Exploring the pathogenicity of Mycoplasma pneumoniae: Focus on community-acquired respiratory distress syndrome toxins.

Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX) is a unique exotoxin produced by Mycoplasma pneumoniae (MP) and has been confirmed to possess ADP-ribosyltransferase (ART) and vacuolating activities. CARDS TX binds to receptors on the surfaces of mammalian cells followed by entry into the cells through clathrin-mediated endocytosis, and exerts cytotoxic effects by undergoing retrograde transport and finally cleavage on endosomes and cellular organelles. In addition, CARDS TX can trigger severe inflammatory reactions resulting in airway dysfunction, producing allergic inflammation and asthma-like conditions. As a newly discovered virulence factor of MP, CARDS TX has been extensively studied in recent years. As resistance to macrolide drugs has increased significantly in recent years and there is no vaccine against MP, the development of a vaccine targeting CARDS TX is considered a potential preventive measure. This review focuses on recent studies and insights into this toxin, providing directions for a better understanding of MP pathogenesis and treatment. IMPORTANCE: A serious hazard to worldwide public health in recent years, Mycoplasma pneumoniae (MP) is a prominent bacterium that causes community-acquired pneumonia (CAP) in hospitalized children. Due to their high prevalence and fatality rates, MP infections often cause both respiratory illnesses and extensive extrapulmonary symptoms. It has recently been shown that MP produces a distinct exotoxin known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). Mycoplasma pneumoniae pneumonia (MPP)-like tissue injury is caused by this toxin because it has both ADP-ribosyltransferase and vacuolating properties. A better knowledge of MP etiology and therapy is provided by this review, which focuses on latest research and insights into this toxin.

Enhancing the cytotoxicity of immunotoxins by facilitating their dissociation from target receptors under the reducing conditions of the endocytic pathway.

Immunotoxins (ITs) are recombinant chimeric proteins that combine a protein toxin with a targeting moiety to facilitate the selective delivery of the toxin to cancer cells. Here, we present a novel strategy to enhance the cytosolic access of ITs by promoting their dissociation from target receptors under the reducing conditions of the endocytic pathway. We engineered monobodySS, a human fibronectin type III domain-based monobody with disulfide bond (SS)-containing paratopes, targeting receptors such as EGFR, EpCAM, Her2, and FAP. MonobodySS exhibited SS-dependent target receptor binding with a significant reduction in binding under reducing conditions. We then created monobodySS-based ITs carrying a 25 kDa fragment of Pseudomonas exotoxin A (PE25), termed monobodySS-PE25. These ITs showed dose-dependent cytotoxicity against target receptor-expressing cancer cells and a wider therapeutic window due to higher efficacy at lower doses compared to controls with SS reduction inhibited. ERSS/28-PE25, with a KD of 28 nM for EGFR, demonstrated superior tumor-killing potency compared to ER/21-PE25, which lacks an SS bond, at equivalent and lower doses. In vivo, ERSS/28-PE25 outperformed ER/21-PE25 in suppressing tumor growth in EGFR-overexpressing xenograft mouse models. This study presents a strategy for developing solid tumor-targeting ITs using SS-containing paratopes to enhance cytosolic delivery and antitumor efficacy.

DNA Repair and Mutagenesis of ADP-Ribosylated DNA by Pierisin.

Pierisin is a DNA-targeting ADP-ribosyltransferase found in cabbage white butterfly (Pieris rapae). Pierisin transfers an ADP-ribosyl moiety to the 2-amino group of the guanine residue in DNA, yielding N2-(ADP-ribos-1-yl)-2'-deoxyguanosine (N2-ADPR-dG). Generally, such chemically modified DNA is recognized as DNA damage and elicits cellular responses, including DNA repair pathways. In Escherichia coli and human cells, it has been experimentally demonstrated that N2-ADPR-dG is a substrate of the nucleotide excision repair system. Although DNA repair machineries can remove most lesions, some unrepaired damages frequently lead to mutagenesis through DNA replication. Replication past the damaged DNA template is called translesion DNA synthesis (TLS). In vitro primer extension experiments have shown that eukaryotic DNA polymerase κ is involved in TLS across N2-ADPR-dG. In many cases, TLS is error-prone and thus a mutagenic process. Indeed, the induction of G:C to T:A and G:C to C:G mutations by N2-ADPR-dG in the hypoxanthine phosphoribosyltransferase gene mutation assay with Chinese hamster cells and supF shuttle vector plasmids assay using human fibroblasts has been reported. This review provides a detailed overview of DNA repair, TLS and mutagenesis of N2-ADPR-dG induced by cabbage butterfly pierisin-1.

General ADP-Ribosylation Mechanism Based on the Structure of ADP-Ribosyltransferase-Substrate Complexes.

