The intracellular bacterial pathogen Legionella pneumophila modulates host cell functions by secreting multiple effectors with diverse biochemical activities. In particular, effectors of the SidE family interfere with host protein ubiquitination in a process that involves production of phosphoribosyl ubiquitin (PR-Ub). Here, we show that effector LnaB converts PR-Ub into ADP-ribosylated ubiquitin, which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (SHxxxE) present in a large family of toxins from diverse bacterial pathogens. Thus, our study sheds light on the mechanisms by which a pathogen maintains ubiquitin homeostasis and identifies a family of enzymes capable of protein AMPylation.

Aging, a complex biological process, plays key roles the development of multiple disorders referred as aging-related diseases involving cardiovascular diseases, stroke, neurodegenerative diseases, cancers, lipid metabolism-related diseases. ADP-ribosylation is a reversible modification onto proteins and nucleic acids to alter their structures and/or functions. Growing evidence support the importance of ADP-ribosylation and ADP-ribosylation-associated enzymes in aging and age-related diseases. In this review, we summarized ADP-ribosylation-associated proteins including ADP-ribosyl transferases, the ADP-ribosyl hydrolyses and ADP-ribose binding domains. Furthermore, we outlined the latest knowledge about regulation of ADP-ribosylation in the pathogenesis and progression of main aging-related diseases, organism aging and cellular senescence, and we also speculated the underlying mechanisms to better disclose this novel molecular network. Moreover, we discussed current issues and provided an outlook for future research, aiming to revealing the unknown bio-properties of ADP-ribosylation, and establishing a novel therapeutic perspective in aging-related diseases and health aging via targeting ADP-ribosylation.

AMPylation is a post-translational modification in which AMP is added to the amino acid side chains of proteins1,2. Here we show that, with ATP as the ligand and actin as the host activator, the effector protein LnaB of Legionella pneumophila exhibits AMPylase activity towards the phosphoryl group of phosphoribose on PRR42-Ub that is generated by the SidE family of effectors, and deubiquitinases DupA and DupB in an E1- and E2-independent ubiquitination process3-7. The product of LnaB is further hydrolysed by an ADP-ribosylhydrolase, MavL, to Ub, thereby preventing the accumulation of PRR42-Ub and ADPRR42-Ub and protecting canonical ubiquitination in host cells. LnaB represents a large family of AMPylases that adopt a common structural fold, distinct from those of the previously known AMPylases, and LnaB homologues are found in more than 20 species of bacterial pathogens. Moreover, LnaB also exhibits robust phosphoryl AMPylase activity towards phosphorylated residues and produces unique ADPylation modifications in proteins. During infection, LnaB AMPylates the conserved phosphorylated tyrosine residues in the activation loop of the Src family of kinases8,9, which dampens downstream phosphorylation signalling in the host. Structural studies reveal the actin-dependent activation and catalytic mechanisms of the LnaB family of AMPylases. This study identifies, to our knowledge, an unprecedented molecular regulation mechanism in bacterial pathogenesis and protein phosphorylation.

ADP-ribosylation plays a significant role in various biological processes including genomic stability maintenance, transcriptional regulation, energy metabolism, and cell death. Using macrodomain pull-down assay with microglia lysates and MALDI-TOF-MS analysis, we identified vimentin as a major protein highly ADP-ribosylated by the poly(ADP-ribose) polymerases-1 (PARP-1) in response to LPS. ABT-888, a potent inhibitor of PARP-1/2 blocks the disassembly and ADP-ribosylation of vimentin. PARP-1 is a highly abundant nuclear protein. Its nuclear functions in repairing DNA damages induced by various stress signals, such as inflammatory stresses, have been well studied. In contrast, limited studies have been done on the cytoplasmic role(s) of PARP-1. Our study focuses on the cytoplasmic role of PARP-1 during microglia activation. Using immunofluorescence microscopy and Western blotting, we showed that a significant amount of PARP-1 is present in the cytosol of microglia cells stimulated and activated by LPS. Live cell imaging showed the translocation of nuclear PARP-1-EGFP to the cytoplasm in vesicular structures upon LPS stimulation. ABT-888 and U0126 can block this translocation. Immunofluorescence staining with various organelle marker antibodies revealed that PARP-1 vesicles show colocalization with Lamin A/C, suggesting they might be derived from the nuclear envelope through nuclear envelope budding. In conclusion, we demonstrated that PARP-1 is translocated from the nucleus to cytoplasm via vesicles upon LPS stimulation and that cytoplasmic PARP-1 causes ADP-ribosylation and disassembly of vimentin filaments during microglia activation induced by LPS.

