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Protein ADP-ribosylation plays important but ill-defined roles in antiviral signalling cascades such as the interferon response. Several viruses of clinical interest, including coronaviruses, express hydrolases that reverse ADP-ribosylation catalysed by host enzymes, suggesting an important role for this modification in host-pathogen interactions. However, which ADP-ribosyltransferases mediate host ADP-ribosylation, what proteins and pathways they target and how these modifications affect viral infection and pathogenesis is currently unclear. Here we show that host ADP-ribosyltransferase activity induced by IFNγ signalling depends on PARP14 catalytic activity and that the PARP9/DTX3L complex is required to uphold PARP14 protein levels via post-translational mechanisms. Both the PARP9/DTX3L complex and PARP14 localise to IFNγ-induced cytoplasmic inclusions containing ADP-ribosylated proteins, and both PARP14 itself and DTX3L are likely targets of PARP14 ADP-ribosylation. We provide evidence that these modifications are hydrolysed by the SARS-CoV-2 Nsp3 macrodomain, shedding light on the intricate cross-regulation between IFN-induced ADP-ribosyltransferases and the potential roles of the coronavirus macrodomain in counteracting their activity. Synopsis: Protein ADP-ribosylation plays important but ill-defined roles in antiviral signaling cascades. This study demonstrates that PARP9, DTX3L and PARP14 regulate IFNγ-induced ADP-ribosylation, and explores an intricate cross-regulation between these factors proposed to be targets of the coronavirus macrodomain. PARP14 catalytic activity is required for IFNγ-induced ADP-ribosylation. PARP9 and DTX3L regulate PARP14 protein stability. PARP9, DTX3L, PARP14 and ADPr co-localize in IFNγ-induced cytoplasmic structures. IFNγ induces PARP14-dependent modification of itself and of DTX3L, which may be hydrolyzed by the SARS-CoV2 macrodomain. Interferon signaling induces cytoplasmic colocalization and cross-regulation of two ADP-ribosyltransferases and a ubiquitin ligase. [ABSTRACT FROM AUTHOR]

ADP-ribosylation is a ubiquitous modification of proteins and other targets, such as nucleic acids, that regulates various cellular functions in all kingdoms of life. Furthermore, these ADP-ribosyltransferases (ARTs) modify a variety of substrates and atoms. It has been almost 60 years since ADP-ribosylation was discovered. Various ART structures have been revealed with cofactors (NAD+ or NAD+ analog). However, we still do not know the molecular mechanisms of ART. It needs to be better understood how ART specifies the target amino acids or bases. For this purpose, more information is needed about the tripartite complex structures of ART, the cofactors, and the substrates. The tripartite complex is essential to understand the mechanism of ADP-ribosyltransferase. This review updates the general ADP-ribosylation mechanism based on ART tripartite complex structures. [ABSTRACT FROM AUTHOR]

Sirtuins (SIRTs) are a family of proteins with enzymatic activity. In particular, they are a family of class III NAD+-dependent histone deacetylases and ADP-ribosyltransferases. NAD+-dependent deac(et)ylase activities catalyzed by sirtuin include ac(et)ylation, propionylation, butyrylation, crotonylation, manylation, and succinylation. Specifically, human SIRT3 is a 399 amino acid protein with two functional domains: a large Rossmann folding motif and NAD+ binding, and a small complex helix and zinc-binding motif. SIRT3 is widely expressed in mitochondria-rich tissues and is involved in maintaining mitochondrial integrity, homeostasis, and function. Moreover, SIRT3 regulates related diseases, such as aging, hepatic, kidney, neurodegenerative and cardiovascular disease, metabolic diseases, and cancer development. In particular, one of the most significant and damaging post-translational modifications is irreversible protein oxidation, i.e. carbonylation. This process is induced explicitly by increased ROS production due to mitochondrial dysfunction. SIRT3 is carbonylated by 4-hydroxynonenal at the level of Cys280. The carbonylation induces conformational changes in the active site, resulting in allosteric inhibition of SIRT3 activity and loss of the ability to deacetylate and regulate antioxidant enzyme activity. Phytochemicals and, in particular, polyphenols, thanks to their strong antioxidant activity, are natural compounds with a positive regulatory action on SIRT3 in various pathologies. Indeed, the enzymatic SIRT3 activity is modulated, for example, by different natural polyphenol classes, including resveratrol and the bergamot polyphenolic fraction. Thus, this review aims to elucidate the mechanisms by which phytochemicals can interact with SIRT3, resulting in post-translational modifications that regulate cellular metabolism. [ABSTRACT FROM AUTHOR]