ADP-ribosylation is a ubiquitous modification of proteins and other targets, such as nucleic acids, that regulates various cellular functions in all kingdoms of life. Furthermore, these ADP-ribosyltransferases (ARTs) modify a variety of substrates and atoms. It has been almost 60 years since ADP-ribosylation was discovered. Various ART structures have been revealed with cofactors (NAD+ or NAD+ analog). However, we still do not know the molecular mechanisms of ART. It needs to be better understood how ART specifies the target amino acids or bases. For this purpose, more information is needed about the tripartite complex structures of ART, the cofactors, and the substrates. The tripartite complex is essential to understand the mechanism of ADP-ribosyltransferase. This review updates the general ADP-ribosylation mechanism based on ART tripartite complex structures.

An epidermal growth factor receptor-targeting immunotoxin based on IgG shows potent antitumor activity against head and neck cancer.

The epidermal growth factor receptor (EGFR) is an important target for cancer therapies. Many head and neck cancer (HNC) cells have been reported to overexpress EGFR; therefore, anti-EGFR therapies have been attempted in patients with HNC. However, its clinical efficacy is limited owing to the development of drug resistance. In this study, we developed an EGFR-targeting immunotoxin consisting of a clinically proven anti-EGFR IgG (cetuximab; CTX) and a toxin fragment (LR-LO10) derived from Pseudomonas exotoxin A (PE) using a novel site-specific conjugation technology (peptide-directed photo-crosslinking reaction), as an alternative option. The immunotoxin (CTX-LR-LO10) showed specific binding to EGFR and properties of a typical IgG, such as stability, interactions with receptors of immune cells, and pharmacokinetics, and inhibited protein synthesis via modification of elongation factor-2. Treatment of EGFR-positive HNC cells with the immunotoxin resulted in apoptotic cell death and the inhibition of cell migration and invasion. The efficacy of CTX-LR-LO10 was evaluated in xenograft mouse models, and the immunotoxin exhibited much stronger tumor suppression than CTX or LR-LO10. Transcriptome analyses revealed that the immunotoxins elicited immune responses and altered the expression of genes related to its mechanisms of action. These results support the notion that CTX-LR-LO10 may serve as a new therapeutic agent targeting EGFR-positive cancers.

PARP14 is regulated by the PARP9/DTX3L complex and promotes interferon γ-induced ADP-ribosylation.

Protein ADP-ribosylation plays important but ill-defined roles in antiviral signalling cascades such as the interferon response. Several viruses of clinical interest, including coronaviruses, express hydrolases that reverse ADP-ribosylation catalysed by host enzymes, suggesting an important role for this modification in host-pathogen interactions. However, which ADP-ribosyltransferases mediate host ADP-ribosylation, what proteins and pathways they target and how these modifications affect viral infection and pathogenesis is currently unclear. Here we show that host ADP-ribosyltransferase activity induced by IFNγ signalling depends on PARP14 catalytic activity and that the PARP9/DTX3L complex is required to uphold PARP14 protein levels via post-translational mechanisms. Both the PARP9/DTX3L complex and PARP14 localise to IFNγ-induced cytoplasmic inclusions containing ADP-ribosylated proteins, and both PARP14 itself and DTX3L are likely targets of PARP14 ADP-ribosylation. We provide evidence that these modifications are hydrolysed by the SARS-CoV-2 Nsp3 macrodomain, shedding light on the intricate cross-regulation between IFN-induced ADP-ribosyltransferases and the potential roles of the coronavirus macrodomain in counteracting their activity.

Pierisin, Cytotoxic and Apoptosis-Inducing DNA ADP-Ribosylating Protein in Cabbage Butterfly.

Pierisin-1 was serendipitously discovered as a strong cytotoxic and apoptosis-inducing protein from pupae of the cabbage butterfly Pieris rapae against cancer cell lines. This 98-kDa protein consists of the N-terminal region (27 kDa) and C-terminal region (71 kDa), and analysis of their biological function revealed that pierisin-1 binds to cell surface glycosphingolipids on the C-terminal side, is taken up into the cell, and is cleaved to N- and C-terminal portions, where the N-terminal portion mono-ADP-ribosylates the guanine base of DNA in the presence of NAD to induce cellular genetic mutation and apoptosis. Unlike other ADP-ribosyltransferases, pieisin-1 was first found to exhibit DNA mono-ADP-ribosylating activity and show anti-cancer activity in vitro and in vivo against various cancer cell lines. Pierisin-1 was most abundantly produced during the transition from the final larval stage to the pupal stage of the cabbage butterfly, and this production was regulated by ecdysteroid hormones. This suggests that pierisn-1 might play a pivotal role in the process of metamorphosis. Moreover, pierisin-1 could contribute as a defense factor against parasitization and microbial infections in the cabbage butterfly. Pierisin-like proteins in butterflies were shown to be present not only among the subtribe Pierina but also among the subtribes Aporiina and Appiadina, and pierisin-2, -3, and -4 were identified in these butterflies. Furthermore, DNA ADP-ribosylating activities were found in six different edible clams. Understanding of the biological nature of pierisin-1 with DNA mono-ADP-ribosylating activity could open up exciting avenues for research and potential therapeutic applications, making it a subject of great interest in the field of molecular biology and biotechnology.

