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The ADP‐ribosylation of proteins is an important post‐translational modification that occurs in a variety of biological processes, including DNA repair, transcription, chromatin biology and long‐term memory formation. Yet no protein modules are known that specifically recognize the ADP‐ribose nucleotide. We provide biochemical and structural evidence that macro domains are high‐affinity ADP‐ribose binding modules. Our structural analysis reveals a conserved ligand binding pocket among the macro domain fold. Consistently, distinct human macro domains retain their ability to bind ADP‐ribose. In addition, some macro domain proteins also recognize poly‐ADP‐ribose as a ligand. Our data suggest an important role for proteins containing macro domains in the biology of ADP‐ribose.

Mouse caspase-11 and human caspase-4 and caspase-5 recognize cytosolic lipopolysaccharide (LPS) to induce pyroptosis by cleaving the pore-forming protein GSDMD 1 , 2 , 3 , 4 – 5 . This non-canonical inflammasome defends against Gram-negative bacteria 6 , 7 . Shigella flexneri , which causes bacillary dysentery, lives freely within the host cytosol where these caspases reside. However, the role of caspase-11-mediated pyroptosis in S .  flexneri infection is unknown. Here we show that caspase-11 did not protect mice from S .  flexneri infection, in contrast to infection with another cytosolic bacterium, Burkholderia thailandensis 8 . S .  flexneri evaded pyroptosis mediated by caspase-11 or caspase 4 (hereafter referred to as caspase-11/4) using a type III secretion system (T3SS) effector, OspC3. OspC3, but not its paralogues OspC1 and 2, covalently modified caspase-11/4; although it used the NAD + donor, this modification was not ADP-ribosylation. Biochemical dissections uncovered an ADP-riboxanation modification on Arg314 and Arg310 in caspase-4 and caspase-11, respectively. The enzymatic activity was shared by OspC1 and 2, whose ankyrin-repeat domains, unlike that of OspC3, could not recognize caspase-11/4. ADP-riboxanation of the arginine blocked autoprocessing of caspase-4/11 as well as their recognition and cleavage of GSDMD. ADP-riboxanation of caspase-11 paralysed pyroptosis-mediated defence in Shigella -infected mice and mutation of ospC3 stimulated caspase-11- and GSDMD-dependent anti- Shigella humoral immunity, generating a vaccine-like protective effect. Our study establishes ADP-riboxanation of arginine as a bacterial virulence mechanism that prevents LPS-induced pyroptosis. This study reports the identification of a new post-translational modification, termed ADP riboxanation, which is mediated by the Shigella effector OspC3 and inactivates the cytosolic LPS sensing pathway of caspase-4 and caspase-11.

In bacteria, archaea, fungi and plants the Trk, Ktr and HKT ion transporters are key components of osmotic regulation, pH homeostasis and resistance to drought and high salinity. These ion transporters are functionally diverse: they can function as Na + or K + channels and possibly as cation/K + symporters. They are closely related to potassium channels both at the level of the membrane protein and at the level of the cytosolic regulatory domains. Here we describe the crystal structure of a Ktr K + transporter, the KtrAB complex from Bacillus subtilis . The structure shows the dimeric membrane protein KtrB assembled with a cytosolic octameric KtrA ring bound to ATP, an activating ligand. A comparison between the structure of KtrAB–ATP and the structures of the isolated full-length KtrA protein with ATP or ADP reveals a ligand-dependent conformational change in the octameric ring, raising new ideas about the mechanism of activation in these transporters. This study reports the X-ray crystal structure of a Ktr K + transporter; the structure of this KtrAB complex reveals how the dimeric membrane protein KtrB interacts with the cytosolic octameric KtrA regulatory protein. Bacterial potassium transporters characterized K + is essential for many physiological processes and must be concentrated in all living cells for their survival. In bacteria, K + uptake is mediated and regulated by SKT (superfamily of K + transporter) proteins. Two papers in this issue of Nature examine the structure and function of SKT proteins from different sub-families. Ming Zhou and colleagues present the electrophysiological and structural characterization of the complex formed by TrkH and its associated RCK protein, TrkA. Their study suggests a mechanism by which ATP-induced conformational changes in TrkA augment TrkH's activity. Joo Morais-Cabral and colleagues determined the X-ray crystal structure of a Ktr K + transporter; the structure of this KtrAB complex reveals how the dimeric membrane protein KtrB interacts with the cytosolic octameric KtrA regulatory protein.

The poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP) protein family generates ADP-ribose (ADPr) modifications onto target proteins using NAD + as substrate. Based on the composition of three NAD + coordinating amino acids, the H-Y-E motif, each PARP is predicted to generate either poly(ADPr) (PAR) or mono(ADPr) (MAR). However, the reaction product of each PARP has not been clearly defined, and is an important priority since PAR and MAR function via distinct mechanisms. Here we show that the majority of PARPs generate MAR, not PAR, and demonstrate that the H-Y-E motif is not the sole indicator of PARP activity. We identify automodification sites on seven PARPs, and demonstrate that MAR and PAR generating PARPs modify similar amino acids, suggesting that the sequence and structural constraints limiting PARPs to MAR synthesis do not limit their ability to modify canonical amino-acid targets. In addition, we identify cysteine as a novel amino-acid target for ADP-ribosylation on PARPs. The poly(ADP-ribose) polymerase family of enzymes control many aspects of cellular signalling by covalently modifying proteins with either poly- or mono-(ADP-ribose). Vyas et al. catalogue the catalytic specificity of this family, and reveal that the majority of these enzymes generate only mono(ADP-ribose).

Taking PAR apart Proteins can be reversibly modified through the addition of repeating, polymerized ADP-ribose (PAR) subunits catalysed by poly(ADP-ribose) polymerase (PARP). Removal of PAR requires a glycohydrolase (PARG), which cleaves the ribose–ribose bond between subunits. Ivan Ahel and colleagues report that bacteria and fungi have a divergent PARG, which is unrelated to other enzymes that cleave PAR. Its structure, in complex with ADP-ribose and with a PARG inhibitor, and the results of mutational analysis suggest that the mechanism used in mammals and bacteria may be conserved. PARP inhibitors are being developed as pharmaceuticals for diseases including cancer, and this work suggests that small, cell-permeable PARG inhibitors might also be possible drug candidates. Post-translational modification of proteins by poly(ADP-ribosyl)ation regulates many cellular pathways that are critical for genome stability, including DNA repair, chromatin structure, mitosis and apoptosis 1 . Poly(ADP-ribose) (PAR) is composed of repeating ADP-ribose units linked via a unique glycosidic ribose–ribose bond, and is synthesized from NAD by PAR polymerases 1 , 2 . PAR glycohydrolase (PARG) is the only protein capable of specific hydrolysis of the ribose–ribose bonds present in PAR chains; its deficiency leads to cell death 3 , 4 . Here we show that filamentous fungi and a number of bacteria possess a divergent form of PARG that has all the main characteristics of the human PARG enzyme. We present the first PARG crystal structure (derived from the bacterium Thermomonospora curvata ), which reveals that the PARG catalytic domain is a distant member of the ubiquitous ADP-ribose-binding macrodomain family 5 , 6 . High-resolution structures of T. curvata PARG in complexes with ADP-ribose and the PARG inhibitor ADP-HPD, complemented by biochemical studies, allow us to propose a model for PAR binding and catalysis by PARG. The insights into the PARG structure and catalytic mechanism should greatly improve our understanding of how PARG activity controls reversible protein poly(ADP-ribosyl)ation and potentially of how the defects in this regulation are linked to human disease.

Structural and biochemical approaches are used to show how RNF146 activity is allosterically regulated by the binding of poly(ADP-ribose) ligand, and how substrate specificity is achieved with protein poly(ADP-ribosyl)ation and ubiquitination occurring in the same protein complex. PARylation-dependent ubiquitination mechanism PARylation is a post-translational modification in which ADP-ribose polymers are covalently attached to protein targets. One of its many cellular functions is to control the ubiquitination and degradation of cell regulators such as Axin and PTEN. Wenqing Xu and colleagues use structural and biochemical approaches to show how the activity of RNF146, an E3 ligase responsible for PARylation-dependent ubiquitination, is regulated by the binding of PAR ligand and how substrate specificity is achieved with PARylation and ubiquitination occurring in the same protein complex. RNF146 represents a new class of RING E3 ligases, the activity of which can be regulated by ligand binding. Protein poly(ADP-ribosyl)ation (PARylation) has a role in diverse cellular processes such as DNA repair, transcription, Wnt signalling, and cell death 1 , 2 , 3 , 4 , 5 , 6 . Recent studies have shown that PARylation can serve as a signal for the polyubiquitination and degradation of several crucial regulatory proteins, including Axin and 3BP2 (refs 7 , 8 , 9 ). The RING-type E3 ubiquitin ligase RNF146 (also known as Iduna) is responsible for PARylation-dependent ubiquitination (PARdU) 10 , 11 , 12 . Here we provide a structural basis for RNF146-catalysed PARdU and how PARdU specificity is achieved. First, we show that iso -ADP-ribose ( iso -ADPr), the smallest internal poly(ADP-ribose) (PAR) structural unit, binds between the WWE and RING domains of RNF146 and functions as an allosteric signal that switches the RING domain from a catalytically inactive state to an active one. In the absence of PAR, the RING domain is unable to bind and activate a ubiquitin-conjugating enzyme (E2) efficiently. Binding of PAR or iso -ADPr induces a major conformational change that creates a functional RING structure. Thus, RNF146 represents a new mechanistic class of RING E3 ligases, the activities of which are regulated by non-covalent ligand binding, and that may provide a template for designing inducible protein-degradation systems. Second, we find that RNF146 directly interacts with the PAR polymerase tankyrase (TNKS). Disruption of the RNF146–TNKS interaction inhibits turnover of the substrate Axin in cells. Thus, both substrate PARylation and PARdU are catalysed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate–TNKS interaction. We propose that the maintenance of unliganded RNF146 in an inactive state may serve to maintain the stability of the RNF146–TNKS complex, which in turn regulates the homeostasis of PARdU activity in the cell.

