. Interestingly, several MMA sites are down-regulated after a few hours of transcriptional arrest. In contrast, the corresponding di-methylation or protein expression levels are not altered, confirming that MMA sites contain regulated functions on their own. Collectively, we present a site-specific MMA data set in human cells and demonstrate for the first time that MMA is a dynamic post-translational modification regulated during transcriptional arrest by a hitherto uncharacterized arginine demethylase.
Subject(s)
Arginine/metabolism , Peptides/isolation & purification , Proteomics/methods , Transcription, Genetic , Binding Sites/drug effects , Cell Line, Tumor , Dactinomycin/pharmacology , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , Methylation , Peptides/chemistry , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Transcription, Genetic/drug effectsABSTRACT
Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a 'head-to-tail' dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damage.
Subject(s)
Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Trans-Activators/chemistry , Trans-Activators/metabolism , Tumor Suppressor Proteins/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Ataxia Telangiectasia Mutated Proteins , Cells, Cultured , Chromosomal Proteins, Non-Histone/analysis , DNA Breaks, Double-Stranded , DNA-Binding Proteins/analysis , Dimerization , Humans , Mice , Models, Molecular , Molecular Sequence Data , Phosphothreonine/metabolism , Protein Interaction Domains and Motifs , Threonine/metabolism , Tumor Suppressor p53-Binding Protein 1ABSTRACT
The MRE11-RAD50-NBS1 (MRN) complex accumulates at sites of DNA double-strand breaks in large chromatin domains flanking the lesion site. The mechanism of MRN accumulation involves direct binding of the Nijmegen breakage syndrome 1 (NBS1) subunit to phosphorylated mediator of the DNA damage checkpoint 1 (MDC1), a large nuclear adaptor protein that interacts directly with phosphorylated H2AX. NBS1 contains an FHA domain and two BRCT domains at its amino terminus. Here, we show that both of these domains participate in the interaction with phosphorylated MDC1. Point mutations in key amino acid residues of either the FHA or the BRCT domains compromise the interaction with MDC1 and lead to defects in MRN accumulation at sites of DNA damage. Surprisingly, only mutation in the FHA domain, but not in the BRCT domains, yields a G2/M checkpoint defect, indicating that MDC1-dependent chromatin accumulation of the MRN complex at sites of DNA breaks is not required for G2/M checkpoint activation.
Subject(s)
Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Chromatin/metabolism , DNA Damage , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Acid Anhydride Hydrolases , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Cell Line , DNA Breaks, Double-Stranded , G2 Phase , Humans , MRE11 Homologue Protein , Mitosis , Molecular Sequence Data , Mutation/genetics , Protein Binding , Protein Structure, Tertiary , Structure-Activity Relationship , Trans-Activators/chemistry , Trans-Activators/metabolismABSTRACT
The chromatin structure is important for recognition and repair of DNA damage. Many DNA damage response proteins accumulate in large chromatin domains flanking sites of DNA double-strand breaks. The assembly of these structures-usually termed DNA damage foci-is primarily regulated by MDC1, a large nuclear mediator/adaptor protein that is composed of several distinct structural and functional domains. Here, we are summarizing the latest discoveries about the mechanisms by which MDC1 mediates DNA damage foci formation, and we are reviewing the considerable efforts taken to understand the functional implication of these structures.
Subject(s)
Chromatin/metabolism , DNA Damage , DNA Repair , Intracellular Signaling Peptides and Proteins/physiology , Nuclear Proteins/physiology , Trans-Activators/physiology , Adaptor Proteins, Signal Transducing , Animals , Cell Cycle Proteins , DNA Breaks, Double-Stranded , DNA Damage/genetics , DNA Repair/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Histones/chemistry , Histones/metabolism , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Mice , Mitosis , Nuclear Proteins/chemistry , Protein Interaction Domains and Motifs , Signal Transduction , Trans-Activators/chemistryABSTRACT
Induction of DNA double-strand breaks (DSBs) in ribosomal DNA (rDNA) repeats is associated with ATM-dependent repression of ribosomal RNA synthesis and large-scale reorganization of nucleolar architecture, but the signaling events that regulate these responses are largely elusive. Here we show that the nucleolar response to rDNA breaks is dependent on both ATM and ATR activity. We further demonstrate that ATM- and NBS1-dependent recruitment of TOPBP1 in the nucleoli is required for inhibition of ribosomal RNA synthesis and nucleolar segregation in response to rDNA breaks. Mechanistically, TOPBP1 recruitment is mediated by phosphorylation-dependent interactions between three of its BRCT domains and conserved phosphorylated Ser/Thr residues at the C-terminus of the nucleolar phosphoprotein Treacle. Our data thus reveal an important cooperation between TOPBP1 and Treacle in the signaling cascade that triggers transcriptional inhibition and nucleolar segregation in response to rDNA breaks.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/metabolism , Carrier Proteins/metabolism , Cell Nucleolus/genetics , DNA, Ribosomal/genetics , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Amino Acid Motifs , Ataxia Telangiectasia Mutated Proteins/genetics , Carrier Proteins/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Nucleolus/metabolism , DNA Breaks, Double-Stranded , DNA, Ribosomal/metabolism , DNA-Binding Proteins/genetics , Humans , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Phosphoproteins/chemistry , Phosphoproteins/genetics , Protein Binding , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolismABSTRACT
ADP-ribosylation is a posttranslational modification (PTM) that affects a variety of cellular processes. In recent years, mass spectrometry (MS)-based proteomics has become a valuable tool for studying ADP-ribosylation. However, studying this PTM in vivo in an unbiased and sensitive manner has remained a difficult challenge. Here, we describe a detailed protocol for unbiased analysis of ADP-ribosylated proteins and their ADP-ribose acceptor sites under physiological conditions. The method relies on the enrichment of mono-ADP-ribosylated peptides using the macrodomain Af1521 in combination with liquid chromatography-high-resolution tandem MS (LC-MS/MS). The 5-day protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture stage all the way through to data processing using the MaxQuant software suite.
