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.

NAD+ is not utilized as a co-factor for DNA ligation by human DNA ligase IV.

As nucleotidyl transferases, formation of a covalent enzyme-adenylate intermediate is a common first step of all DNA ligases. While it has been shown that eukaryotic DNA ligases utilize ATP as the adenylation donor, it was recently reported that human DNA ligase IV can also utilize NAD+ and, to a lesser extent ADP-ribose, as the source of the adenylate group and that NAD+, unlike ATP, enhances ligation by supporting multiple catalytic cycles. Since this unexpected finding has significant implications for our understanding of the mechanisms and regulation of DNA double strand break repair, we attempted to confirm that NAD+ and ADP-ribose can be used as co-factors by human DNA ligase IV. Here, we provide evidence that NAD+ does not enhance ligation by pre-adenylated DNA ligase IV, indicating that this co-factor is not utilized for re-adenylation and subsequent cycles of ligation. Moreover, we find that ligation by de-adenylated DNA ligase IV is dependent upon ATP not NAD+ or ADP-ribose. Thus, we conclude that human DNA ligase IV cannot use either NAD+ or ADP-ribose as adenylation donor for ligation.

Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins.

Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the ADP-ribose binding domain. This forces the proximal ribose to rotate within the binding pocket and, as a consequence, improves engineered Af1521 ADPr-binding affinity. Its use in our proteomic ADP-ribosylome workflow increases the ADP-ribosylated protein identification rates and yields greater ADP-ribosylome coverage. Furthermore, generation of an engineered Af1521 Fc fusion protein confirms the improved detection of cellular ADP-ribosylation by immunoblot and immunofluorescence. Thus, this engineered isoform of Af1521 can also serve as a valuable tool for the analysis of cellular ADP-ribosylation under in vivo conditions.

Functional Interplay between Histone H2B ADP-Ribosylation and Phosphorylation Controls Adipogenesis.

Although ADP-ribosylation of histones by PARP-1 has been linked to genotoxic stress responses, its role in physiological processes and gene expression has remained elusive. We found that NAD+-dependent ADP-ribosylation of histone H2B-Glu35 by small nucleolar RNA (snoRNA)-activated PARP-1 inhibits AMP kinase-mediated phosphorylation of adjacent H2B-Ser36, which is required for the proadipogenic gene expression program. The activity of PARP-1 on H2B requires NMNAT-1, a nuclear NAD+ synthase, which directs PARP-1 catalytic activity to Glu and Asp residues. ADP-ribosylation of Glu35 and the subsequent reduction of H2B-Ser36 phosphorylation inhibits the differentiation of adipocyte precursors in cultured cells. Parp1 knockout in preadipocytes in a mouse lineage-tracing genetic model increases adipogenesis, leading to obesity. Collectively, our results demonstrate a functional interplay between H2B-Glu35 ADP-ribosylation and H2B-Ser36 phosphorylation that controls adipogenesis.

Selective profiling of N- and C-terminal nucleotide-binding sites in a TRPM2 channel.

Transient receptor potential melastatin 2 (TRPM2) is a homotetrameric Ca2+-permeable cation channel important for the immune response, body temperature regulation, and insulin secretion, and is activated by cytosolic Ca2+ and ADP ribose (ADPR). ADPR binds to two distinct locations, formed by large N- and C-terminal cytosolic domains, respectively, of the channel protein. In invertebrate TRPM2 channels, the C-terminal site is not required for channel activity but acts as an active ADPR phosphohydrolase that cleaves the activating ligand. In vertebrate TRPM2 channels, the C-terminal site is catalytically inactive but cooperates with the N-terminal site in channel activation. The precise functional contributions to channel gating and the nucleotide selectivities of the two sites in various species have not yet been deciphered. For TRPM2 of the sea anemone Nematostella vectensis (nvTRPM2), catalytic activity is solely attributable to the C-terminal site. Here, we show that nvTRPM2 channel gating properties remain unaltered upon deletion of the C-terminal domain, indicating that the N-terminal site is single-handedly responsible for channel gating. Exploiting such functional independence of the N- and C-terminal sites, we selectively measure their affinity profiles for a series of ADPR analogues, as reflected by apparent affinities for channel activation and catalysis, respectively. Using site-directed mutagenesis, we confirm that the same N-terminal site observed in vertebrate TRPM2 channels was already present in ancient cnidarians. Finally, by characterizing the functional effects of six amino acid side chain truncations in the N-terminal site, we provide first insights into the mechanistic contributions of those side chains to TRPM2 channel gating.

