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Modern Pathology : An Official Journal... Jul 2023PARP inhibitors (PARPi) are increasingly used in breast cancer therapy, including high-grade triple-negative breast cancer (TNBC) treatment. Varying treatment responses...
PARP inhibitors (PARPi) are increasingly used in breast cancer therapy, including high-grade triple-negative breast cancer (TNBC) treatment. Varying treatment responses and PARPi resistance with relapse currently pose limitations to the efficacy of PARPi therapy. The pathobiological reasons why individual patients respond differently to PARPi are poorly understood. In this study, we analyzed expression of PARP1, the main target of PARPi, in normal breast tissue, breast cancer, and its precursor lesions using human breast cancer tissue microarrays covering a total of 824 patients, including more than 100 TNBC cases. In parallel, we analyzed nuclear adenosine diphosphate (ADP)-ribosylation as a marker of PARP1 activity and TRIP12, an antagonist of PARPi-induced PARP1 trapping. Although we found PARP1 expression to be generally increased in invasive breast cancer, PARP1 protein levels and nuclear ADP-ribosylation were lower in higher tumor grade and TNBC samples than non-TNBCs. Cancers with low levels of PARP1 and low levels of nuclear ADP-ribosylation were associated with significantly reduced overall survival. This effect was even more pronounced in cases with high levels of TRIP12. These results indicate that PARP1-dependent DNA repair capacity may be compromised in aggressive breast cancers, potentially fueling enhanced accumulation of mutations. Moreover, the results revealed a subset of breast cancers with low PARP1, low nuclear ADP-ribosylation, and high TRIP12 levels, which may compromise their response to PARPi, suggesting a combination of markers for PARP1 abundance, enzymatic activity, and trapping capabilities might aid patient stratification for PARPi therapy.
Topics: Humans; Poly (ADP-Ribose) Polymerase-1; Triple Negative Breast Neoplasms; Neoplasm Recurrence, Local; ADP-Ribosylation; Mutation; Carrier Proteins; Ubiquitin-Protein Ligases
PubMed: 36990278
DOI: 10.1016/j.modpat.2023.100167 -
Biological Chemistry Mar 2019The functionality of eukaryotic translation elongation factor 2 (eEF2) is modulated by phosphorylation, eEF2 is simultaneously the molecular target of ADP-ribosylating...
The functionality of eukaryotic translation elongation factor 2 (eEF2) is modulated by phosphorylation, eEF2 is simultaneously the molecular target of ADP-ribosylating toxins. We analyzed the interplay between phosphorylation and diphthamide-dependent ADP-ribosylation. Phosphorylation does not require diphthamide, eEF2 without it still becomes phosphorylated. ADP-ribosylation not only modifies the H715 diphthamide but also inhibits phosphorylation of S595 located in proximity to H715, and stimulates phosphorylation of T56. S595 can be phosphorylated by CDK2 and CDK1 which affects EEF2K-mediated T56-phosphorylation. Thus, ADP-ribosylation and S595-phosphorylation by kinases occur within the same vicinity and both trigger T56-phosphorylation. Diphthamide is surface-accessible permitting access to ADP-ribosylating enzymes, the adjacent S595 side chain extends into the interior. This orientation is incompatible with phosphorylation, neither allowing kinase access nor phosphate attachment. S595 phosphorylation must therefore be accompanied by structural alterations affecting the interface to ADP-ribosylating toxins. In agreement with that, replacement of S595 with Ala, Glu or Asp prevents ADP-ribosylation. Phosphorylation (starvation) as well as ADP-ribosylation (toxins) inhibit protein synthesis, both affect the S595/H715 region of eEF2, both trigger T57-phosphorylation eliciting similar transcriptional responses. Phosphorylation is short lived while ADP-ribosylation is stable. Thus, phosphorylation of the S595/H715 'modifier region' triggers transient interruption of translation while ADP-ribosylation arrests irreversibly.
Topics: ADP-Ribosylation; Elongation Factor 2 Kinase; Humans; MCF-7 Cells; Models, Molecular; Phosphorylation
PubMed: 30218597
DOI: 10.1515/hsz-2018-0280 -
Journal of Molecular Cell Biology Jan 2024Mono-ADP-ribosylation (MARylation) is a post-translational modification that regulates a variety of biological processes, including DNA damage repair, cell...
