ADP-ribosylation is a reversible post-translational modification with wide-ranging biological functions in all kingdoms of life. A variety of enzymes use NAD(+) to transfer either single or multiple ADP-ribose (ADPr) moieties onto distinct amino acid substrates, often in response to DNA damage or other stresses. Poly-ADPr-glycohydrolase readily reverses poly-ADP-ribosylation induced by the DNA-damage sensor PARP1 and other enzymes, but it does not remove the most proximal ADPr linked to the target amino acid. Searches for enzymes capable of fully reversing cellular mono-ADP-ribosylation back to the unmodified state have proved elusive, which leaves a gap in the understanding of this modification. Here, we identify a family of macrodomain enzymes present in viruses, yeast and animals that reverse cellular ADP-ribosylation by acting on mono-ADP-ribosylated substrates. Our discoveries establish the complete reversibility of PARP-catalyzed cellular ADP-ribosylation as a regulatory modification.

Adenosine diphosphate (ADP)-ribosylation is a post-translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP-ribosylation reactions are the poly(ADPribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP-ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP-ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP-ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP-interacting protein that removes mono(ADP-ribosyl)ation on glutamate amino acid residues in PARP-modified proteins. X-ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl-(ADP-ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP-ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans.

ADP-ribosylation is a common posttranslational modification that may have antiviral properties and impact innate immunity. To regulate this activity, macrodomain proteins enzymatically remove covalently attached ADP-ribose from protein targets. All members of the Coronavirinae, a subfamily of positive-sense RNA viruses, contain a highly conserved macrodomain within nonstructural protein 3 (nsp3). However, its function or targets during infection remain unknown. We identified several macrodomain mutations that greatly reduced nsp3’s de-ADP-ribosylation activity in vitro. Next, we created recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) strains with these mutations. These mutations led to virus attenuation and a modest reduction of viral loads in infected mice, despite normal replication in cell culture. Further, macrodomain mutant virus elicited an early, enhanced interferon (IFN), interferon-stimulated gene (ISG), and proinflammatory cytokine response in mice and in a human bronchial epithelial cell line. Using a coinfection assay, we found that inclusion of mutant virus in the inoculum protected mice from an otherwise lethal SARS-CoV infection without reducing virus loads, indicating that the changes in innate immune response were physiologically significant. In conclusion, we have established a novel function for the SARS-CoV macrodomain that implicates ADP-ribose in the regulation of the innate immune response and helps to demonstrate why this domain is conserved in CoVs.

SummaryThe discovery and study of toxin-antitoxin (TA) systems helps us advance our understanding of the strategies prokaryotes employ to regulate cellular processes related to the general stress response, such as defense against phages, growth control, biofilm formation, persistence, and programmed cell death. Here we identify and characterize a TA system found in various bacteria, including the global pathogen Mycobacterium tuberculosis. The toxin of the system (DarT) is a domain of unknown function (DUF) 4433, and the antitoxin (DarG) a macrodomain protein. We demonstrate that DarT is an enzyme that specifically modifies thymidines on single-stranded DNA in a sequence-specific manner by a nucleotide-type modification called ADP-ribosylation. We also show that this modification can be removed by DarG. Our results provide an example of reversible DNA ADP-ribosylation, and we anticipate potential therapeutic benefits by targeting this enzyme-enzyme TA system in bacterial pathogens such as M. tuberculosis.

The poly(ADP-ribose) polymerases (PARPs) are a major family of enzymes capable of modifying proteins by ADP-ribosylation. Due to the large size and diversity of this family, PARPs affect almost every aspect of cellular life and have fundamental roles in DNA repair, transcription, heat shock and cytoplasmic stress responses, cell division, protein degradation, and much more. In the past decade, our understanding of the PARP enzymatic mechanism and activation, as well as regulation of ADP-ribosylation signals by the readers and erasers of protein ADP-ribosylation, has been significantly advanced by the emergence of new structural data, reviewed herein, which allow for better understanding of the biological roles of this widespread post-translational modification.

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Contraction of the large Diversity in the Ig Ab repertoire is achieved through V(D)J recombination, a lineage-specific process that is highly regulated during B cell development. IgH rearrangement in pro-B cells begins with D H to J H rearrangement followed by rearrangement of a V H gene segment to D H J H . The IgH is assembled before the L chains, and Ig rearrangement precedes Ig rearrangement. Strict regulation of accessibility allows for the lineage-specific and developmentally ordered rearrangement of V(D)J gene segments. The mechanisms controlling V H to D H J H rearrangement are exceptionally complex because Ͼ100 functional murine V H genes span a 2.5 Mb region. Likewise, the 96 functional V genes cover 3.1 Mb. The question arises of how all the V genes acquire access to the small J cluster (Ͻ2 kb) in either the Igh or Ig loci. Three-dimensional fluorescence in situ hybridization studies have demonstrated that the Igh and Ig loci undergo significant contraction to position gene segments in proximity for rearrangement at the appropriate time for rearrangement (1-4). In pro-B cells, the V H genes are brought into close proximity to D H gene segments via multiple loop structures, thus facilitating rearrangement through these long-range chromosomal interactions (4). The contraction and looping of the loci raises the question of which nuclear factors could be controlling these interactions. One potential nuclear protein that participates in long-range chromosomal interactions is CCCTC-binding factor (CTCF). 3CTCF is a ubiquitously expressed 11-zinc finger nuclear protein that is associated with all known vertebrate insulators (5). Chromatin insulators or boundary elements create distinct chromosomal domains preventing outside influences on the insulated region (5). The enhancer-blocking activity of CTCF prevents the interactions between enhancers and promoters separated by the insulator. Global mapping of CTCF binding has shown that CTCF binds in regions that could separate different chromosomal domains, consistent with the idea that CTCF may insulate the spread of repressive chromatin modifications into neighboring active domains (6 -8). One of the ways that CTCF may function as an insulator and regulate gene expression is through the facilitation of long-range intrachromosomal and interchromosomal looping (9 -11).CTCF has been reported to associate with a number of factors including YY1 (12), the chromodomain helicase CHD8 (13), and cohesin subunits (14 -17). Cohesin proteins have an established role of facilitating cohesion of sister chromatids during cell division (18)

Poly(ADP‐ribosyl)ation is involved in the regulation of a variety of cellular pathways, including, but not limited to, transcription, chromatin, DNA damage and other stress signalling. Similar to other tightly regulated post‐translational modifications, poly(ADP‐ribosyl)ation employs ‘writers’, ‘readers’ and ‘erasers’ to confer regulatory functions. The generation of poly(ADP‐ribose) is catalyzed by poly(ADP‐ribose) polymerase enzymes, which use NAD+ as a cofactor to sequentially transfer ADP‐ribose units generating long polymers, which, in turn, can affect protein function or serve as a recruitment platform for additional factors. Historically, research has focused on poly(ADP‐ribose) generation pathways, with knowledge about PAR recognition and degradation lagging behind. Over recent years, several discoveries have significantly furthered our understanding of poly(ADP‐ribose) recognition and, even more so, of poly(ADP‐ribose) degradation. In this review, we summarize current knowledge about the protein modules recognizing poly(ADP‐ribose) and discuss the newest developments on the complete reversibility of poly(ADP‐ribosyl)ation.