Both ADP-Ribosyl-Binding and Hydrolase Activities of the Alphavirus nsP3 Macrodomain Affect Neurovirulence in Mice.

Macrodomain (MD), a highly conserved protein fold present in a subset of plus-strand RNA viruses, binds to and hydrolyzes ADP-ribose (ADPr) from ADP-ribosylated proteins. ADPr-binding by the alphavirus nonstructural protein 3 (nsP3) MD is necessary for the initiation of virus replication in neural cells, whereas hydrolase activity facilitates replication complex amplification. To determine the importance of these activities for pathogenesis of alphavirus encephalomyelitis, mutations were introduced into the nsP3 MD of Sindbis virus (SINV), and the effects on ADPr binding and hydrolase activities, virus replication, immune responses, and disease were assessed. Elimination of ADPr-binding and hydrolase activities (G32E) severely impaired in vitro replication of SINV in neural cells and in vivo replication in the central nervous systems of 2-week-old mice with reversion to wild type (WT) (G) or selection of a less compromising change (S) during replication. SINVs with decreased binding and hydrolase activities (G32S and G32A) or with hydrolase deficiency combined with better ADPr-binding (Y114A) were less virulent than WT virus. Compared to the WT, the G32S virus replicated less well in both the brain and spinal cord, induced similar innate responses, and caused less severe disease with full recovery of survivors, whereas the Y114A virus replicated well, induced higher expression of interferon-stimulated and NF-κB-induced genes, and was cleared more slowly from the spinal cord with persistent paralysis in survivors. Therefore, MD function was important for neural cell replication both in vitro and in vivo and determined the outcome from alphavirus encephalomyelitis in mice.IMPORTANCE Viral encephalomyelitis is an important cause of long-term disability, as well as acute fatal disease. Identifying viral determinants of outcome helps in assessing disease severity and developing new treatments. Mosquito-borne alphaviruses infect neurons and cause fatal disease in mice. The highly conserved macrodomain of nonstructural protein 3 binds and can remove ADP-ribose (ADPr) from ADP-ribosylated proteins. To determine the importance of these functions for virulence, recombinant mutant viruses were produced. If macrodomain mutations eliminated ADPr-binding or hydrolase activity, viruses did not grow. If the binding and hydrolase activities were impaired, the viruses grew less well than the wild-type virus, induced similar innate responses, and caused less severe disease, and most of the infected mice recovered. If binding was improved, but hydrolase activity was decreased, the virus replicated well and induced greater innate responses than did the WT, but clearance from the nervous system was impaired, and mice remained paralyzed. Therefore, macrodomain function determined the outcome of alphavirus encephalomyelitis.

Curcumin diminishes cisplatin-induced apoptosis and mitochondrial oxidative stress through inhibition of TRPM2 channel signaling pathway in mouse optic nerve.