ADP-ribosyltransferases PARP1 and PARP2 play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins. Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations. Recently, a study showed that Histone PARylation Factor (HPF1) forms a joint active site with PARP1/2. The interaction of HPF1 with PARP1/2 alters the modification site from Aspartate/Glutamate to Serine, which has been shown to be a key ADP-ribosylation event in the context of DNA damage. Therefore, disruption of PARP1/2-HPF1 interaction could be an alternative strategy for drug development to block the PARP1/2 activity. In this study, we describe a FRET based high-throughput screening assay to screen inhibitor libraries against PARP-HPF1 interaction. We optimized the conditions for FRET signal and verified the interaction by competing the FRET pair in multiple ways. The assay is robust and easy to automate. Validatory screening showed the robust performance of the assay, and we discovered two compounds Dimethylacrylshikonin and Alkannin, with µM inhibition potency against PARP1/2-HPF1 interaction. The assay will facilitate the discovery of inhibitors against HPF1-PARP1/2 complex and to develop potentially new effective anticancer agents. [ABSTRACT FROM AUTHOR]

An NAD+ featuring an adenosyl 4′-azido functions as a general substrate for poly-ADP-ribose polymerases. Its derived mono- and poly-ADP-ribosylated proteins can be adequately recognized by distinct ADP-ribosylation-specific readers. This molecule represents the first ribose-functionalized NAD+ with versatile activities across different ADP-ribosyltransferases and provides insight into developing new probes for ADP-ribosylation. [ABSTRACT FROM AUTHOR]

ADP-ribose is a versatile modification that plays a critical role in diverse cellular processes. The addition of this modification is catalyzed by ADP-ribosyltransferases, among which notable poly(ADP-ribose) polymerase (PARP) enzymes are intimately involved in the maintenance of genome integrity. The role of ADP-ribose modifications during DNA damage repair is of significant interest for the proper development of PARP inhibitors targeted toward the treatment of diseases caused by genomic instability. More specifically, inhibitors promoting PARP persistence on DNA lesions, termed PARP "trapping," is considered a desirable characteristic. In this review, we discuss key classes of proteins involved in ADP-ribose signaling (writers, readers, and erasers) with a focus on those involved in the maintenance of genome integrity. An overview of factors that modulate PARP1 and PARP2 persistence at sites of DNA lesions is also discussed. Finally, we clarify aspects of the PARP trapping model in light of recent studies that characterize the kinetics of PARP1 and PARP2 recruitment at sites of lesions. These findings suggest that PARP trapping could be considered as the continuous recruitment of PARP molecules to sites of lesions, rather than the physical stalling of molecules. Recent studies and novel research tools have elevated the level of understanding of ADP-ribosylation, marking a coming-ofage for this interesting modification. [ABSTRACT FROM AUTHOR]

