Preserving ester-linked modifications reveals glutamate and aspartate mono-ADP-ribosylation by PARP1 and its reversal by PARG.

Ester-linked post-translational modifications, including serine and threonine ubiquitination, have gained recognition as important cellular signals. However, their detection remains a significant challenge due to the chemical lability of the ester bond. This is the case even for long-known modifications, such as ADP-ribosylation on aspartate and glutamate, whose role in PARP1 signaling has recently been questioned. Here, we present easily implementable methods for preserving ester-linked modifications. When combined with a specific and sensitive modular antibody and mass spectrometry, these approaches reveal DNA damage-induced aspartate/glutamate mono-ADP-ribosylation. This previously elusive signal represents an initial wave of PARP1 signaling, contrasting with the more enduring nature of serine mono-ADP-ribosylation. Unexpectedly, we show that the poly-ADP-ribose hydrolase PARG is capable of reversing ester-linked mono-ADP-ribosylation in cells. Our methodology enables broad investigations of various ADP-ribosylation writers and, as illustrated here for noncanonical ubiquitination, it paves the way for exploring other emerging ester-linked modifications.

Specificity of DNA ADP-Ribosylation Reversal by NADARs.

Recent discoveries establish DNA and RNA as bona fide substrates for ADP-ribosylation. NADAR ("NAD- and ADP-ribose"-associated) enzymes reverse guanine ADP-ribosylation and serve as antitoxins in the DarT-NADAR operon. Although NADARs are widespread across prokaryotes, eukaryotes, and viruses, their specificity and broader physiological roles remain poorly understood. Using phylogenetic and biochemical analyses, we further explore de-ADP-ribosylation activity and antitoxin functions of NADAR domains. We demonstrate that different subfamilies of NADAR proteins from representative E. coli strains and an E. coli-infecting phage retain biochemical activity while displaying specificity in providing protection from toxic guanine ADP-ribosylation in cells. Furthermore, we identify a myxobacterial enzyme within the YbiA subfamily that functions as an antitoxin for its associated DarT-unrelated ART toxin, which we termed YarT, thus presenting a hitherto uncharacterised ART-YbiA toxin-antitoxin pair. Our studies contribute to the burgeoning field of DNA ADP-ribosylation, supporting its physiological relevance within and beyond bacterial toxin-antitoxin systems. Notably, the specificity and confinement of NADARs to non-mammals infer their potential as highly specific targets for antimicrobial drugs with minimal off-target effects.

Deregulated DNA ADP-ribosylation impairs telomere replication.

The recognition that DNA can be ADP ribosylated provides an unexpected regulatory level of how ADP-ribosylation contributes to genome stability, epigenetics and immunity. Yet, it remains unknown whether DNA ADP-ribosylation (DNA-ADPr) promotes genome stability and how it is regulated. Here, we show that telomeres are subject to DNA-ADPr catalyzed by PARP1 and removed by TARG1. Mechanistically, we show that DNA-ADPr is coupled to lagging telomere DNA strand synthesis, forming at single-stranded DNA present at unligated Okazaki fragments and on the 3' single-stranded telomere overhang. Persistent DNA-linked ADPr, due to TARG1 deficiency, eventually leads to telomere shortening. Furthermore, using the bacterial DNA ADP-ribosyl-transferase toxin to modify DNA at telomeres directly, we demonstrate that unhydrolyzed DNA-linked ADP-ribose compromises telomere replication and telomere integrity. Thus, by identifying telomeres as chromosomal targets of PARP1 and TARG1-regulated DNA-ADPr, whose deregulation compromises telomere replication and integrity, our study highlights and establishes the critical importance of controlling DNA-ADPr turnover for sustained genome stability.

Legionella metaeffector MavL reverses ubiquitin ADP-ribosylation via a conserved arginine-specific macrodomain.