Background: Cisplatin (CiSP), a chemotherapeutic agent, is widely used to treat several types of cancers. However, its clinical use is limited due to adverse side effects caused by excessive production of reactive oxygen species (ROS) and death of neurons. The transient receptor potential (TRP) melastatin 2 (TRPM2) cation channel is activated by ADP-ribose (ADPR) and ROS. The protective effect of curcumin (CURCU) against CiSP-induced apoptosis and mitochondrial ROS through inhibition of TRP channels in several types of neuron except optic nerve, was recently reported. The aim of the current study is to clarify the protective effect of CURCU on CiSP-induced mitochondrial oxidative injury and TRPM2 activation in the mice optic nerve and SH-SY5Y human derived neuronal cells.Material and methods: The SH-SY5Y cells and mice were divided into four groups: Control, CURCU, CiSP, and CURCU + CiSP. The mice were treated for 14 days and the cells were incubated with CiSP and CURCU for 24 h.Results: CURCU and PARP-1 inhibitor (PJ34) treatments ameliorated CiSP-induced mitochondrial membrane depolarization, mitochondrial and cytosolic ROS levels and neuronal death in the optic nerve. In the patch-clamp of SH-SY5Y cells and laser confocal microscopy experiments of optic nerve, CURCU and TRPM2 blocker treatments also decreased ADPR-induced TRPM2 currents and cytosolic free calcium ion (Ca2+) concentration, suggesting a suppression of Ca2+ influx and neuronal death.Conclusion: CURCU prevents CiSP-induced optic nerve oxidative injury and cell death by suppressing mitochondrial ROS production via regulating TRPM2 signaling pathways. CURCU may serve as a potential therapeutic target against CiSP-induced toxicity in the optic nerve of CiSP-treated patients.

Forced Self-Modification Assays as a Strategy to Screen MonoPARP Enzymes.

Mono(ADP-ribosylation) (MARylation) and poly(ADP-ribosylation) (PARylation) are posttranslational modifications found on multiple amino acids. There are 12 enzymatically active mono(ADP-ribose) polymerase (monoPARP) enzymes and 4 enzymatically active poly(ADP-ribose) polymerase (polyPARP) enzymes that use nicotinamide adenine dinucleotide (NAD+) as the ADP-ribose donating substrate to generate these modifications. While there are approved drugs and clinical trials ongoing for the enzymes that perform PARylation, MARylation is gaining recognition for its role in immune function, inflammation, and cancer. However, there is a lack of chemical probes to study the function of monoPARPs in cells and in vivo. An important first step to generating chemical probes for monoPARPs is to develop biochemical assays to enable hit finding, and determination of the potency and selectivity of inhibitors. Complicating the development of enzymatic assays is that it is poorly understood how monoPARPs engage their substrates. To overcome this, we have developed a family-wide approach to developing robust high-throughput monoPARP assays where the enzymes are immobilized and forced to self-modify using biotinylated-NAD+, which is detected using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) readout. Herein we describe the development of assays for 12 monoPARPs and 3 polyPARPs and apply them to understand the potency and selectivity of a focused library of inhibitors across this family.

Functional importance of NUDT9H domain and N-terminal ADPR-binding pocket in two species variants of vertebrate TRPM2 channels.

There are at least two different principles of how ADP-ribose (ADPR) induces activation of TRPM2 channels. In human TRPM2, gating requires the C-terminal NUDT9H domain as ADPR-binding module, whereas in sea anemone, NUDT9H is dispensable and binding of ADPR occurs N-terminally. Zebrafish TRPM2 needs both, the N-terminal ADPR-binding pocket and NUDT9H. Our aim was to pinpoint the relative functional contributions of NUDT9H and the N-terminal ADPR-binding pocket in zebrafish TRPM2, to identify fundamental mechanisms of ADPR-directed gating. We show that the NUDT9H domains of human and zebrafish TRPM2 are interchangeable since chimeras generate ADPR-sensitive channels. A point mutation at a highly conserved position within NUDT9H induces loss-of-function in both vertebrate channels. The substrate specificity of zebrafish TRPM2 corresponds to that of sea anemone TRPM2, indicating gating by the proposed N-terminal ADPR-binding pocket. However, a point mutation in this region abolishes ADPR activation also in human TRPM2. These findings provide functional evidence for an uniform N-terminal ADPR-binding pocket in TRPM2 of zebrafish and sea anemone with modified function in human TRPM2. The structural importance of NUDT9H in vertebrate TRPM2 can be associated with a single amino acid residue which is not directly involved in the binding of ADPR.