Mono-ADP-ribosylation (MARylation) is a post-translational modification that regulates a variety of biological processes, including DNA damage repair, cell proliferation, metabolism, and stress and immune responses. In mammals, MARylation is mainly catalyzed by ADP-ribosyltransferases (ARTs), which consist of two groups: ART cholera toxin-like (ARTCs) and ART diphtheria toxin-like (ARTDs, also known as PARPs). The human ARTC (hARTC) family is composed of four members: two active mono-ADP-ARTs (hARTC1 and hARTC5) and two enzymatically inactive enzymes (hARTC3 and hARTC4). In this study, we systematically examined the homology, expression, and localization pattern of the hARTC family, with a particular focus on hARTC1. Our results showed that hARTC3 interacted with hARTC1 and promoted the enzymatic activity of hARTC1 by stabilizing hARTC1. We also identified vesicle-associated membrane protein-associated protein B (VAPB) as a new target of hARTC1 and pinpointed Arg50 of VAPB as the ADP-ribosylation site. Furthermore, we demonstrated that knockdown of hARTC1 impaired intracellular calcium homeostasis, highlighting the functional importance of hARTC1-mediated VAPB Arg50 ADP-ribosylation in regulating calcium homeostasis. In summary, our study identified a new target of hARTC1 in the endoplasmic reticulum and suggested that ARTC1 plays a role in regulating calcium signaling.
Topics: Animals; Humans; Calcium; ADP-Ribosylation; ADP Ribose Transferases; Protein Processing, Post-Translational; Homeostasis; Mammals; Vesicular Transport Proteins
PubMed: 37381178
DOI: 10.1093/jmcb/mjad043 -
Frontiers in Immunology 2021Murine T cells express the GPI-anchored ADP-ribosyltransferase 2.2 (ARTC2.2) on the cell surface. In response to T cell activation or extracellular NAD or ATP-mediated...
Murine T cells express the GPI-anchored ADP-ribosyltransferase 2.2 (ARTC2.2) on the cell surface. In response to T cell activation or extracellular NAD or ATP-mediated gating of the P2X7 ion channel ARTC2.2 is shed from the cell surface as a soluble enzyme. Shedding alters the target specificity of ARTC2.2 from cell surface proteins to secreted proteins. Here we demonstrate that shed ARTC2.2 potently ADP-ribosylates IFN-γ in addition to other cytokines. Using mass spectrometry, we identify arginine 128 as the target site of ADP-ribosylation. This residue has been implicated to play a key role in binding of IFN-γ to the interferon receptor 1 (IFNR1). Indeed, binding of IFN-γ to IFNR1 blocks ADP-ribosylation of IFN-γ. Moreover, ADP-ribosylation of IFN-γ inhibits the capacity of IFN-γ to induce STAT1 phosphorylation in macrophages and upregulation of the proteasomal subunit ß5i and the proteasomal activator PA28-α in podocytes. Our results show that ADP-ribosylation inhibits the signaling functions of IFN-γ and point to a new regulatory mechanism for controlling signaling by IFN-γ.
Topics: ADP Ribose Transferases; ADP-Ribosylation; Animals; Interferon-gamma; Mice; Mice, Inbred C57BL; Signal Transduction
PubMed: 33763084
DOI: 10.3389/fimmu.2021.642545 -
Nucleic Acids Research Aug 2023Nucleic acid ADP-ribosylation has been established as a novel modification found in a wide diversity of prokaryotic and eukaryotic organisms. tRNA 2'-phosphotransferase...
Nucleic acid ADP-ribosylation has been established as a novel modification found in a wide diversity of prokaryotic and eukaryotic organisms. tRNA 2'-phosphotransferase 1 (TRPT1/TPT1/KptA) possesses ADP-ribosyltransferase (ART) activity and is able to ADP-ribosylate nucleic acids. However, the underlying molecular mechanism remains elusive. Here, we determined crystal structures of TRPT1s in complex with NAD+ from Homo sapiens, Mus musculus and Saccharomyces cerevisiae. Our results revealed that the eukaryotic TRPT1s adopt common mechanisms for both NAD+ and nucleic acid substrate binding. The conserved SGR motif induces a significant conformational change in the donor loop upon NAD+ binding to facilitate the catalytic reaction of ART. Moreover, the nucleic acid-binding residue redundancy provides structural flexibility to accommodate different nucleic acid substrates. Mutational assays revealed that TRPT1s employ different catalytic and nucleic acid-binding residues to perform nucleic acid ADP-ribosylation and RNA 2'-phosphotransferase activities. Finally, cellular assays revealed that the mammalian TRPT1 is able to promote endocervical HeLa cell survival and proliferation. Together, our results provide structural and biochemical insights into the molecular mechanism of TRPT1 for nucleic acid ADP-ribosylation.
