Development and characterization of recombinant ADP-ribose binding reagents that allow simultaneous detection of mono and poly ADP-ribose.

ADP-ribosylation (ADPRylation) is a post-translational modification (PTM) of proteins mediated by the activity of a variety of ADP-ribosyltransferase (ART) enzymes, such as the Poly (ADP-ribose) Polymerase (PARP) family of proteins. This PTM is diverse in both form and biological functions, which makes it a highly interesting modification, but difficult to study due to limitations in reagents available to detect the diversity of ADPRylation. Recently we developed a set of recombinant antibody-like ADP-ribose (ADPR) binding proteins using naturally occurring ADPR binding domains (ARBDs), including macrodomains and WWE domains, functionalized by fusion to the constant "Fc" region of rabbit immunoglobulin. Herein, we present an expansion of this biological toolkit, where we have replaced the rabbit Fc sequence with the sequence from two other species, mouse and goat. These new reagents are based on a previously characterized set of naturally occurring ARBDs with known specificity. Characterization of the new reagents demonstrates that they can be detected in a species-dependent manner by secondary immunological tools, recognize specific ADPR moieties, and can be used for simultaneous detection of mono ADPR and poly ADPR at single-cell resolution in various antibody-based assays. The expansion of this toolkit will allow for more multiplexed assessments of the complexity of ADPRylation biology in many biological systems.

Crystal structure and biochemical activity of the macrodomain from rubella virus p150.

Rubella virus encodes a nonstructural polyprotein with RNA polymerase, methyltransferase, and papain-like cysteine protease activities, along with a putative macrodomain of unknown function. Macrodomains bind ADP-ribose adducts, a post-translational modification that plays a key role in host-virus conflicts. Some macrodomains can also remove the mono-ADP-ribose adduct or degrade poly-ADP-ribose chains. Here, we report high-resolution crystal structures of the macrodomain from rubella virus nonstructural protein p150, with and without ADP-ribose binding. The overall fold is most similar to macroD-type macrodomains from various nonviral species. The specific composition and structure of the residues that coordinate ADP-ribose in the rubella virus macrodomain are most similar to those of macrodomains from alphaviruses. Isothermal calorimetry shows that the rubella virus macrodomain binds ADP-ribose in solution. Enzyme assays show that the rubella virus macrodomain can hydrolyze both mono- and poly-ADP-ribose adducts. Site-directed mutagenesis identifies Asn39 and Cys49 required for mono-ADP-ribosylhydrolase (de-MARylation) activity.IMPORTANCERubella virus remains a global health threat. Rubella infections during pregnancy can cause serious congenital pathology, for which no antiviral treatments are available. Our work demonstrates that, like alpha- and coronaviruses, rubiviruses encode a mono-ADP-ribosylhydrolase with a structurally conserved macrodomain fold to counteract MARylation by poly (ADP-ribose) polymerases (PARPs) in the host innate immune response. Our structural data will guide future efforts to develop novel antiviral therapeutics against rubella or infections with related viruses.

Macro1 domain residue F156: A hallmark of SARS-CoV-2 de-MARylation specificity.

SARS-CoV-2 is a large, enveloped and positive sense single stranded RNA virus. Its genome codes for 16 non-structural proteins. The largest protein of this complex is nsp3, that contains a well conserved Macro1 domain. Viral Macro domains were shown to bind to mono-ADP-ribose (MAR) and poly-ADP-ribose (PAR) in their free form or conjugated to protein substrates. They carry ADP-ribose hydrolase activities implicated in the regulation of innate immunity. SARS-CoV-2 and SARS-CoV show widely different induction and handling of the host interferon response. Herein, we have conducted a mutational study on the key amino-acid residue F156 in SARS-CoV-2, pinpointed by bioinformatic and structural studies, and its cognate residue N157 in SARS-CoV. Our data suggest that the exchange of these residues slightly modifies ADP-ribose binding, but drastically impacts de-MARylation activity. Alanine substitutions at this position hampers PAR binding, abolishes MAR hydrolysis of SARS-CoV-2, and reduces by 70% this activity in the case of SARS-CoV.