Tankyrase 1 and 2 are ADP-ribosyltransferases that catalyze formation of polyADP-Ribose (PAR) onto themselves and their binding partners. Tankyrase protein levels are regulated by the PAR-binding E3 ligase RNF146, which promotes K48-linked polyubiquitylation and proteasomal degradation of tankyrase and its partners. We identified a novel interaction between tankyrase and a distinct class of E3 ligases: the RING-UIM (Ubiquitin-Interacting Motif) family. We show that RNF114 and RNF166 bind and stabilize monoubiquitylated tankyrase and promote K11-linked diubiquitylation. This action competes with RNF146-mediated degradation, leading to stabilization of tankyrase and its binding partner, Angiomotin, a cancer cell signaling protein. Moreover, we identify multiple PAR-binding E3 ligases that promote ubiquitylation of tankyrase and induce stabilization or degradation. Discovery of K11 ubiquitylation that opposes degradation, along with identification of multiple PAR-binding E3 ligases that ubiquitylate tankyrase, provide insights into mechanisms of tankyrase regulation and may offer additional uses for tankyrase inhibitors in cancer therapy. The poly(ADP-ribosyl)transferases, tankyrase 1 and 2, are regulated by RNF146-mediated K48-linked polyubiquitylation and degradation. Here the authors show that this is opposed by K11-linked diubiquitylation by RING-UIM E3 ligases RNF114 and 166 and further impacted by several PAR-binding E3 ligases. [ABSTRACT FROM AUTHOR]

The transfer of ADP–ribose (ADPr) from nicotinamide adenine dinucleotide (NAD+) to target proteins is mediated by a class of human diphtheria toxin-like ADP-ribosyltransferases (ARTDs; previously referred to as poly-ADP–ribose polymerases or PARPs) and the removal of ADPr is catalyzed by a family of glycohydrolases. Although thousands of potential ADPr modification sites have been identified using high-throughput mass-spectrometry, relatively little is known about the sequence specificity encoded near the modification site. Herein, we present a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method that facilitates the in vitro analysis of proximal factors that guide ARTD target selection. We identify a minimal 5-mer peptide sequence that is necessary and sufficient to drive glutamate/aspartate targeting using PARP14 while highlighting the importance of the adjacent residues in PARP14 targeting. We measure the stability of the resultant ester bond and show that non-enzymatic removal is pH and temperature dependent, sequence independent, and occurs within hours. Finally, we use the ADPr–peptides to highlight differential activities within the glycohydrolase family and their sequence preferences. Our results highlight (1) the utility of MALDI-TOF in analyzing proximal ARTD–substrate interactions and (2) the importance of peptide sequences in governing ADPr transfer and removal. [ABSTRACT FROM AUTHOR]

Cells have developed different strategies to cope with viral infections. Key to initiating a defense response against viruses is the ability to distinguish foreign molecules from their own. One central mechanism is the perception of foreign nucleic acids by host proteins which, in turn, initiate an efficient immune response. Nucleic acid sensing pattern recognition receptors have evolved, each targeting specific features to discriminate viral from host RNA. These are complemented by several RNA-binding proteins that assist in sensing of foreign RNAs. There is increasing evidence that the interferon-inducible ADP-ribosyltransferases (ARTs; PARP9—PARP15) contribute to immune defense and attenuation of viruses. However, their activation, subsequent targets, and precise mechanisms of interference with viruses and their propagation are still largely unknown. Best known for its antiviral activities and its role as RNA sensor is PARP13. In addition, PARP9 has been recently described as sensor for viral RNA. Here we will discuss recent findings suggesting that some PARPs function in antiviral innate immunity. We expand on these findings and integrate this information into a concept that outlines how the different PARPs might function as sensors of foreign RNA. We speculate about possible consequences of RNA binding with regard to the catalytic activities of PARPs, substrate specificity and signaling, which together result in antiviral activities. [ABSTRACT FROM AUTHOR]

The chemical modification of cellular macromolecules by the transfer of ADP-ribose unit(s), known as ADP-ribosylation, is an ancient homeostatic and stress response control system. Highly conserved across the evolution, ADP-ribosyltransferases and ADP-ribosylhydrolases control ADP-ribosylation signalling and cellular responses. In addition to proteins, both prokaryotic and eukaryotic transferases can covalently link ADP-ribosylation to different conformations of nucleic acids, thus highlighting the evolutionary conservation of archaic stress response mechanisms. Here, we report several structural and functional aspects of DNA ADP-ribosylation modification controlled by the prototype DarT and DarG pair, which show ADP-ribosyltransferase and hydrolase activity, respectively. DarT/DarG is a toxin–antitoxin system conserved in many bacterial pathogens, for example in Mycobacterium tuberculosis, which regulates two clinically important processes for human health, namely, growth control and the anti-phage response. The chemical modulation of the DarT/DarG system by selective inhibitors may thus represent an exciting strategy to tackle resistance to current antimicrobial therapies. [ABSTRACT FROM AUTHOR]