Hepatocellular carcinoma is one of the most common malignancies, and its prognosis and treatment outcome cannot be accurately predicted. ADP-ribosylation (ADPR) is a post-translationa modification of proteins involved in protein trafficking and immune response. Therefore, it is necessary to explore the ADPR-related genes associated with the prognosis and therapeutic efficacy of hepatocellular carcinoma treatments.
We downloaded the data of hepatocellular carcinoma samples to identify ADPR-related genes as prognostic markers, and established a novel ADPR-related index (ADPRI) based on univariate and multivariate COX regression analyses. Patients\' prognosis, clinical features, somatic variant, tumor immune microenvironment, chemotherapeutic response and immunotherapeutic response were systematically analyzed. Finally, the role of ARFIP2 in hepatocellular carcinoma cells was preliminarily explored in vitro.
The ADPRI consisting of four ADPR related genes (ARL8B, ARFIP2, PARP12, ADPRHL1) was established to be a reliable predictor of survival in patients with hepatocellular carcinoma and was validated using external datasets. Compared with the low ADPRI group, the high ADPRI group presented higher levels of mutation frequency, immune infiltration and patients in high ADPRI group benefit more from immune checkpoint inhibitor treatment. In addition, we predicted some natural small molecule drugs as potential therapeutic targets for hepatocellular carcinoma. Finally, Knockdown of ARFIP2 inhibits the proliferation and migration of hepatocellular carcinoma cells by inducing the G1/S phase cell cycle arrest in HCC cells.
The ADPRI can be used to accurately predict the prognosis and immunotherapeutic response of hepatocellular carcinoma patients and providing valuable insights for future precision treatment of patients with hepatocellular carcinoma.

Poly(ADP-ribosyl)ation (PARylation) is a critical posttranslational modification that plays a vital role in maintaining genomic stability via a variety of molecular mechanisms, including activation of replication stress and the DNA damage response. The nudix hydrolase NUDT16 was recently identified as a phosphodiesterase that is responsible for removing ADP-ribose units and that plays an important role in DNA repair. However, the roles of NUDT16 in coordinating replication stress and cell cycle progression remain elusive. Here, we report that SETD3, which is a member of the SET-domain containing protein (SETD) family, is a novel substrate for NUDT16, that its protein levels fluctuate during cell cycle progression, and that its stability is strictly regulated by NUDT16-mediated dePARylation. Moreover, our data indicated that the E3 ligase CHFR is responsible for the recognition and degradation of endogenous SETD3 in a PARP1-mediated PARylation-dependent manner. Mechanistically, we revealed that SETD3 associates with BRCA2 and promotes its recruitment to stalled replication fork and DNA damage sites upon replication stress or DNA double-strand breaks, respectively. Importantly, depletion of SETD3 in NUDT16-deficient cells did not further exacerbate DNA breaks or enhance the sensitivity of cancer cells to IR exposure, suggesting that the NUDT16-SETD3 pathway may play critical roles in the induction of tolerance to radiotherapy. Collectively, these data showed that NUDT16 functions as a key upstream regulator of SETD3 protein stability by reversing the ADP-ribosylation of SETD3, and NUDT16 participates in the resolution of replication stress and facilitates HR repair.