PARP12 regulates mouse oocyte meiotic maturation.

Poly(ADP-ribosyl)ation (PARylation) is an important post-translational modification of proteins that involves the transfer of ADP-ribose moieties, and plays important roles in many biological processes including DNA repair, gene expression, RNA processing, ribosome biogenesis, and protein translation. Though it is accepted that PARylation is crucial for oocyte maturation, little is known about how Mono(ADP-ribosyl)ation (MARylation) regulates this process. Here, we report that Parp12, a mon(ADP-ribosyl) transferase of poly(ADP-ribosyl) polymerase (PARP) family, was highly expressed at all stages of oocytes during meiotic maturation. At germinal vesicle (GV) stage, PARP12 was mainly distributed in cytoplasm. Interestingly, PARP12 formed granular aggregation near to spindle poles during metaphase I (MI) and metaphase II (MII). PARP12 depletion results in abnormal spindle organization and chromosome misalignment in mouse oocytes. Chromosome aneuploidy frequency in PARP12 knockdown oocytes was significantly increased. Importantly, PARP12 knockdown triggers activation of spindle assembly checkpoint as shown by active BUBR1 in PARP12-KD MI oocytes. Besides, F actin was significantly attenuated in PARP12-KD MI oocytes which may affect the asymmetric division process. Transcriptomic analysis demonstrated that PARP12 depletion disrupts transcriptome homeostasis. Collectively, our results showed that the maternally expressed mono(ADPribosyl) transferases PARP12 was essential for oocyte meiotic maturation in mouse.

IFN-Induced PARPs-Sensors of Foreign Nucleic Acids?

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.

ADP-Ribosylation in Antiviral Innate Immune Response.

Adenosine diphosphate (ADP)-ribosylation is a reversible post-translational modification catalyzed by ADP-ribosyltransferases (ARTs). ARTs transfer one or more ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to the target substrate and release the nicotinamide (Nam). Accordingly, it comes in two forms: mono-ADP-ribosylation (MARylation) and poly-ADP-ribosylation (PARylation). ADP-ribosylation plays important roles in many biological processes, such as DNA damage repair, gene regulation, and energy metabolism. Emerging evidence demonstrates that ADP-ribosylation is implicated in host antiviral immune activity. Here, we summarize and discuss ADP-ribosylation modifications that occur on both host and viral proteins and their roles in host antiviral response.

Structurally distinct PARP7 inhibitors provide new insights into the function of PARP7 in regulating nucleic acid-sensing and IFN-ß signaling.

The mono-ADP-ribosyltransferase PARP7 has emerged as a key negative regulator of cytosolic NA-sensors of the innate immune system. We apply a rational design strategy for converting a pan-PARP inhibitor into a potent selective PARP7 inhibitor (KMR-206). Consistent with studies using the structurally distinct PARP7 inhibitor RBN-2397, co-treatment of mouse embryonic fibroblasts with KMR-206 and NA-sensor ligands synergistically induced the expression of the type I interferon, IFN-ß. In mouse colon carcinoma (CT-26) cells, KMR-206 alone induced IFN-ß. Both KMR-206 and RBN-2397 increased PARP7 protein levels in CT-26 cells, demonstrating that PARP7's catalytic activity regulates its own protein levels. Curiously, treatment with saturating doses of KMR-206 and RBN-2397 achieved different levels of PARP7 protein, which correlated with the magnitude of type I interferon gene expression. These latter results have important implications for the mechanism of action of PARP7 inhibitors and highlights the usefulness of having structurally distinct chemical probes for the same target.

PARP10 Mediates Mono-ADP-Ribosylation of Aurora-A Regulating G2/M Transition of the Cell Cycle.