ADP‐ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra‐ and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP‐ribosylation is catalyzed by ADP‐ribosyltransferases (ARTs), which transfer ADP‐ribose from NAD+ onto substrates. The modification, which occurs as mono‐ or poly‐ADP‐ribosylation, is reversible due to the action of different ADP‐ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP‐ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP‐ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP‐ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP‐ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono‐ and poly‐ADP‐ribose mediated cellular processes. [ABSTRACT FROM AUTHOR]

The ARH family of ADP-ribose-acceptor hydrolases consists of three 39-kDa members (ARH1-3), with similarities in amino acid sequence. ARH1 was identified based on its ability to cleave ADP-ribosyl-arginine synthesized by cholera toxin. Mammalian ADP-ribosyltransferases (ARTCs) mimicked the toxin reaction, with ARTC1 catalyzing the synthesis of ADP-ribosyl-arginine. ADP-ribosylation of arginine was stereospecific, with β-NAD+ as substrate and, α-anomeric ADP-ribose-arginine the reaction product. ARH1 hydrolyzed α-ADP-ribose-arginine, in addition to α-NAD+ and O-acetyl-ADP-ribose. Thus, ADP-ribose attached to oxygen-containing or nitrogen-containing functional groups was a substrate. Arh1 heterozygous and knockout (KO) mice developed tumors. Arh1-KO mice showed decreased cardiac contractility and developed myocardial fibrosis. In addition to Arh1-KO mice showed increased ADP-ribosylation of tripartite motif-containing protein 72 (TRIM72), a membrane-repair protein. ARH3 cleaved ADP-ribose from ends of the poly(ADP-ribose) (PAR) chain and released the terminal ADP-ribose attached to (serine)protein. ARH3 also hydrolyzed α-NAD+ and O-acetyl-ADP-ribose. Incubation of Arh3-KO cells with H2O2 resulted in activation of poly-ADP-ribose polymerase (PARP)-1, followed by increased nuclear PAR, increased cytoplasmic PAR, leading to release of Apoptosis Inducing Factor (AIF) from mitochondria. AIF, following nuclear translocation, stimulated endonucleases, resulting in cell death by Parthanatos. Human ARH3-deficiency is autosomal recessive, rare, and characterized by neurodegeneration and early death. Arh3-KO mice developed increased brain infarction following ischemia-reperfusion injury, which was reduced by PARP inhibitors. Similarly, PARP inhibitors improved survival of Arh3-KO cells treated with H2O2. ARH2 protein did not show activity in the in vitro assays described above for ARH1 and ARH3. ARH2 has a restricted tissue distribution, with primary involvement of cardiac and skeletal muscle. Overall, the ARH family has unique functions in biological processes and different enzymatic activities. [ABSTRACT FROM AUTHOR]

Clostridioides bacteria are responsible for life threatening infections. Here, we show that in addition to actin, the binary toxins CDT, C2I, and Iota from Clostridioides difficile, botulinum, and perfrigens, respectively, ADP-ribosylate the actin-related protein Arp2 of Arp2/3 complex and its additional components ArpC1, ArpC2, and ArpC4/5. The Arp2/3 complex is composed of seven subunits and stimulates the formation of branched actin filament networks. This activity is inhibited after ADP-ribosylation of Arp2. Translocation of the ADP-ribosyltransferase component of CDT toxin into human colon carcinoma Caco2 cells led to ADP-ribosylation of cellular Arp2 and actin followed by a collapse of the lamellipodial extensions and F-actin network. Exposure of isolated mouse colon pieces to CDT toxin induced the dissolution of the enterocytes leading to luminal aggregation of cellular debris and the collapse of the mucosal organization. Thus, we identify the Arp2/3 complex as hitherto unknown target of clostridial ADP-ribosyltransferases. [ABSTRACT FROM AUTHOR]