ADP-ribosylation is a reversible post-translational modification involved in various cellular activities. Removal of ADP-ribosylation requires (ADP-ribosyl)hydrolases, with macrodomain enzymes being a major family in this category. The pathogen Legionella pneumophila mediates atypical ubiquitination of host targets using the SidE effector family in a process that involves ubiquitin ADP-ribosylation on arginine 42 as an obligatory step. Here, we show that the Legionella macrodomain effector MavL regulates this pathway by reversing the arginine ADP-ribosylation, likely to minimize potential detrimental effects caused by the modified ubiquitin. We determine the crystal structure of ADP-ribose-bound MavL, providing structural insights into recognition of the ADP-ribosyl group and catalytic mechanism of its removal. Further analyses reveal DUF4804 as a class of MavL-like macrodomain enzymes whose representative members show unique selectivity for mono-ADP-ribosylated arginine residue in synthetic substrates. We find such enzymes are also present in eukaryotes, as exemplified by two previously uncharacterized (ADP-ribosyl)hydrolases in Drosophila melanogaster. Crystal structures of several proteins in this class provide insights into arginine specificity and a shared mode of ADP-ribose interaction distinct from previously characterized macrodomains. Collectively, our study reveals a new regulatory layer of SidE-catalyzed ubiquitination and expands the current understanding of macrodomain enzymes.

Synthesis and Macrodomain Binding of Gln-carba-ADPr-peptide.

Mono-ADP-ribosylation is a dynamic post-translational modification (PTM) with important roles in cell signalling. This modification occurs on a wide variety of amino acids, and one of the canonical modification sites within proteins is the side chain of glutamic acid. Given the transient nature of this modification (acylal linkage) and the high sensitivity of ADP-ribosylated glutamic acid, stabilized isosteres are required for structural and biochemical studies. Here, we report the synthesis of a mimic of ADP-ribosylated peptide derived from histone H2B that contains carba-ADP-ribosylated glutamine as a potential mimic for Glu-ADPr. We synthesized a cyclopentitol-ribofuranosyl derivative of 5'-phosphoribosylated Fmoc-glutamine and used this in the solid-phase synthesis of the carba-ADPr-peptide mimicking the ADP-ribosylated N-terminal tail of histone H2B. Binding studies with isothermal calorimetry demonstrate that the macrodomains of human MacroD2 and TARG1 bind to carba-ADPr-peptide in the same way as ADPr-peptides containing the native ADP-riboside moiety connected to the side chain of glutamine in the same peptide sequence.

Deciphering the immunological and prognostic features of hepatocellular carcinoma through ADP-ribosylation-related genes analysis and identify potential therapeutic target ARFIP2.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

NUDT16 regulates CtIP PARylation to dictate homologous recombination repair.

CtIP initiates DNA end resection and mediates homologous recombination (HR) repair. However, the underlying mechanisms of CtIP regulation and how the control of its regulation affects DNA repair remain incompletely characterized. In this study, NUDT16 loss decreases CtIP protein levels and impairs CtIP recruitment to double-strand breaks (DSBs). Furthermore, overexpression of a catalytically inactive NUDT16 mutant is unable to rescue decreased CtIP protein and impaired CtIP recruitment to DSBs. In addition, we identified a novel posttranslational modification of CtIP by ADP-ribosylation that is targeted by a PAR-binding E3 ubiquitin ligase, RNF146, leading to CtIP ubiquitination and degradation. These data suggest that the hydrolase activity of NUDT16 plays a major role in controlling CtIP protein levels. Notably, ADP-ribosylation of CtIP is required for its interaction with NUDT16, its localization at DSBs, and for HR repair. Interestingly, NUDT16 can also be ADP-ribosylated. The ADP-ribosylated NUDT16 is critical for CtIP protein stability, CtIP recruitment to DSBs, and HR repair in response to DNA damage. In summary, we demonstrate that NUDT16 and its PARylation regulate CtIP stability and CtIP recruitment to DSBs, providing new insights into our understanding of the regulation of CtIP-mediated DNA end resection in the HR repair pathway.

The recruitment of ACF1 and SMARCA5 to DNA lesions relies on ADP-ribosylation dependent chromatin unfolding.

ADP-ribosylation signaling orchestrates the recruitment of various repair actors and chromatin remodeling processes promoting access to lesions during the early stages of the DNA damage response. The chromatin remodeler complex ACF, composed of the ATPase subunit SMARCA5/SNF2H and the cofactor ACF1/BAZ1A, is among the factors that accumulate at DNA lesions in an ADP-ribosylation dependent manner. In this work, we show that each subunit of the ACF complex accumulates to DNA breaks independently from its partner. Furthermore, we demonstrate that the recruitment of SMARCA5 and ACF1 to sites of damage is not due to direct binding to the ADP-ribose moieties but due to facilitated DNA binding at relaxed ADP-ribosylated chromatin. Therefore, our work provides new insights regarding the mechanisms underlying the timely accumulation of ACF1 and SMARCA5 to DNA lesions, where they contribute to efficient DNA damage resolution.