Intracellular mono-ADP-ribosyltransferases (mono-ARTs) catalyze the covalent attachment of a single ADP-ribose molecule to protein substrates, thus regulating their functions. PARP10 is a soluble mono-ART involved in the modulation of intracellular signaling, metabolism and apoptosis. PARP10 also participates in the regulation of the G1- and S-phase of the cell cycle. However, the role of this enzyme in G2/M progression is not defined. In this study, we found that genetic ablation, protein depletion and pharmacological inhibition of PARP10 cause a delay in the G2/M transition of the cell cycle. Moreover, we found that the mitotic kinase Aurora-A, a previously identified PARP10 substrate, is actively mono-ADP-ribosylated (MARylated) during G2/M transition in a PARP10-dependent manner. Notably, we showed that PARP10-mediated MARylation of Aurora-A enhances the activity of the kinase in vitro. Consistent with an impairment in the endogenous activity of Aurora-A, cells lacking PARP10 show a decreased localization of the kinase on the centrosomes and mitotic spindle during G2/M progression. Taken together, our data provide the first evidence of a direct role played by PARP10 in the progression of G2 and mitosis, an event that is strictly correlated to the endogenous MARylation of Aurora-A, thus proposing a novel mechanism for the modulation of Aurora-A kinase activity.

Research Progress on Mono-ADP-Ribosyltransferases in Human Cell Biology.

ADP-ribosylation is a well-established post-translational modification that is inherently connected to diverse processes, including DNA repair, transcription, and cell signaling. The crucial roles of mono-ADP-ribosyltransferases (mono-ARTs) in biological processes have been identified in recent years by the comprehensive use of genetic engineering, chemical genetics, and proteomics. This review provides an update on current methodological advances in the study of these modifiers. Furthermore, the review provides details on the function of mono ADP-ribosylation. Several mono-ARTs have been implicated in the development of cancer, and this review discusses the role and therapeutic potential of some mono-ARTs in cancer.

Intracellular mono-ADP-ribosyltransferases at the host-virus interphase.

The innate immune system, the primary defense mechanism of higher organisms against pathogens including viruses, senses pathogen-associated molecular patterns (PAMPs). In response to PAMPs, interferons (IFNs) are produced, allowing the host to react swiftly to viral infection. In turn the expression of IFN-stimulated genes (ISGs) is induced. Their products disseminate the antiviral response. Among the ISGs conserved in many species are those encoding mono-ADP-ribosyltransferases (mono-ARTs). This prompts the question whether, and if so how, mono-ADP-ribosylation affects viral propagation. Emerging evidence demonstrates that some mono-ADP-ribosyltransferases function as PAMP receptors and modify both host and viral proteins relevant for viral replication. Support for mono-ADP-ribosylation in virus-host interaction stems from the findings that some viruses encode mono-ADP-ribosylhydrolases, which antagonize cellular mono-ARTs. We summarize and discuss the evidence linking mono-ADP-ribosylation and the enzymes relevant to catalyze this reversible modification with the innate immune response as part of the arms race between host and viruses.

ADP-Ribosylation Post-Translational Modification: An Overview with a Focus on RNA Biology and New Pharmacological Perspectives.

Cellular functions are regulated through the gene expression program by the transcription of new messenger RNAs (mRNAs), alternative RNA splicing, and protein synthesis. To this end, the post-translational modifications (PTMs) of proteins add another layer of complexity, creating a continuously fine-tuned regulatory network. ADP-ribosylation (ADPr) is an ancient reversible modification of cellular macromolecules, regulating a multitude of key functional processes as diverse as DNA damage repair (DDR), transcriptional regulation, intracellular transport, immune and stress responses, and cell survival. Additionally, due to the emerging role of ADP-ribosylation in pathological processes, ADP-ribosyltransferases (ARTs), the enzymes involved in ADPr, are attracting growing interest as new drug targets. In this review, an overview of human ARTs and their related biological functions is provided, mainly focusing on the regulation of ADP-ribosyltransferase Diphtheria toxin-like enzymes (ARTD)-dependent RNA functions. Finally, in order to unravel novel gene functional relationships, we propose the analysis of an inventory of human gene clusters, including ARTDs, which share conserved sequences at 3' untranslated regions (UTRs).

Ribosome ADP-ribosylation: A mechanism for maintaining protein homeostasis in cancers.