Sirtfood is a new concept food that compounds diets that can target sirtuins (SIRTs). SIRTs are nicotinamide adenine dinucleotide (NAD+)-dependent deacylases and ADP-ribosyltransferases (enzymes). SIRTs are mediators of calorie restriction (CR) and their activation can achieve some effects similar to CR. SIRTs play essential roles in ameliorating obesity and age-related metabolic diseases. Food ingredients such as resveratrol, piceatannol, anthocyanidin, and quinine are potential modulators of SIRTs. SIRT modulators are involved in autophagy, apoptosis, aging, inflammation, and energy homeostasis. Sirtfood proponents believe that natural Sirtfood recipes exert significant health effects. [ABSTRACT FROM AUTHOR]

NAD+ and ADP-ribose (ADPr)-containing molecules are at the interface of virus–host conflicts across life encompassing RNA processing, restriction, lysogeny/dormancy and functional hijacking. We objectively defined the central components of the NAD+–ADPr networks involved in these conflicts and systematically surveyed 21,191 completely sequenced viral proteomes representative of all publicly available branches of the viral world to reconstruct a comprehensive picture of the viral NAD+–ADPr systems. These systems have been widely and repeatedly exploited by positive-strand RNA and DNA viruses, especially those with larger genomes and more intricate life-history strategies. We present evidence that ADP-ribosyltransferases (ARTs), ADPr-targeting Macro, NADAR and Nudix proteins are frequently packaged into virions, particularly in phages with contractile tails (Myoviruses), and deployed during infection to modify host macromolecules and counter NAD+-derived signals involved in viral restriction. Genes encoding NAD+–ADPr-utilizing domains were repeatedly exchanged between distantly related viruses, hosts and endo-parasites/symbionts, suggesting selection for them across the virus world. Contextual analysis indicates that the bacteriophage versions of ADPr-targeting domains are more likely to counter soluble ADPr derivatives, while the eukaryotic RNA viral versions might prefer macromolecular ADPr adducts. Finally, we also use comparative genomics to predict host systems involved in countering viral ADP ribosylation of host molecules. [ABSTRACT FROM AUTHOR]

Many important bacterial pathogens utilize virulence factors that attach an ADP-ribose moiety to target host molecules. These enzymes, referred to as mono-ADP-ribosyltransferases (mARTs), have been extensively studied in human pathogens, and prominent examples include the Cholera toxin from Vibrio cholerae, the Pertussis toxin from Bordetella pertussis, and the Diphtheria toxin from Corynebacterium diphtheriae. In contrast, the characterization of mART enzymes as virulence factors in phytopathogens is a relatively new area of research. Several mARTs produced by the bacterial plant pathogen Pseudomonas syringae have been shown to play a key role in suppressing pathogen-triggered immunity and/or effector-triggered immunity, and similarly, the SirTMs from Phytophthora species may contribute to oomycete resistance to host immune responses. Recently, a novel mART toxin called Scabin was identified in the potato common scab pathogen Streptomyces scabiei. Unlike most mARTs, Scabin targets guanosine residues in DNA, and in vitro studies have shown that it preferentially modifies potato genomic DNA, suggesting that it may function in modulating host gene expression during pathogen infection. [ABSTRACT FROM AUTHOR]

NAD+ is a versatile biomolecule acting as a master regulator and substrate in various cellular processes, including redox regulation, metabolism, and various signaling pathways. In this article, we concisely and critically review the role of NAD+ in mechanisms promoting genome maintenance. Numerous NAD+-dependent reactions are involved in the preservation of genome stability, the cellular DNA damage response, and other pathways regulating nucleic acid metabolism, such as gene expression and cell proliferation pathways. Of note, NAD+ serves as a substrate to ADP-ribosyltransferases, sirtuins, and potentially also eukaryotic DNA ligases, all of which regulate various aspects of DNA integrity, damage repair, and gene expression. Finally, we critically analyze recent developments in the field as well as discuss challenges associated with therapeutic actions intended to raise NAD+ levels. [ABSTRACT FROM AUTHOR]

