Virus–Host Interplay Between Poly (ADP-Ribose) Polymerase 1 and Oncogenic Gammaherpesviruses

The gammaherpesviruses, include the Epstein–Barr virus, Kaposi’s sarcoma-associated herpesvirus, and murine gammaherpesvirus 68. They establish latent infection in the B lymphocytes and are associated with various lymphoproliferative diseases and tumors. The poly (ADP-ribose) polymerase-1 (PARP1), also called ADP-ribosyltransferase diphtheria-toxin-like 1 (ARTD1) is a nuclear enzyme that catalyzes the transfer of the ADP-ribose moiety to its target proteins and participates in important cellular activities, such as the DNA-damage response, cell death, transcription, chromatin remodeling, and inflammation. In gammaherpesvirus infection, PARP1 acts as a key regulator of the virus life cycle: lytic replication and latency. These viruses also develop various strategies to regulate PARP1, facilitating their replication. This review summarizes the roles of PARP1 in the viral life cycle as well as the viral modulation of host PARP1 activity and discusses the implications. Understanding the interactions between the PARP1 and oncogenic gammaherpesviruses may lead to the identification of effective therapeutic targets for the associated diseases.

Mono-ADP-ribosylation sites of human CD73 inhibit its adenosine-generating enzymatic activity

CD73-derived adenosine plays a major role in damage-induced tissue responses by inhibiting inflammation. Damage-associated stimuli, such as hypoxia and mechanical stress, induce the cellular release of ATP and NAD+ and upregulate the expression of the nucleotide-degrading purinergic ectoenzyme cascade, including adenosine-generating CD73. Extracellular NAD+ also serves as substrate for mono-ADP-ribosylation of cell surface proteins, which in human cells is mediated by ecto-ADP-ribosyltransferase 1 (ARTC1). Here we explored, whether human CD73 enzymatic activity is regulated by mono-ADP-ribosylation, using recombinant human CD73 in the presence of ARTC1 with etheno-labelled NAD+ as substrate. Multi-colour immunoblotting with an anti-etheno-adenosine antibody showed ARTC1-mediated transfer of ADP-ribose together with the etheno label to CD73. HPLC analysis of the enzymatic activity of in vitro-ribosylated CD73 revealed strong inhibition of adenosine generation in comparison to non-ribosylated CD73. Mass spectrometry of in vitro-ribosylated CD73 identified six ribosylation sites. 3D model analysis indicated that three of them (R328, R354, R545) can interfere with CD73 enzymatic activity. Our study identifies human CD73 as target for ARTC1-mediated mono-ADP-ribosylation, which can profoundly modulate its adenosine-generating activity. Thus, in settings with enhanced release of NAD+ as substrate for ARTC1, assessment of CD73 protein expression in human tissues may not be predictive of adenosine formation resulting in anti-inflammatory activity.

Crystal structures and functional analysis of the ZnF5-WWE1-WWE2 region of PARP13/ZAP define a new mode of engaging poly(ADP-ribose)

PARP13/ZAP acts against multiple viruses through recognizing and promoting degradation of cytoplasmic viral mRNA. PARP13 has four N-terminal Zn-finger motifs that bind CG-rich nucleotide sequences, and a C-terminal ADP ribosyltransferase fold similar to other PARPs. A central region predicted to contain a fifth Zn-finger and two tandem WWE domains is implicated in binding poly(ADP-ribose); however, there are limited insights into the structure and function of this PARP13 region (ZnF5-WWE1-WWE2). Here, we present crystal structures of ZnF5-WWE1-WWE2 from mouse PARP13 in complex with ADP-ribose and with ATP. ZnF5-WWE1-WWE2 crystallized as a dimer with major contacts formed between WWE1 and WWE2 originating from different monomers, indicative of a more compact monomeric arrangement of the tandem WWE domains. Solution scattering experiments and biophysical analysis indicated a monomer in solution, suggesting that the crystal dimer represents domain swapping that could potentially represent a PARP13 conformation assumed when signaling viral RNA detection. The crystal structure and binding studies demonstrate that WWE2 interacts with ADP-ribose and ATP, whereas WWE1 does not have a functional binding site. The shape of the WWE2 binding pocket disfavors interaction with the ribose-ribose linkage of poly(ADP-ribose). Binding studies with poly(ADP-ribose) ligands indicate that WWE2 serves as an anchor for preferential binding to the terminal end of poly(ADP-ribose), and the composite structure of ZnF5-WWE1-WWE2 forms an extended surface to engage polymer chains of ADP-ribose. This model represents a novel mode of poly(ADP-ribose) recognition and provides a structural framework for investigating poly(ADP-ribose) impact on PARP13 function.