Key nuclear processes in eukaryotes, including DNA replication, repair, and gene regulation, require extensive chromatin remodeling catalyzed by energy-consuming enzymes. It remains unclear how the ATP demands of such processes are met in response to rapid stimuli. We analyzed this question in the context of the massive gene regulation changes induced by progestins in breast cancer cells and found that ATP is generated in the cell nucleus via the hydrolysis of poly(ADP-ribose) to ADP-ribose. In the presence of pyrophosphate, ADP-ribose is used by the pyrophosphatase NUDIX5 to generate nuclear ATP. The nuclear source of ATP is essential for hormone-induced chromatin remodeling, transcriptional regulation, and cell proliferation.

Platelet-activating factor (PAF) is a potent mediator of inflammation that plays a crucial role in atherosclerosis. The purpose of this study was to evaluate the effect of a dietary supplement containing mainly plant extracts on PAF actions and metabolism in healthy volunteers. A double-blind, placebo-controlled, 8 weeks’ duration study was performed. Healthy volunteers were randomly allocated into the supplement or the placebo group and fifty-eight of them completed the study. The supplement contained plant extracts (Aloe gel, grape juice, Polygonum cuspidatum) and vitamins. The activities of PAF metabolic enzymes: the two isoforms of acetyl-CoA:lyso-PAF acetyltransferase, cytidine 5’-diphospho-choline:1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-cholinephosphotransferase) and platelet-activating factor–acetylhydrolase (PAF-AH) in leucocytes and lipoprotein associated phospholipase-A2 in plasma were measured along with several markers of endothelial function. Platelet aggregation against PAF, ADP and thrombin receptor activating peptide was measured in human platelet-rich plasma by light transmission aggregometry. No difference was observed on soluble vascular cell adhesion molecule-1, sP-selectin and IL-6 levels at the beginning or during the study period between the two groups. Concerning PAF metabolism enzymes’ activity, no difference was observed at baseline between the groups. PAF-AH activity was only increased in the supplement group at 4 and 8 weeks compared with baseline levels. In addition, supplement consumption led to lower platelet sensitivity against PAF and ADP compared with baseline levels. However, a trial effect was only observed when platelets were stimulated by PAF. In conclusion, supplementation with plant extracts and vitamins ameliorates platelet aggregation primarily against PAF and secondarily against ADP and affects PAF catabolism by enhancing PAF-acetylhydrolase activity in healthy subjects.

The poly(ADP-ribose) polymerase (PARP) family comprises 17 enzymes, which generate poly(ADP-ribose) and/or mono(ADP-ribose) (MAR) that can modify target protein function and can function as a signalling scaffold. These modifications may have various roles in cancer and, as discussed in this Opinion article, inhibitors of MARylation in particular may warrant investigation as anticancer drugs. Poly(ADP-ribose) polymerases (PARPs) modify target proteins post-translationally with poly(ADP-ribose) (PAR) or mono(ADP-ribose) (MAR) using NAD + as substrate. The best-studied PARPs generate PAR modifications and include PARP1 and the tankyrase PARP5A, both of which are targets for cancer therapy with inhibitors in either clinical trials or preclinical development. There are 15 additional PARPs, most of which modify proteins with MAR, and their biology is less well understood. Recent data identify potentially cancer-relevant functions for these PARPs, which indicates that we need to understand more about these PARPs to effectively target them.

ADP-ribosylation (ADPRylation) is a reversible post-translation modification resulting in the covalent attachment of ADP-ribose (ADPR) moieties on substrate proteins. Naturally occurring protein motifs and domains, including WWEs, PBZs, and macrodomains, act as ‘readers’ for protein-linked ADPR. Although recombinant, antibody-like ADPR detection reagents containing these readers have facilitated the detection of ADPR, they are limited in their ability to capture the dynamic nature of ADPRylation. Herein, we describe and characterize a set of poly(ADP-ribose) (PAR) Trackers (PAR-Ts)—optimized dimerization-dependent or split-protein reassembly PAR sensors in which a naturally occurring PAR binding domain, WWE, was fused to both halves of dimerization-dependent GFP (ddGFP) or split Nano Luciferase (NanoLuc), respectively. We demonstrate that these new tools allow the detection and quantification of PAR levels in extracts, living cells, and living tissues with greater sensitivity, as well as temporal and spatial precision. Importantly, these sensors detect changes in cellular ADPR levels in response to physiological cues (e.g., hormone-dependent induction of adipogenesis without DNA damage), as well as xenograft tumor tissues in living mice. Our results indicate that PAR Trackers have broad utility for detecting ADPR in many different experimental and biological systems.