In nature, the majority of known RNA-protein interactions are transient. Our recent study has depicted a novel mechanism known as RNAylation, which covalently links proteins and RNAs. This novel modification bridges the realms of RNA and protein modifications. This review specifically explores RNAylation catalyzed by bacteriophage T4 ADP-ribosyltransferase ModB, with a focus on its protein targets and RNA substrates in the context of Escherichia coli -bacteriophage T4 interaction. Additionally, we discuss the biological significance of RNAylation and present perspectives on RNAylation as a versatile bioconjugation strategy for RNAs and proteins. [Display omitted] • RNAs and proteins are covalently linked in vivo by an RNAylation reaction. • RNAylation is catalyzed by the bacteriophage T4 ADP-ribosyltransferase ModB. • NAD-capped RNAs are the substrates of ModB for RNAylation. • Ribosomal proteins are specifically RNAylated in vivo. • RNAylation may be used as a bioconjugation strategy to engineer RNA-protein conjugates. [ABSTRACT FROM AUTHOR]

ADP‐ribosylation is a post‐translational modification catalyzed by writer enzymes – ADP‐ribosyltransferases. The modification is part of many signaling events, can modulate the function and stability of target proteins, and often results in the recruitment of reader proteins that bind to the ADP‐ribosyl groups. Erasers are integral actors in these signaling events and reverse the modification. ADP‐ribosylation can be targeted with therapeutics and many inhibitors against writers exist, with some being in clinical use. Inhibitors against readers and erasers are sparser and development of these has gained momentum only in recent years. Drug discovery has been hampered by the lack of specific tools, however many significant advances in the methods have recently been reported. We discuss assays used in the field with a focus on methods allowing efficient identification of small molecule inhibitors and profiling against enzyme families. While human proteins are focused, the methods can be also applied to bacterial toxins and virus encoded erasers that can be targeted to treat infectious diseases in the future. [ABSTRACT FROM AUTHOR]

Signaling cascades provide integrative and interactive frameworks that allow the cell to respond to signals from its environment and/or from within the cell itself. The dynamic regulation of mammalian cell signaling pathways is often modulated by cascades of protein post-translational modifications (PTMs). ADP-ribosylation is a PTM that is catalyzed by ADP-ribosyltransferases and manifests as mono- (MARylation) or poly- (PARylation) ADP-ribosylation depending on the addition of one or multiple ADP-ribose units to protein substrates. ADP-ribosylation has recently emerged as an important cell regulator that impacts a plethora of cellular processes, including many intracellular signaling events. Here, we provide an overview of the interplay between the intracellular diphtheria toxin-like ADP-ribosyltransferase (ARTD) family members and five selected signaling pathways (including NF-κB, JAK/STAT, Wnt-β-catenin, MAPK, PI3K/AKT), which are frequently described to control or to be controlled by ADP-ribosyltransferases and how these interactions impact the cellular responses. [ABSTRACT FROM AUTHOR]

Intracellular ADP‐ribosyltransferases catalyze mono‐ and poly‐ADP‐ribosylation and affect a broad range of biological processes. The mono‐ADP‐ribosyltransferase PARP10 is involved in signaling and DNA repair. Previous studies identified OUL35 as a selective, cell permeable inhibitor of PARP10. We have further explored the chemical space of OUL35 by synthesizing and investigating structurally related analogs. Key synthetic steps were metal‐catalyzed cross‐couplings and functional group modifications. We identified 4‐(4‐cyanophenoxy)benzamide and 3‐(4‐carbamoylphenoxy)benzamide as PARP10 inhibitors with distinct selectivities. Both compounds were cell permeable and interfered with PARP10 toxicity. Moreover, both revealed some inhibition of PARP2 but not PARP1, unlike clinically used PARP inhibitors, which typically inhibit both enzymes. Using crystallography and molecular modeling the binding of the compounds to different ADP‐ribosyltransferases was explored regarding selectivity. Together, these studies define additional compounds that interfere with PARP10 function and thus expand our repertoire of inhibitors to further optimize selectivity and potency. [ABSTRACT FROM AUTHOR]