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Pathogens (Basel, Switzerland) May 2023Poly ADP-ribose polymerases (PARPs) catalyze ADP-ribosylation, a subclass of post-translational modification (PTM). Mono-ADP-ribose (MAR) moieties bind to target...
Poly ADP-ribose polymerases (PARPs) catalyze ADP-ribosylation, a subclass of post-translational modification (PTM). Mono-ADP-ribose (MAR) moieties bind to target molecules such as proteins and nucleic acids, and are added as part of the process which also leads to formation of polymer chains of ADP-ribose. ADP-ribosylation is reversible; its removal is carried out by ribosyl hydrolases such as PARG (poly ADP-ribose glycohydrolase), TARG (terminal ADP-ribose protein glycohydrolase), macrodomain, etc. In this study, the catalytic domain of tankyrase was expressed in bacteria and purified. The tankyrase PARP catalytic domain was found to be enzymatically active, as demonstrated by an in vitro poly ADP-ribosylation (PARylation) experiment. Using in vitro ADP-ribosylation assay, we further demonstrate that the chikungunya virus (CHIKV) nsp3 (non-structural protein 3) macrodomain inhibits ADP-ribosylation in a time-dependent way. We have also demonstrated that transfection of the CHIKV nsP3 macrodomain increases the CHIKV viral titer in mosquito cells, suggesting that ADP-ribosylation may play a significant role in viral replication.
PubMed: 37242388
DOI: 10.3390/pathogens12050718 -
Angewandte Chemie (International Ed. in... Apr 2022Post-translational modification (PTM) with ADP-ribose and poly(ADP-ribose) using nicotinamide adenine dinucleotide (NAD ) as substrate is involved in the regulation of...
Post-translational modification (PTM) with ADP-ribose and poly(ADP-ribose) using nicotinamide adenine dinucleotide (NAD ) as substrate is involved in the regulation of numerous cellular pathways in eukaryotes, notably the response to DNA damage caused by cellular stress. Nevertheless, due to intrinsic properties of NAD e.g., high polarity and associated poor cell passage, these PTMs are difficult to characterize in cells. Here, two new NAD derivatives are presented, which carry either a fluorophore or an affinity tag and, in combination with developed methods for mild cell delivery, allow studies in living human cells. We show that this approach allows not only the imaging of ADP-ribosylation in living cells but also the proteome-wide analysis of cellular adaptation by protein ADP-ribosylation as a consequence of environmental changes such as H O -induced oxidative stress or the effect of the approved anti-cancer drug olaparib. Our results therefore pave the way for further functional and clinical studies of the ADP-ribosylated proteome in living cells in health and disease.
Topics: ADP-Ribosylation; Adenosine Diphosphate Ribose; Humans; NAD; Protein Processing, Post-Translational; Proteome
PubMed: 35188710
DOI: 10.1002/anie.202200977 -
Viruses Mar 2020Macrodomains, enzymes that remove ADP-ribose from proteins, are encoded by several families of RNA viruses and have recently been shown to counter innate immune... (Review)
Review
Macrodomains, enzymes that remove ADP-ribose from proteins, are encoded by several families of RNA viruses and have recently been shown to counter innate immune responses to virus infection. ADP-ribose is covalently attached to target proteins by poly-ADP-ribose polymerases (PARPs), using nicotinamide adenine dinucleotide (NAD+) as a substrate. This modification can have a wide variety of effects on proteins including alteration of enzyme activity, protein-protein interactions, and protein stability. Several PARPs are induced by interferon (IFN) and are known to have antiviral properties, implicating ADP-ribosylation in the host defense response and suggesting that viral macrodomains may counter this response. Recent studies have demonstrated that viral macrodomains do counter the innate immune response by interfering with PARP-mediated antiviral defenses, stress granule formation, and pro-inflammatory cytokine production. Here, we will describe the known functions of the viral macrodomains and review recent literature demonstrating their roles in countering PARP-mediated antiviral responses.