Bi-allelic ADPRHL2 Mutations Cause Neurodegeneration with Developmental Delay, Ataxia, and Axonal Neuropathy.

ADP-ribosylation is a reversible posttranslational modification used to regulate protein function. ADP-ribosyltransferases transfer ADP-ribose from NAD+ to the target protein, and ADP-ribosylhydrolases, such as ADPRHL2, reverse the reaction. We used exome sequencing to identify five different bi-allelic pathogenic ADPRHL2 variants in 12 individuals from 8 families affected by a neurodegenerative disorder manifesting in childhood or adolescence with key clinical features including developmental delay or regression, seizures, ataxia, and axonal (sensori-)motor neuropathy. ADPRHL2 was virtually absent in available affected individuals' fibroblasts, and cell viability was reduced upon hydrogen peroxide exposure, although it was rescued by expression of wild-type ADPRHL2 mRNA as well as treatment with a PARP1 inhibitor. Our findings suggest impaired protein ribosylation as another pathway that, if disturbed, causes neurodegenerative diseases.

PARPs in genome stability and signal transduction: implications for cancer therapy.

The poly(ADP-ribose) polymerase (PARP) superfamily of enzymes catalyses the ADP-ribosylation (ADPr) of target proteins by using nicotinamide adenine dinucleotide (NAD+) as a donor. ADPr reactions occur either in the form of attachment of a single ADP-ribose nucleotide unit on target proteins or in the form of ADP-ribose chains, with the latter called poly(ADP-ribosyl)ation. PARPs regulate many cellular processes, including the maintenance of genome stability and signal transduction. In this review, we focus on the PARP family members that possess the ability to modify proteins by poly(ADP-ribosyl)ation, namely PARP1, PARP2, Tankyrase-1, and Tankyrase-2. Here, we detail the cellular functions of PARP1 and PARP2 in the regulation of DNA damage response and describe the function of Tankyrases in Wnt-mediated signal transduction. Furthermore, we discuss how the understanding of these pathways has provided some major breakthroughs in the treatment of human cancer.

MacroH2A histone variants limit chromatin plasticity through two distinct mechanisms.

MacroH2A histone variants suppress tumor progression and act as epigenetic barriers to induced pluripotency. How they impart their influence on chromatin plasticity is not well understood. Here, we analyze how the different domains of macroH2A proteins contribute to chromatin structure and dynamics. By solving the crystal structure of the macrodomain of human macroH2A2 at 1.7 Å, we find that its putative binding pocket exhibits marked structural differences compared with the macroH2A1.1 isoform, rendering macroH2A2 unable to bind ADP-ribose. Quantitative binding assays show that this specificity is conserved among vertebrate macroH2A isoforms. We further find that macroH2A histones reduce the transient, PARP1-dependent chromatin relaxation that occurs in living cells upon DNA damage through two distinct mechanisms. First, macroH2A1.1 mediates an isoform-specific effect through its ability to suppress PARP1 activity. Second, the unstructured linker region exerts an additional repressive effect that is common to all macroH2A proteins. In the absence of DNA damage, the macroH2A linker is also sufficient for rescuing heterochromatin architecture in cells deficient for macroH2A.

Vitamin B3 in Health and Disease: Toward the Second Century of Discovery.

This introductory chapter briefly reviews the history, chemistry, and biochemistry of NAD (the term NAD as it is used here refers to both oxidized and reduced forms of the molecule) consuming ADP-ribose transfer enzymes as components of the involvement of vitamin B3 in health and disease.

Monitoring Poly(ADP-Ribosyl)ation in Response to DNA Damage in Live Cells Using Fluorescently Tagged Macrodomains.