The dePARylase NUDT16 promotes radiation resistance of cancer cells by blocking SETD3 for degradation via reversing its ADP-ribosylation.

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.

Molecular basis of threonine ADP-ribosylation of ubiquitin by bacterial ARTs.

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.

ARTC1-mediated VAPB ADP-ribosylation regulates calcium homeostasis.

Mono-ADP-ribosylation (MARylation) is a post-translational modification that regulates a variety of biological processes, including DNA damage repair, cell proliferation, metabolism, and stress and immune responses. In mammals, MARylation is mainly catalyzed by ADP-ribosyltransferases (ARTs), which consist of two groups: ART cholera toxin-like (ARTCs) and ART diphtheria toxin-like (ARTDs, also known as PARPs). The human ARTC (hARTC) family is composed of four members: two active mono-ADP-ARTs (hARTC1 and hARTC5) and two enzymatically inactive enzymes (hARTC3 and hARTC4). In this study, we systematically examined the homology, expression, and localization pattern of the hARTC family, with a particular focus on hARTC1. Our results showed that hARTC3 interacted with hARTC1 and promoted the enzymatic activity of hARTC1 by stabilizing hARTC1. We also identified vesicle-associated membrane protein-associated protein B (VAPB) as a new target of hARTC1 and pinpointed Arg50 of VAPB as the ADP-ribosylation site. Furthermore, we demonstrated that knockdown of hARTC1 impaired intracellular calcium homeostasis, highlighting the functional importance of hARTC1-mediated VAPB Arg50 ADP-ribosylation in regulating calcium homeostasis. In summary, our study identified a new target of hARTC1 in the endoplasmic reticulum and suggested that ARTC1 plays a role in regulating calcium signaling.

Solid-Phase Synthesis and Biological Evaluation of Peptides ADP-Ribosylated at Histidine.

The transfer of an adenosine diphosphate (ADP) ribose moiety to a nucleophilic side chain by consumption of nicotinamide adenine dinucleotide is referred to as ADP-ribosylation, which allows for the spatiotemporal regulation of vital processes such as apoptosis and DNA repair. Recent mass-spectrometry based analyses of the "ADP-ribosylome" have identified histidine as ADP-ribose acceptor site. In order to study this modification, a fully synthetic strategy towards α-configured N(τ)- and N(π)-ADP-ribosylated histidine-containing peptides has been developed. Ribofuranosylated histidine building blocks were obtained via Mukaiyama-type glycosylation and the building blocks were integrated into an ADP-ribosylome derived peptide sequence using fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase peptide synthesis. On-resin installation of the ADP moiety was achieved using phosphoramidite chemistry, and global deprotection provided the desired ADP-ribosylated oligopeptides. The stability under various chemical conditions and resistance against (ADP-ribosyl) hydrolase-mediated degradation has been investigated to reveal that the constructs are stable under various chemical conditions and non-degradable by any of the known ADP-ribosylhydrolases.

DELTEX E3 ligases ubiquitylate ADP-ribosyl modification on nucleic acids.

Although ubiquitylation had traditionally been considered limited to proteins, the discovery of non-proteinaceous substrates (e.g. lipopolysaccharides and adenosine diphosphate ribose (ADPr)) challenged this perspective. Our recent study showed that DTX2 E3 ligase efficiently ubiquitylates ADPr. Here, we show that the ADPr ubiquitylation activity is also present in another DELTEX family member, DTX3L, analysed both as an isolated catalytic fragment and the full-length PARP9:DTX3L complex, suggesting that it is a general feature of the DELTEX family. Since structural predictions show that DTX3L possesses single-stranded nucleic acids binding ability and given the fact that nucleic acids have recently emerged as substrates for ADP-ribosylation, we asked whether DELTEX E3s might catalyse ubiquitylation of an ADPr moiety linked to nucleic acids. Indeed, we show that DTX3L and DTX2 are capable of ubiquitylating ADP-ribosylated DNA and RNA synthesized by PARPs, including PARP14. Furthermore, we demonstrate that the Ub-ADPr-nucleic acids conjugate can be reversed by two groups of hydrolases, which remove either the whole adduct (e.g. SARS-CoV-2 Mac1 or PARP14 macrodomain 1) or just the Ub (e.g. SARS-CoV-2 PLpro). Overall, this study reveals ADPr ubiquitylation as a general function of the DELTEX family E3s and presents the evidence of reversible ubiquitylation of ADP-ribosylated nucleic acids.

Crystal structure of bacterial ubiquitin ADP-ribosyltransferase CteC reveals a substrate-recruiting insertion.