Structural organization of erythrocyte membrane microdomains and their relation with malaria susceptibility

Cholesterol-rich microdomains are membrane compartments characterized by specific lipid and protein composition. These dynamic assemblies are involved in several biological processes, including infection by intracellular pathogens. This work provides a comprehensive analysis of the composition of human erythrocyte membrane microdomains. Based on their floating properties, we also categorized the microdomain-associated proteins into clusters. Interestingly, erythrocyte microdomains include the vast majority of the proteins known to be involved in invasion by the malaria parasite Plasmodium falciparum. We show here that the Ecto-ADP-ribosyltransferase 4 (ART4) and Aquaporin 1 (AQP1), found within one specific cluster, containing the essential host determinant CD55, are recruited to the site of parasite entry and then internalized to the newly formed parasitophorous vacuole membrane. By generating null erythroid cell lines, we showed that one of these proteins, ART4, plays a role in P. falciparum invasion. We also found that genetic variants in both ART4 and AQP1 are associated with susceptibility to the disease in a malaria-endemic population.

ADP-ribosyl transferase activity and gamma radiation cytotoxicity of Pseudomonas aeruginosa exotoxin A

This work explores the ADP-ribosyltransferase activity of Pseudomonas (P.) aeruginosa exotoxin A using the guanyl hydrazone derivative, nitrobenzylidine aminoguanidine (NBAG) and the impact of gamma radiation on its efficacy. Unlike the conventional detection methods, NBAG was used as the acceptor of ADP ribose moiety instead of wheat germ extract elongation factor 2. Exotoxin A was extracted from P. aeruginosa clinical isolates and screened for toxA gene using standard PCR. NBAG was synthesized using aminoguanidine bicarbonate and 4-nitrobenzaldehyde and its identity has been confirmed by UV, FTIR, Mass and 13C-NMR spectroscopy. The ADP-ribosyl transferase activity of exotoxin A on NBAG in the presence of Nicotinamide adenine dinucleotide (NAD+) was recorded using UV spectroscopy and HPLC. In vitro ADP-ribosyl transferase activity of exotoxin A protein extract was also explored by monitoring its cytotoxicity on Hep-2 cells using sulforhodamine B cytotoxicity assay. Bacterial broths were irradiated at 5, 10, 15, 24 Gy and exotoxin A protein extract activity were assessed post exposure. Exotoxin A extract exerted an ADP-ribosyltransferase ability which was depicted by the appearance of a new ʎmax after the addition of exotoxin A to NBAG/NAD+ mixture, fragmentation of NAD+ and development of new peaks in HPLC chromatograms. Intracellular enzyme activity was confirmed by the prominent cytotoxic effects of exotoxin A extract on cultured cells. In conclusion, the activity of Exotoxin A can be monitored via its ADP-ribosyltransferase activity and low doses of gamma radiation reduced its activity. Therefore, coupling radiotherapy with exotoxin A in cancer therapy should be carefully monitored.

Structural and Functional Characterization of Legionella pneumophila Effector MavL

Legionella pneumophila is a Gram-negative intracellular pathogen that causes Legionnaires' disease in elderly or immunocompromised individuals. This bacterium relies on the Dot/Icm (Defective in organelle trafficking/Intracellular multiplication) Type IV Secretion System (T4SS) and a large (>330) set of effector proteins to colonize the host cell. The structural variability of these effectors allows them to disrupt many host processes. Herein, we report the crystal structure of MavL to 2.65 Å resolution. MavL adopts an ADP-ribosyltransferase (ART) fold and contains the distinctive ligand-binding cleft of ART proteins. Indeed, MavL binds ADP-ribose with Kd of 13 µM. Structural overlay of MavL with poly-(ADP-ribose) glycohydrolases (PARGs) revealed a pair of aspartate residues in MavL that align with the catalytic glutamates in PARGs. MavL also aligns with ADP-ribose “reader” proteins (proteins that recognize ADP-ribose). Since no glycohydrolase activity was observed when incubated in the presence of ADP-ribosylated PARP1, MavL may play a role as a signaling protein that binds ADP-ribose. An interaction between MavL and the mammalian ubiquitin-conjugating enzyme UBE2Q1 was revealed by yeast two-hybrid and co-immunoprecipitation experiments. This work provides structural and molecular insights to guide biochemical studies aimed at elucidating the function of MavL. Our findings support the notion that ubiquitination and ADP-ribosylation are global modifications exploited by L. pneumophila.