Topics: Animals; Humans; Mice; Adenosine Diphosphate Ribose; ADP Ribose Transferases; ADP-Ribosylation; HeLa Cells; NAD; Nucleic Acids; Phosphotransferases (Alcohol Group Acceptor); Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37334830
DOI: 10.1093/nar/gkad525 -
Journal of Proteome Research Feb 2023We have developed the underrepresented post-translational modification (PTM) database (urPTMdb), a PTM gene set database to accelerate the discovery of enriched protein...
We have developed the underrepresented post-translational modification (PTM) database (urPTMdb), a PTM gene set database to accelerate the discovery of enriched protein modifications in experimental data. urPTMdb provides curated lists of proteins reported to be substrates of underrepresented modifications. Their enrichment in proteomics datasets can reveal unexpected PTM regulations. urPTMdb can be implemented in existing workflows, or used in TeaProt, an online Shiny tool that integrates upstream transcription factor enrichment analysis with downstream pathway analysis through an easy-to-use interactive interface. TeaProt annotates user-uploaded data with drug-gene interactions, subcellular localizations, phenotypic functions, gene-disease associations, and enzyme-gene interactions. TeaProt enables gene set enrichment analysis (GSEA) to discover enrichments in gene sets from various resources, including MSigDB, CHEA, and urPTMdb. We demonstrate the utility of urPTMdb and TeaProt through the analysis of a previously published Western diet-induced remodeling of the tongue proteome, which revealed altered cellular processes associated with energy metabolism, interferon alpha/gamma response, adipogenesis, HMGylation substrate enrichment, and transcription regulation through PPARG and CEBPA. Additionally, we analyzed the interactome of ADP-ribose glycohydrolase TARG1, a key enzyme that removes mono-ADP-ribosylation. This analysis identified an enrichment of ADP-ribosylation, ribosomal proteins, and proteins localized in the nucleoli and endoplasmic reticulum. TeaProt and urPTMdb are accessible at https://tea.coffeeprot.com/.
Topics: Proteomics; Protein Processing, Post-Translational; ADP-Ribosylation; Adenosine Diphosphate Ribose; Proteome
PubMed: 35759515
DOI: 10.1021/acs.jproteome.2c00048 -
Pathogens (Basel, Switzerland) May 2023ADP-ribosylation is a reversible post-translational protein modification, which is evolutionarily conserved in prokaryotic and eukaryotic organisms. It governs critical... (Review)
Review
ADP-ribosylation is a reversible post-translational protein modification, which is evolutionarily conserved in prokaryotic and eukaryotic organisms. It governs critical cellular functions, including, but not limited to cellular proliferation, differentiation, RNA translation, and genomic repair. The addition of one or multiple ADP-ribose moieties can be catalysed by poly(ADP-ribose) polymerase (PARP) enzymes, while in eukaryotic organisms, ADP-ribosylation can be reversed through the action of specific enzymes capable of ADP-ribose signalling regulation. In several lower eukaryotic organisms, including parasites, ADP-ribosylation is thought to be important for infection establishment. encompasses several human disease-causing pathogens, including , , and the genus. These parasites are the etiological agents of Chagas disease, African trypanosomiasis (sleeping sickness), and leishmaniasis, respectively. Currently, licenced medications for these infections are outdated and often result in harmful side effects, and can be inaccessible to those carrying infections, due to them being classified as neglected tropical diseases (NTDs), meaning that many infected individuals will belong to already marginalised communities in countries already facing socioeconomic challenges. Consequently, funding to develop novel therapeutics for these infections is overlooked. As such, understanding the molecular mechanisms of infection, and how ADP-ribosylation facilitates infection establishment by these organisms may allow the identification of potential molecular interventions that would disrupt infection. In contrast to the complex ADP-ribosylation pathways in eukaryotes, the process of is more linear, with the parasites only expressing one PARP enzyme, compared to the, at least, 17 genes that encode human PARP enzymes. If this simplified pathway can be understood and exploited, it may reveal new avenues for combatting infection. This review will focus on the current state of knowledge on the importance of ADP-ribosylation in during infection establishment in human hosts, and the potential therapeutic options that disrupting ADP-ribosylation may offer to combat .
PubMed: 37242378
DOI: 10.3390/pathogens12050708 -
The FEBS Journal Dec 2017Adenosine diphosphate (ADP)-ribosylation is a chemical modification of macromolecules that plays an important role in regulation of quintessential biological processes...