Topics: ADP-Ribosylation; Adenosine Diphosphate Ribose; Cytoplasmic Granules; Humans; Interferons; Mutation; Poly(ADP-ribose) Polymerases; Protein Domains; RNA Virus Infections; RNA Viruses; Viral Nonstructural Proteins; Virus Replication
PubMed: 32244383
DOI: 10.3390/v12040384 -
Naunyn-Schmiedeberg's Archives of... Apr 2022Numerous studies in airways, ileum, and urinary bladder have demonstrated that relaxation by β-adrenoceptor agonists has lower potency and/or efficacy when contraction... (Review)
Review
Does coupling to ADP ribosylation factor 6 explain differences between muscarinic and other receptors in interaction with β-adrenoceptor-mediated smooth muscle relaxation?
Numerous studies in airways, ileum, and urinary bladder have demonstrated that relaxation by β-adrenoceptor agonists has lower potency and/or efficacy when contraction was elicited by muscarinic receptor agonists as compared to other G-protein-coupled receptors, KCl, or basal tone, but the molecular mechanisms behind this relative resistance remain unclear. A paper by Huang et al. in this issue demonstrates that NAV2729, an inhibitor of ADP ribosylation factor 6, inhibits contraction of isolated blood vessels elicited by muscarinic receptor agonists, but not by α-adrenoceptor agonists or KCl. Against this background, we discuss the role of ADP ribosylation factor 6 in cellular responses to G-protein-coupled receptor stimulation. While ADP ribosylation factor 6 apparently is the only promising molecular explanation for the relative resistance of smooth muscle contraction elicited by muscarinic agonists, the existing data are insufficient for a robust conclusion.
Topics: ADP-Ribosylation Factor 6; Adrenergic beta-Agonists; Muscarinic Agonists; Muscle Contraction; Muscle Relaxation; Receptors, Adrenergic
PubMed: 35175382
DOI: 10.1007/s00210-022-02221-7 -
Cell Reports Feb 2020ADP-ribosylation of proteins is crucial for fundamental cellular processes. Despite increasing examples of DNA ADP-ribosylation, the impact of this modification on DNA...
ADP-ribosylation of proteins is crucial for fundamental cellular processes. Despite increasing examples of DNA ADP-ribosylation, the impact of this modification on DNA metabolism and cell physiology is unknown. Here, we show that the DarTG toxin-antitoxin system from enteropathogenic Escherichia coli (EPEC) catalyzes reversible ADP-ribosylation of single-stranded DNA (ssDNA). The DarT toxin recognizes specific sequence motifs. EPEC DarG abrogates DarT toxicity by two distinct mechanisms: removal of DNA ADP-ribose (ADPr) groups and DarT sequestration. Furthermore, we investigate how cells recognize and deal with DNA ADP-ribosylation. We demonstrate that DNA ADPr stalls replication and is perceived as DNA damage. Removal of ADPr from DNA requires the sequential activity of two DNA repair pathways, with RecF-mediated homologous recombination likely to transfer ADP-ribosylation from single- to double-stranded DNA (dsDNA) and subsequent nucleotide excision repair eliminating the lesion. Our work demonstrates that these DNA repair pathways prevent the genotoxic effects of DNA ADP-ribosylation.
Topics: ADP-Ribosylation; Adenosine Diphosphate Ribose; DNA Repair; DNA Replication; DNA, Bacterial; DNA-Binding Proteins; Enteropathogenic Escherichia coli; Escherichia coli Proteins; Homologous Recombination; Microbial Viability; Models, Biological; SOS Response, Genetics
PubMed: 32023456
DOI: 10.1016/j.celrep.2020.01.014 -
The Journal of Pathology. Clinical... Jul 2023ADP-ribosylation (ADPR) of proteins is catalyzed by ADP-ribosyltransferases, which are targeted by inhibitors (i.e. poly(ADP-ribose) polymerase inhibitors [PARPi])....