ADP-ribosylation (ADPr) is a dynamically reversible post-translational modification (PTM) driven primarily by ADP-ribosyltransferases (ADPRTs or ARTs), which have ADP-ribosyl transfer activity. ADPr modification is involved in signaling pathways, DNA damage repair, metabolism, immunity, and inflammation. In recent years, several studies have revealed that new targets or treatments for tumors, cardiovascular diseases, neuromuscular diseases and infectious diseases can be explored by regulating ADPr. Here, we review the recent research progress on ART-mediated ADP-ribosylation and the latest findings in the diagnosis and treatment of related diseases.

Ubiquitination plays essential roles in eukaryotic cellular processes. The effector protein CteC from Chromobacterium violaceum blocks host ubiquitination by mono-ADP-ribosylation of ubiquitin (Ub) at residue T66. However, the structural basis for this modification is unknown. Here we report three crystal structures of CteC in complexes with Ub, NAD+ or ADP-ribosylated Ub, which represent different catalytic states of CteC in the modification. CteC adopts a special \'D-E\' catalytic motif for catalysis and binds NAD+ in a half-ligand binding mode. The specific recognition of Ub by CteC is determined by a relatively separate Ub-targeting domain and a long loop L6, not the classic ADP-ribosylating turn-turn loop. Structural analyses with biochemical results reveal that CteC represents a large family of poly (ADP-ribose) polymerase (PARP)-like ADP-ribosyltransferases, which harbors chimeric features from the R-S-E and H-Y-E classes of ADP-ribosyltransferases. The family of CteC-like ADP-ribosyltransferases has a common \'D-E\' catalytic consensus and exists extensively in bacteria and eukaryotic microorganisms.

O-linked N-acetylglucosamine (O-GlcNAc) glycosylation, a prevalent protein post-translational modification (PTM) that occurs intracellularly, has been shown to crosstalk with phosphorylation and ubiquitination. However, it is unclear whether it interplays with other PTMs. Here we studied its relationship with ADP-ribosylation, which involves decorating target proteins with the ADP-ribose moiety. We discovered that the poly(ADP-ribosyl)ation \"eraser\", ADP-ribose glycohydrolase (PARG), is O-GlcNAcylated at Ser26, which is in close proximity to its nuclear localization signal. O-GlcNAcylation of PARG promotes nuclear localization and chromatin association. Upon DNA damage, O-GlcNAcylation augments the recruitment of PARG to DNA damage sites and interacting with proliferating cell nuclear antigen (PCNA). In hepatocellular carcinoma (HCC) cells, PARG O-GlcNAcylation enhances the poly(ADP-ribosyl)ation of DNA damage-binding protein 1 (DDB1) and attenuates its auto-ubiquitination, thereby stabilizing DDB1 and allowing it to degrade its downstream targets, such as c-Myc. We further demonstrated that PARG-S26A, the O-GlcNAc-deficient mutant, promoted HCC in mouse xenograft models. Our findings thus reveal that PARG O-GlcNAcylation inhibits HCC, and we propose that O-GlcNAc glycosylation may crosstalk with many other PTMs.

ADP-ribosylation is a post-translational modification of proteins that plays a key role in various cellular processes, including DNA repair. Recently, significant progress has been made in understanding the mechanism and function of ADP-ribosylation in DNA repair. ADP-ribosylation can regulate the recruitment and activity of DNA repair proteins by facilitating protein-protein interactions and regulating protein conformations. Moreover, ADP-ribosylation can influence additional post-translational modifications (PTMs) of proteins involved in DNA repair, such as ubiquitination, methylation, acetylation, phosphorylation, and SUMOylation. The interaction between ADP-ribosylation and these additional PTMs can fine-tune the activity of DNA repair proteins and ensure the proper execution of the DNA repair process. In addition, PARP inhibitors have been developed as a promising cancer therapeutic strategy by exploiting the dependence of certain cancer types on the PARP-mediated DNA repair pathway. In this paper, we review the progress of ADP-ribosylation in DNA repair, discuss the crosstalk of ADP-ribosylation with additional PTMs in DNA repair, and summarize the progress of PARP inhibitors in cancer therapy.