Adenosine diphosphate (ADP)-ribosylation is a chemical modification of macromolecules that plays an important role in regulation of quintessential biological processes such as DNA repair, transcription, chromatin remodelling, stress response, apoptosis, bacterial metabolism and many others. ADP-ribosylation is carried out by ADP-ribosyltransferase proteins, such as poly (ADP-ribose) polymerases (PARPs) that transfer either monomer or polymers of ADP-ribose onto the molecular targets by using nicotinamide adenine dinucleotide (NAD ) as a cofactor. Traditionally, proteins have been described as primary targets of ADP-ribosylation; however, there has been growing evidence that DNA may be a common target as well. Here, we show using biochemical studies that PARP3, a DNA damage-activated ADP-ribosyltransferase, can mono-ADP-ribosylate double-stranded DNA ends. ADP-ribosylation of DNA mediated by PARP3 attaches a single mono-ADP-ribose moiety to the phosphate group at the terminal ends of DNA. We further show that mono ADP-ribosylation at DNA ends can be efficiently reversed by several cellular hydrolases (PARG, MACROD2, TARG1 and ARH3). This suggests that mono ADP-ribosylated DNA adducts can be efficiently removed in cells by several mechanisms.
Topics: ADP-Ribosylation; Adenosine Diphosphate Ribose; Animals; Cell Cycle Proteins; DNA; DNA Breaks; DNA Repair; Humans; Hydrolases; Models, Genetic; NAD; Poly(ADP-ribose) Polymerases
PubMed: 29054115
DOI: 10.1111/febs.14297 -
Nature Communications May 2021Androgen signaling through the androgen receptor (AR) directs gene expression in both normal and prostate cancer cells. Androgen regulates multiple aspects of the AR...
Androgen signaling through the androgen receptor (AR) directs gene expression in both normal and prostate cancer cells. Androgen regulates multiple aspects of the AR life cycle, including its localization and post-translational modification, but understanding how modifications are read and integrated with AR activity has been difficult. Here, we show that ADP-ribosylation regulates AR through a nuclear pathway mediated by Parp7. We show that Parp7 mono-ADP-ribosylates agonist-bound AR, and that ADP-ribosyl-cysteines within the N-terminal domain mediate recruitment of the E3 ligase Dtx3L/Parp9. Molecular recognition of ADP-ribosyl-cysteine is provided by tandem macrodomains in Parp9, and Dtx3L/Parp9 modulates expression of a subset of AR-regulated genes. Parp7, ADP-ribosylation of AR, and AR-Dtx3L/Parp9 complex assembly are inhibited by Olaparib, a compound used clinically to inhibit poly-ADP-ribosyltransferases Parp1/2. Our study reveals the components of an androgen signaling axis that uses a writer and reader of ADP-ribosylation to regulate protein-protein interactions and AR activity.
Topics: ADP-Ribosylation; Adenocarcinoma; Antineoplastic Agents; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Male; Metribolone; Neoplasm Proteins; Phthalazines; Piperazines; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Protein Isoforms; Protein Processing, Post-Translational; Receptors, Androgen; Signal Transduction; Survival Analysis
PubMed: 33976187
DOI: 10.1038/s41467-021-23055-6 -
Mono(ADP-ribosyl)ation Enzymes and NAD Metabolism: A Focus on Diseases and Therapeutic Perspectives.Cells Jan 2021Mono(ADP-ribose) transferases and mono(ADP-ribosyl)ating sirtuins use NAD to perform the mono(ADP-ribosyl)ation, a simple form of post-translational modification of... (Review)
Review
Mono(ADP-ribose) transferases and mono(ADP-ribosyl)ating sirtuins use NAD to perform the mono(ADP-ribosyl)ation, a simple form of post-translational modification of proteins and, in some cases, of nucleic acids. The availability of NAD is a limiting step and an essential requisite for NAD consuming enzymes. The synthesis and degradation of NAD, as well as the transport of its key intermediates among cell compartments, play a vital role in the maintenance of optimal NAD levels, which are essential for the regulation of NAD-utilizing enzymes. In this review, we provide an overview of the current knowledge of NAD metabolism, highlighting the functional liaison with mono(ADP-ribosyl)ating enzymes, such as the well-known ARTD10 (also named PARP10), SIRT6, and SIRT7. To this aim, we discuss the link of these enzymes with NAD metabolism and chronic diseases, such as cancer, degenerative disorders and aging.
Topics: ADP-Ribosylation; Animals; Biosynthetic Pathways; Disease; Enzymes; Humans; NAD; Sirtuins
PubMed: 33440786
DOI: 10.3390/cells10010128