A recent study suggests that ribosome MARylation in cancers maintains proteostasis by reducing protein synthesis and preventing toxic protein aggregation. Mechanistically, NMNAT-2 is a cytosolic NAD+ synthase that supports the catalytic activity of PARP-16, which mediates ribosomal proteins MARylation. Ribosomal protein MARylation regulates polysomes assembly or function through the 3'-untranslated region (3'-UTR) stem-loop secondary structures in mRNAs, resulting in reduced protein synthesis and preventing toxic protein aggregation, thus supporting the growth of cancer cells during accelerated cell growth. When PARP-16 or NMNAT-2 is deleted, the stem-loop element in the 3'-UTRs of mRNAs increases polysome loading, enhances protein synthesis, promotes toxic protein aggregation, leading to inhibited cancer cell growth. Collectively, ribosome MARylation provides us with an exciting and scientific direction for us to understand cancers.

Chemical genetic methodologies for identifying protein substrates of PARPs.

Poly-ADP-ribose-polymerases (PARPs) are a family of 17 enzymes that regulate a diverse range of cellular processes in mammalian cells. PARPs catalyze the transfer of ADP-ribose from NAD+ to target molecules, most prominently amino acids on protein substrates, in a process known as ADP-ribosylation. Identifying the direct protein substrates of individual PARP family members is an essential first step for elucidating the mechanism by which PARPs regulate a particular pathway in cells. Two distinct chemical genetic (CG) strategies have been developed for identifying the direct protein substrates of individual PARP family members. In this review, we discuss the design principles behind these two strategies and how target identification has provided novel insight into the cellular function of individual PARPs and PARP-mediated ADP-ribosylation.

ADP-ribosyltransferases, an update on function and nomenclature.

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.

Noncanonical mono(ADP-ribosyl)ation of zinc finger SZF proteins counteracts ubiquitination for protein homeostasis in plant immunity.

Protein ADP-ribosylation is a reversible post-translational modification that transfers ADP-ribose from NAD+ onto acceptor proteins. Poly(ADP-ribosyl)ation (PARylation), catalyzed by poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolases (PARGs), which remove the modification, regulates diverse cellular processes. However, the chemistry and physiological functions of mono(ADP-ribosyl)ation (MARylation) remain elusive. Here, we report that Arabidopsis zinc finger proteins SZF1 and SZF2, key regulators of immune gene expression, are MARylated by the noncanonical ADP-ribosyltransferase SRO2. Immune elicitation promotes MARylation of SZF1/SZF2 via dissociation from PARG1, which has an unconventional activity in hydrolyzing both poly(ADP-ribose) and mono(ADP-ribose) from acceptor proteins. MARylation antagonizes polyubiquitination of SZF1 mediated by the SH3 domain-containing proteins SH3P1/SH3P2, thereby stabilizing SZF1 proteins. Our study uncovers a noncanonical ADP-ribosyltransferase mediating MARylation of immune regulators and underpins the molecular mechanism of maintaining protein homeostasis by the counter-regulation of ADP-ribosylation and polyubiquitination to ensure proper immune responses.

Ribosome ADP-ribosylation inhibits translation and maintains proteostasis in cancers.

Defects in translation lead to changes in the expression of proteins that can serve as drivers of cancer formation. Here, we show that cytosolic NAD+ synthesis plays an essential role in ovarian cancer by regulating translation and maintaining protein homeostasis. Expression of NMNAT-2, a cytosolic NAD+ synthase, is highly upregulated in ovarian cancers. NMNAT-2 supports the catalytic activity of the mono(ADP-ribosyl) transferase (MART) PARP-16, which mono(ADP-ribosyl)ates (MARylates) ribosomal proteins. Depletion of NMNAT-2 or PARP-16 leads to inhibition of MARylation, increased polysome association and enhanced translation of specific mRNAs, aggregation of their translated protein products, and reduced growth of ovarian cancer cells. Furthermore, MARylation of the ribosomal proteins, such as RPL24 and RPS6, inhibits polysome assembly by stabilizing eIF6 binding to ribosomes. Collectively, our results demonstrate that ribosome MARylation promotes protein homeostasis in cancers by fine-tuning the levels of protein synthesis and preventing toxic protein aggregation.

Characterization of PARP6 Function in Knockout Mice and Patients with Developmental Delay.