Poly(ADP-ribosyl)ation (PARylation) is a dynamic posttranslational modification that is added and removed rapidly at sites of DNA damage. PARylation is important for numerous aspects of DNA repair including chromatin decondensation and protein recruitment. Visualization of PARylation levels after DNA damage induction is generally obtained using traditional immunofluorescent techniques on fixed cells, which results in limited temporal resolution. Here, we describe a microscopy-based method to track ADP-ribosylation at break sites. This method relies on DNA damage induction using a 405 nm FRAP laser on Hoechst-treated cells expressing GFP-tagged PAR-binding proteins, such as macrodomains where the recruitment of the PAR-binder to sites of DNA damage gives an indication of PARylation levels.

Assessment of Intracellular Auto-Modification Levels of ARTD10 Using Mono-ADP-Ribose-Specific Macrodomains 2 and 3 of Murine Artd8.

Mono-ADP-ribosylation is a posttranslational modification, which is catalyzed in cells by certain members of the ADP-ribosyltransferase diphtheria toxin-like family (ARTD) of ADP-ribosyltransferases (aka PARP enzymes). It involves the transfer of a single residue of ADP-ribose (ADPr) from the cofactor NAD+ onto substrate proteins. Although 12 of the 17 members of the ARTD family have been defined as mono-ARTDs in in vitro assays, relatively little is known about their exact cellular functions. A major challenge is the detection of mono-ADP-ribosylated (MARylated) proteins in cells as no antibodies are available that detect exclusively MARylated proteins. As an alternative to classical antibodies, the MAR-specific binding domains macro2 and macro3 of Artd8 can be utilized alone or in combination, to demonstrate intracellular auto-modification levels of ARTD10 in cells in both co-immunoprecipitation and co-localization experiments. Here we demonstrate that different macrodomain constructs of human ARTD8 and murine Artd8, alone or in combination, exert differences with regard to their interaction with ARTD10 in cells. Precisely, while the macrodomains of murine Artd8 interacted with ARTD10 in cells in a MARylation-dependent manner, the macrodomains of human ARTD8 interacted with ARTD10 independent of its catalytic activity. Moreover, we show that a combination of macro2 and macro3 of murine Artd8 was recruited more efficiently to ARTD10 during co-localization experiments compared to the single domains. Therefore, murine Artd8 macrodomain constructs can serve as a tool to evaluate intracellular ARTD10 auto-modification levels using the described methods, while the human ARTD8 macrodomains are less suited because of ADPr-independent binding to ARTD10. Protocols for co-immunoprecipitation and co-localization experiments are described in detail.

In Vitro Techniques for ADP-Ribosylated Substrate Identification.

ADP-ribosylation is a post-translational modification of proteins that has required the development of specific technical approaches for the full definition of its physiological roles and regulation. The identification of the enzymes and specific substrates of this reaction is an instrumental step toward these aims. Here we describe a method for the separation of ADP-ribosylated proteins based on the use of the ADP-ribose-binding macro domain of the thermophilic protein Af1521, coupled to mass spectrometry analysis for protein identification. This method foresees the coupling of the macro domain to resin, an affinity-based pull-down assay, coupled to a specificity step resulting from the clearing of cell lysates with a mutated macro domain unable to bind ADP-ribose. By this method both mono- and poly-ADP-ribosylated proteins have been identified.

Biochemical and Biophysical Assays of PAR-WWE Domain Interactions and Production of iso-ADPr for PAR-Binding Analysis.