Subject(s)
Adenosine Diphosphate Ribose/metabolism , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Animals , Humans , Protein Processing, Post-Translational/genetics , Protein Processing, Post-Translational/physiology , Proteome/metabolism , Proteomics/methods , SoftwareABSTRACT
Although protein ADP-ribosylation is involved in diverse biological processes, it has remained a challenge to identify ADP-ribose acceptor sites. Here, we present an experimental workflow for sensitive and unbiased analysis of endogenous ADP-ribosylation sites, capable of detecting more than 900 modification sites in mammalian cells and mouse liver. In cells, we demonstrate that Lys residues, besides Glu, Asp and Arg residues, are the dominant in vivo targets of ADP-ribosylation during oxidative stress. In normal liver tissue, we find Arg residues to be the predominant modification site. The cellular distribution and biological processes that involve ADP-ribosylated proteins are different in cultured cells and liver tissue, in the latter of which the majority of sites were found to be in cytosolic and mitochondrial protein networks primarily associated with metabolism. Collectively, we describe a robust methodology for the assessment of the role of ADP-ribosylation and ADP-ribosyltransferases in physiological and pathological states.
ABSTRACT
Intrinsically disordered proteins can phase separate from the soluble intracellular space, and tend to aggregate under pathological conditions. The physiological functions and molecular triggers of liquid demixing by phase separation are not well understood. Here we show in vitro and in vivo that the nucleic acid-mimicking biopolymer poly(ADP-ribose) (PAR) nucleates intracellular liquid demixing. PAR levels are markedly induced at sites of DNA damage, and we provide evidence that PAR-seeded liquid demixing results in rapid, yet transient and fully reversible assembly of various intrinsically disordered proteins at DNA break sites. Demixing, which relies on electrostatic interactions between positively charged RGG repeats and negatively charged PAR, is amplified by aggregation-prone prion-like domains, and orchestrates the earliest cellular responses to DNA breakage. We propose that PAR-seeded liquid demixing is a general mechanism to dynamically reorganize the soluble nuclear space with implications for pathological protein aggregation caused by derailed phase separation.
Subject(s)
Poly Adenosine Diphosphate Ribose/chemistry , Proteins/chemistry , Proteins/metabolism , Cell Line, Tumor , Cloning, Molecular , DNA Damage , Gene Expression Regulation/physiology , Humans , Protein Conformation , Protein Structure, Tertiary , Proteins/geneticsABSTRACT
BACKGROUND: Suicide gene therapy employing the prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV-TK)/ ganciclovir (GCV) has proven to be effective in killing experimental brain tumors. In contrast, glioma patients treated with HSV-TK/ GCV did not show significant treatment benefit, most likely due to insufficient transgene delivery to tumor cells. Therefore, this study aimed at developing a strategy for real-time noninvasive in vivo monitoring of the activity of a therapeutic gene in brain tumor cells. METHODS: The HSV-TK gene was fused to the firefly luciferase (Luc) gene and the fusion construct HSV-TK-Luc was expressed in U87MG human malignant glioma cells. Nude mice with subcutaneous gliomas stably expressing HSV-TK-Luc were subjected to GCV treatment and tumor response to therapy was monitored in vivo by serial bioluminescence imaging. Bioluminescent signals over time were compared with tumor volumes determined by caliper. RESULTS: Transient and stable expression of the HSV-TK-Luc fusion protein in U87MG glioma cells demonstrated close correlation of both enzyme activities. Serial optical imaging of tumor bearing mice detected in all cases GCV induced death of tumor cells expressing the fusion protein and proved that bioluminescence can be reliably used for repetitive and noninvasive quantification of HSV-TK/ GCV mediated cell kill in vivo. CONCLUSION: This approach may represent a valuable tool for the in vivo evaluation of gene therapy strategies for treatment of malignant disease.
ABSTRACT
Phosphoinositides (PIPs) play key roles in signaling and disease. Using high-resolution quantitative mass spectrometry, we identified PIP-interacting proteins and profiled their binding specificities toward all seven PIP variants. This analysis revealed 405 PIP-binding proteins, which is greater than the total number of phospho- or ubiquitin-binding domains. Translocation and inhibitor assays of identified PIP-binding proteins confirmed that our methodology targets direct interactors. The PIP interactome encompasses proteins from diverse cellular compartments, prominently including the nucleus. Our data set revealed a consensus motif for PI(3,4,5)P3-interacting pleckstrin homology (PH) domains, which enabled in silico identification of phosphoinositide interactors. Members of the dedicator of cytokinesis family C exhibited specificity toward both PI(3,4,5)P3 and PI(4,5)P2. Structurally, this dual specificity is explained by a decreased number of positively charged residues in the L1 subdomain compared with DOCK1. The presented PIP-binding proteome and its specificity toward individual PIPs should be a valuable resource for the community.
Subject(s)
Mass Spectrometry/methods , Phosphatidylinositols/metabolism , Proteome/metabolism , Amino Acid Motifs , Amino Acid Sequence , Computer Simulation , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Molecular Sequence Data , Protein Binding , Protein Interaction Mapping , Protein Structure, Tertiary , Proteome/chemistry , TransfectionABSTRACT
Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in trans signalling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identify TCOF1 (also known as Treacle), a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in trans in the presence of distant chromosome breaks.