PARP6, a member of a family of enzymes (17 in humans) known as poly-ADP-ribose polymerases (PARPs), is a neuronally enriched PARP. While previous studies from our group show that Parp6 is a regulator of dendrite morphogenesis in rat hippocampal neurons, its function in the nervous system in vivo is poorly understood. Here, we describe the generation of a Parp6 loss-of-function mouse model for examining the function of Parp6 during neurodevelopment in vivo. Using CRISPR-Cas9 mutagenesis, we generated a mouse line that expressed a Parp6 truncated variant (Parp6TR) in place of Parp6WT. Unlike Parp6WT, Parp6TR is devoid of catalytic activity. Homozygous Parp6TR do not exhibit obvious neuromorphological defects during development, but nevertheless die perinatally. This suggests that Parp6 catalytic activity is important for postnatal survival. We also report PARP6 mutations in six patients with several neurodevelopmental disorders, including microencephaly, intellectual disabilities, and epilepsy. The most severe mutation in PARP6 (C563R) results in the loss of catalytic activity. Expression of Parp6C563R in hippocampal neurons decreases dendrite morphogenesis. To gain further insight into PARP6 function in neurons we also performed a BioID proximity labeling experiment in hippocampal neurons and identified several microtubule-binding proteins (e.g., MAP-2) using proteomics. Taken together, our results suggest that PARP6 is an essential microtubule-regulatory gene in mice, and that the loss of PARP6 catalytic activity has detrimental effects on neuronal function in humans.

Uncovering the Invisible: Mono-ADP-ribosylation Moved into the Spotlight.

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.

PARP10 Multi-Site Auto- and Histone MARylation Visualized by Acid-Urea Gel Electrophoresis.

Poly-ADP-ribose polymerase (PARP)-family ADP-ribosyltransferases function in various signaling pathways, predominantly in the nucleus and cytosol. Although PARP inhibitors are in clinical practice for cancer therapy, the enzymatic activities of individual PARP family members are yet insufficiently understood. We studied PARP10, a mono-ADP-ribosyltransferase and potential drug target. Using acid-urea gel electrophoresis, we found that the isolated catalytic domain of PARP10 auto-ADP-ribosylates (MARylates) at eight or more acceptor residues. We isolated individual species with either singular or several modifications and then analyzed them by mass spectrometry. The results confirmed multi-site MARylation in a random order and identified four acceptor residues. The mutagenesis of singular acceptor residues had a minor impact on the overall auto-MARylation level and no effect on the MARylation of histone H3.1. Together, our results suggest that PARP10 automodification may have functions in the regulation of intramolecular or partner binding events, rather than of its enzymatic catalysis. This contributes to a better understanding of PARP10 functions, and, in the long run, to gauging the consequences of PARP inhibitor actions.

Behavioural Characterisation of Macrod1 and Macrod2 Knockout Mice.

Adenosine diphosphate ribosylation (ADP-ribosylation; ADPr), the addition of ADP-ribose moieties onto proteins and nucleic acids, is a highly conserved modification involved in a wide range of cellular functions, from viral defence, DNA damage response (DDR), metabolism, carcinogenesis and neurobiology. Here we study MACROD1 and MACROD2 (mono-ADP-ribosylhydrolases 1 and 2), two of the least well-understood ADPr-mono-hydrolases. MACROD1 has been reported to be largely localized to the mitochondria, while the MACROD2 genomic locus has been associated with various neurological conditions such as autism, attention deficit hyperactivity disorder (ADHD) and schizophrenia; yet the potential significance of disrupting these proteins in the context of mammalian behaviour is unknown. Therefore, here we analysed both Macrod1 and Macrod2 gene knockout (KO) mouse models in a battery of well-defined, spontaneous behavioural testing paradigms. Loss of Macrod1 resulted in a female-specific motor-coordination defect, whereas Macrod2 disruption was associated with hyperactivity that became more pronounced with age, in combination with a bradykinesia-like gait. These data reveal new insights into the importance of ADPr-mono-hydrolases in aspects of behaviour associated with both mitochondrial and neuropsychiatric disorders.