The poly(ADP-ribose) polymerase (PARP) family of proteins utilize NAD+ as the substrate to modify protein acceptors with either mono(ADP-ribose) (MAR) or poly(ADP-ribose) (PAR). MAR and PAR have been shown to regulate distinct cellular processes. Iso-ADP-ribose (iso-ADPr) is the smallest internal PAR structural unit containing the characteristic ribose-ribose glycosidic bond formed during poly(ADP-ribosyl)ation. The WWE domain of RNF146 specifically recognizes the iso-ADPr moiety in PAR but does not interact with MAR. This provides a way to distinguish PAR from MAR modification and to isolate PARylated proteins. Iso-ADPr can be used to detect the PAR-specific binding properties of interested proteins. Here we describe the detailed method to generate and purify iso-ADPr and its use in PAR-binding analysis through isothermal titration calorimetry (ITC) analysis.

Generating Protein-Linked and Protein-Free Mono-, Oligo-, and Poly(ADP-Ribose) In Vitro.

ADP-ribosylation is a covalent posttranslational modification of proteins that is catalyzed by various types of ADP-ribosyltransferase (ART) enzymes, including members of the poly(ADP-ribose) polymerase (PARP) family. ADP-ribose (ADPR) modifications can occur as mono(ADP-ribosyl)ation, oligo(ADP-ribosyl)ation, or poly(ADP-ribosyl)ation, depending on the particular ART enzyme catalyzing the reaction, as well as the specific reaction conditions. Understanding the biology of ADP-ribosylation requires facile and robust means of generating and detecting the modification in all of its forms. Here we describe how to generate protein-linked mono(ADP-ribose), oligo(ADP-ribose), and poly(ADP-ribose) (MAR, OAR, and PAR, respectively) in vitro as an automodification of PARPs 1 or 3. First, epitope-tagged PARP-1 (a PARP polyenzyme) and PARP-3 (a PARP monoenzyme) are expressed individually in insect cells using baculovirus expression vectors, and purified using immunoaffinity chromatography. Second, the purified recombinant PARPs are incubated individually in the presence of different concentrations of NAD+ (as a donor of ADPR groups) and sheared DNA (to activate their catalytic activities) resulting in various forms of auto-ADP-ribosylation. Third, the products are confirmed using ADPR detection reagents that can distinguish among MAR, OAR, and PAR. Finally, if desired, the OAR and PAR can be deproteinized. The protein-linked and free MAR, OAR, and PAR generated in these reactions can be used as standards, substrates, or binding partners in a variety of ADPR-related assays.

Mono-ADP-Ribosylation Catalyzed by Arginine-Specific ADP-Ribosyltransferases.

Methods are described for determination of arginine-specific mono-ADP-ribosyltransferase activity of purified proteins and intact cells by monitoring the transfer of ADP-ribose from NAD+ to a model substrate, e.g., arginine, agmatine, and peptide (human neutrophil peptide-1 [HNP1]), and for the nonenzymatic hydrolysis of ADP-ribose-arginine to ornithine, a noncoded amino acid. In addition, preparation of purified ADP-ribosylarginine is included as a control substrate for ADP-ribosylation reactions.

Monitoring the Sensitivity of T Cell Populations Towards NAD+ Released During Cell Preparation.

Mouse T cells express the toxin-related ecto-ADP-ribosyltransferase ARTC2 that catalyzes the posttranslational ADP-ribosylation of cell surface proteins by transferring the ADP-ribose group of its substrate nicotinamide adenine dinucleotide (NAD+) to arginine residues of its target proteins. One well known target of ARTC2 is the ATP-gated P2X7 ion channel. ADP-ribosylation of P2X7 induces gating of the channel, calcium influx, ecto-domain shedding, phosphatidylserine externalization, and finally cell death. Previous studies have shown that the ARTC2 substrate NAD+ is released during T cell preparation. Since P2X7 is differentially expressed among T cell subpopulations, preparation-related ADP-ribosylation has a strong impact on the vitality of T cells that express high levels of P2X7. With this chapter we provide a protocol to monitor the consequences of preparation-related P2X7 ADP-ribosylation on T cells using regulatory T cells as generic T cell subpopulation known to express high levels of P2X7. However, this protocol could be easily adapted to other T cell populations.