ADP-ribosylation is a post-translational modification involved in regulation of diverse cellular pathways. Interestingly, many pathogens have been identified to utilize ADP-ribosylation as a way for host manipulation. A recent study found that CteC, an effector from the bacterial pathogen Chromobacterium violaceum, hinders host ubiquitin (Ub) signaling pathways via installing mono-ADP-ribosylation on threonine 66 of Ub. However, the molecular basis of substrate recognition by CteC is not well understood. In this article, we probed the substrate specificity of this effector at protein and residue levels. We also determined the crystal structure of CteC in complex with NAD+, which revealed a canonical mono-ADP-ribosyltransferase fold with an additional insertion domain. The AlphaFold-predicted model differed significantly from the experimentally determined structure, even in regions not used in crystal packing. Biochemical and biophysical studies indicated unique features of the NAD+ binding pocket, while showing selectivity distinction between Ub and structurally close Ub-like modifiers and the role of the insertion domain in substrate recognition. Together, this study provides insights into the enzymatic specificities and the key structural features of a novel bacterial ADP-ribosyltransferase involved in host-pathogen interaction.

ADP-ribosylation: An emerging direction for disease treatment.

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.

PARP7-mediated ADP-ribosylation of FRA1 promotes cancer cell growth by repressing IRF1- and IRF3-dependent apoptosis.

PARP7 was reported to promote tumor growth in a cell-autonomous manner and by repressing the antitumor immune response. Nevertheless, the molecular mechanism of how PARP7-mediated ADP-ribosylation exerts these effects in cancer cells remains elusive. Here, we identified PARP7 as a nuclear and cysteine-specific mono-ADP-ribosyltransferase that modifies targets critical for regulating transcription, including the AP-1 transcription factor FRA1. Loss of FRA1 ADP-ribosylation via PARP7 inhibition by RBN-2397 or mutation of the ADP-ribosylation site C97 increased FRA1 degradation by the proteasome via PSMC3. The reduction in FRA1 protein levels promoted IRF1- and IRF3-dependent cytokine as well as proapoptotic gene expression, culminating in CASP8-mediated apoptosis. Furthermore, high PARP7 expression was indicative of the PARP7 inhibitor response in FRA1-positive lung and breast cancer cells. Collectively, our findings highlight the connected roles of PARP7 and FRA1 and emphasize the clinical potential of PARP7 inhibitors for FRA1-driven cancers.

A ribose-functionalized NAD+ with versatile activity for ADP-ribosylation.

An NAD+ featuring an adenosyl 4'-azido functions as a general substrate for poly-ADP-ribose polymerases. Its derived mono- and poly-ADP-ribosylated proteins can be adequately recognized by distinct ADP-ribosylation-specific readers. This molecule represents the first ribose-functionalized NAD+ with versatile activities across different ADP-ribosyltransferases and provides insight into developing new probes for ADP-ribosylation.

Synthetic Dual Cysteine-ADP Ribosylated Peptides from the Androgen Receptor are Recognized by the DTX3L/PARP9 Complex.

Androgen signaling in prostate cancer cells involves multisite cysteine ADP-ribosylation of the androgen receptor (AR) by PARP7. The AR modification is read by ADP-ribosyl binding macrodomains in PARP9, but the reason that multiple cysteines are modified is unknown. Here, we use synthetic peptides to show that dual ADP-ribosylation of closely spaced cysteines mediates recognition by the DTX3L/PARP9 complex. Mono and dual ADP-ribosylated cysteine peptides were prepared using a novel solid-phase synthetic strategy utilizing a key, Boc-protected, ribofuranosylcysteine building block. This synthetic strategy allowed us to synthesize fluorescently labeled peptides containing a dual ADP-ribosylation motif. It was found that the DTX3L/PARP9 complex recognizes the dual ADP-ribosylated AR peptide (Kd = 80.5 nM) with significantly higher affinity than peptides with a single ADP-ribose. Moreover, oligomerization of the DTX3L/PARP9 complex proved crucial for ADP-ribosyl-peptide interaction since a deletion mutant of the complex that prevents its oligomer formation dramatically reduced peptide binding. Our data show that features of the substrate modification and the reader contribute to the efficiency of the interaction and imply that multivalent interactions are important for AR-DTX3L/PARP9 assembly.

The Complex Network of ADP-Ribosylation and DNA Repair: Emerging Insights and Implications for Cancer Therapy.

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.