Interplay between ADP-ribosyltransferases and essential cell signaling pathways controls cellular responses

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.

Genomic epidemiology of rifampicin ADP-ribosyltransferase (Arr) in the Bacteria domain

Arr is an ADP-ribosyltransferase enzyme primarily reported in association with rifamycin resistance, which has been used to treat tuberculosis in addition to Gram-positive infections and, recently, pan-resistant Gram-negative bacteria. The arr gene was initially identified on the Mycolicibacterium smegmatis chromosome and later on Proteobacteria plasmids. This scenario raised concerns on the distribution and spread of arr, considering the Bacteria domain. Based on 198,082 bacterial genomes/metagenomes, we performed in silico analysis, including phylogenetic reconstruction of Arr in different genomic contexts. Besides, new arr alleles were evaluated by in vitro analysis to assess their association with rifampin resistance phenotype. The arr gene was prevalent in thousands of chromosomes and in hundreds of plasmids from environmental and clinical bacteria, mainly from the phyla Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes. Furthermore, this gene was identified in other and new genomic contexts. Interestingly, Arr sequences associated with rifampin resistance were distributed across all phylogeny, indicating that, despite the diversity, their association with rifampin resistance phenotype were maintained. In fact, we found that the key residues were highly conserved. In addition, other analyzes have raised evidence of another Arr function, which is related to guanidine metabolism. Finally, this scenario as a whole also suggested the Actinobacteria phylum as a potential ancestral source of arr within the Bacteria domain.

The DarTG toxin-antitoxin system provides phage defense by ADP-ribosylating viral DNA

Toxin-antitoxin (TA) systems are broadly distributed, yet poorly conserved, genetic elements whose biological functions are unclear and controversial. Some TA systems may provide bacteria with immunity to infection by their ubiquitous viral predators, the bacteriophage. To identify TA systems that protect E. coli MG1655 against phage, we searched for those frequently encoded near known phage defense genes in bacterial genomes. Two of the systems tested provide strong protection against phage infection and are homologs of DarTG, a recently discovered family of TA systems whose biological functions and natural activating conditions were unclear. We demonstrate that phage infection triggers the release of DarT toxin, a DNA ADP-ribosyltransferase, to modify viral DNA and prevent replication, thereby blocking the production of mature virions. Phages can evolve to overcome DarTG defense either through mutations to their DNA polymerase or to an anti-DarT factor, gp61.2, encoded by many T-even phages. Collectively, our results indicate that phage defense may be a common function for TA systems and reveal the mechanism by which DarTG systems inhibit phage infection.

Legionella pneumophila modulates host energy metabolism by ADP-ribosylation of ADP/ATP translocases

The intracellular pathogen Legionella pneumophila delivers more than 330 effectors into host cells by its Dot/Icm secretion system. Those effectors direct the biogenesis of the Legionella-containing vacuole (LCV) that permits its intracellular survival and replication. It has long been documented that the LCV is associated with mitochondria and a number of Dot/Icm effectors have been shown to target to this organelle. Yet, the biochemical function and host cell target of most of these effectors remain unknown. Here, we found that the Dot/Icm substrate Ceg3 (Lpg0080) is a mono-ADP-ribosyltransferase that localizes to the mitochondria in host cells where it attacks ADP/ATP translocases by ADP-ribosylation, and blunts their ADP/ATP exchange activity. The modification occurs on the second arginine residue in the -RRRMMM- element, which is conserved among all known ADP/ATP carriers from different organisms. Our results reveal modulation of host energy metabolism as a virulence mechanism for L. pneumophila.