ADP-ribosylation (ADPR) of proteins is catalyzed by ADP-ribosyltransferases, which are targeted by inhibitors (i.e. poly(ADP-ribose) polymerase inhibitors [PARPi]). Although renal cell carcinoma (RCC) cells are sensitive in vitro to PARPi, studies on the association between ADPR levels and somatic loss of function mutations in DNA damage repair genes are currently missing. Here we observed, in two clear cell RCC (ccRCC) patient cohorts (n = 257 and n = 241) stained with an engineered ADP-ribose binding macrodomain (eAf1521), that decreased cytoplasmic ADPR (cyADPR) levels significantly correlated with late tumor stage, high-ISUP (the International Society of Urological Pathology) grade, presence of necrosis, dense lymphocyte infiltration, and worse patient survival (p < 0.01 each). cyADPR proved to be an independent prognostic factor (p = 0.001). Comparably, absence of nuclear ADPR staining in ccRCC correlated with absence of PARP1 staining (p < 0.01) and worse patient outcome (p < 0.05). In papillary RCC the absence of cyADPR was also significantly associated with tumor progression and worse patient outcome (p < 0.05 each). To interrogate whether the ADPR status could be associated with genetic alterations in DNA repair, chromatin remodeling, and histone modulation, we performed DNA sequence analysis and identified a significant association of increased ARID1A mutations in ccRCC compared with ccRCC (31% versus 4%; p < 0.05). Collectively, our data suggest the prognostic value of nuclear and cytoplasmic ADPR levels in RCC that might be further influenced by genetic alterations.
Topics: Humans; Carcinoma, Renal Cell; Adenosine Diphosphate Ribose; Prognosis; ADP-Ribosylation; Histones; Kidney Neoplasms
PubMed: 36999983
DOI: 10.1002/cjp2.320 -
Cells Mar 2021Adenosine diphosphate (ADP)-ribosylation is a nicotinamide adenine dinucleotide (NAD)-dependent post-translational modification that is found on proteins as well as on... (Review)
Review
Adenosine diphosphate (ADP)-ribosylation is a nicotinamide adenine dinucleotide (NAD)-dependent post-translational modification that is found on proteins as well as on nucleic acids. While ARTD1/PARP1-mediated poly-ADP-ribosylation has extensively been studied in the past 60 years, comparably little is known about the physiological function of mono-ADP-ribosylation and the enzymes involved in its turnover. Promising technological advances have enabled the development of innovative tools to detect NAD and NAD/NADH (H for hydrogen) ratios as well as ADP-ribosylation. These tools have significantly enhanced our current understanding of how intracellular NAD dynamics contribute to the regulation of ADP-ribosylation as well as to how mono-ADP-ribosylation integrates into various cellular processes. Here, we discuss the recent technological advances, as well as associated new biological findings and concepts.
Topics: ADP-Ribosylation; Adenosine Diphosphate Ribose; Humans; NAD; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Protein Processing, Post-Translational
PubMed: 33808662
DOI: 10.3390/cells10030680 -
Cells Oct 2019The transfer of adenosine diphosphate (ADP)-ribose unit(s) from nicotinamide adenine dinucleotide (NAD) to acceptor proteins is known as ADP-ribosylation. This... (Review)
Review
The transfer of adenosine diphosphate (ADP)-ribose unit(s) from nicotinamide adenine dinucleotide (NAD) to acceptor proteins is known as ADP-ribosylation. This post-translational modification (PTM) unavoidably alters protein functions and signaling networks, thereby impacting cell behaviors and tissue outcomes. As a ubiquitous mechanism, ADP-ribosylation affects multiple tissues, including bones, as abnormal ADP-ribosylation compromises bone development and remodeling. In this review, we describe the effects of ADP-ribosylation in bone development and maintenance, and highlight the underlying mechanisms.
Topics: ADP-Ribosylation; Adenosine Diphosphate Ribose; Adipocytes; Animals; Bone Development; Bone Diseases; Bone and Bones; Cell Line; Humans; Mice; Osteoblasts; Osteoclasts; Poly(ADP-ribose) Polymerases
PubMed: 31590342
DOI: 10.3390/cells8101201 -
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