A deep learning method to predict bacterial ADP-ribosyltransferase toxins.

MOTIVATION: ADP-ribosylation is a critical modification involved in regulating diverse cellular processes, including chromatin structure regulation, RNA transcription, and cell death. Bacterial ADP-ribosyltransferase toxins (bARTTs) serve as potent virulence factors that orchestrate the manipulation of host cell functions to facilitate bacterial pathogenesis. Despite their pivotal role, the bioinformatic identification of novel bARTTs poses a formidable challenge due to limited verified data and the inherent sequence diversity among bARTT members. RESULTS: We proposed a deep learning-based model, ARTNet, specifically engineered to predict bARTTs from bacterial genomes. Initially, we introduced an effective data augmentation method to address the issue of data scarcity in training ARTNet. Subsequently, we employed a data optimization strategy by utilizing ART-related domain subsequences instead of the primary full sequences, thereby significantly enhancing the performance of ARTNet. ARTNet achieved a Matthew's correlation coefficient (MCC) of 0.9351 and an F1-score (macro) of 0.9666 on repeated independent test datasets, outperforming three other deep learning models and six traditional machine learning models in terms of time efficiency and accuracy. Furthermore, we empirically demonstrated the ability of ARTNet to predict novel bARTTs across domain superfamilies without sequence similarity. We anticipate that ARTNet will greatly facilitate the screening and identification of novel bARTTs from bacterial genomes. AVAILABILITY AND IMPLEMENTATION: ARTNet is publicly accessible at http://www.mgc.ac.cn/ARTNet/. The source code of ARTNet is freely available at https://github.com/zhengdd0422/ARTNet/.

ART1 knockdown decreases the IL-6-induced proliferation of colorectal cancer cells.

Colorectal cancer (CRC) is a worldwide health concern. Chronic inflammation is a risk factor for CRC, and interleukin-6 (IL-6) plays a pivotal role in this process. Arginine-specific mono-ADP-ribosyltransferase-1 (ART1) positively regulates inflammatory cytokines. ART1 knockdown reduces the level of glycoprotein 130 (gp130), a key transducer in the IL-6 signalling pathway. However, the relationship between ART1 and IL-6 and the resulting effects on IL-6-induced proliferation in CRC cells remain unclear. The aims of this study were to investigate the effects of ART1 knockdown on IL-6-induced cell proliferation in vitro and use an in vivo murine model to observe the growth of transplanted tumours. The results showed that compared with the control, ART1-sh cancer cells induced by IL-6 exhibited reduced viability, a lower rate of colony formation, less DNA synthesis, decreased protein levels of gp130, c-Myc, cyclin D1, Bcl-xL, and a reduced p-STAT3/STAT3 ratio (P < 0.05). Moreover, mice transplanted with ART1-sh CT26 cells that had high levels of IL-6 displayed tumours with smaller volumes (P < 0.05). ART1 and gp130 were colocalized in CT26, LoVo and HCT116 cells, and their expression was positively correlated in human CRC tissues. Overall, ART1 may serve as a promising regulatory factor for IL-6 signalling and a potential therapeutic target for human CRC.

Clostridium botulinum C3bot mediated effects on cytokine-induced psoriasis-like phenotype in full-thickness skin model.

Clostridium botulinum C3 exoenzyme (C3bot) exclusively inhibits RhoA, B and C by ADP-ribosylation and is therefore used as a cell-permeable tool for investigating the cellular role of these Rho-GTPases. Rho-GTPases represent a molecular switch integrating different receptor signalling to downstream cascades including transcriptional cascades that regulate various cellular processes, such as regulation of actin cytoskeleton and cell proliferation. C3bot-induced inhibition of RhoA leads to reorganization of the actin cytoskeleton, morphological changes, and inhibition of cell proliferation as well as modulation of inflammatory response. In this study, we characterized the C3bot-mediated effects on a full-thickness skin model exhibiting a psoriasis-like phenotype through the addition of cytokines. Indeed, after the addition of cytokines, a decrease in epidermal thickness, parakeratosis, and induction of IL-6 was detected. In the next step, it was studied whether C3bot caused a reduction in the cytokine-induced psoriasis-like phenotypes. Basal addition of C3bot after cytokine induction of the full-thickness skin models caused less epidermal thinning and reduced IL-6 abundance. Simultaneous basal incubation with cytokines and C3bot, IL-6 abundance was inhibited, but epidermal thickness was only moderately affected. When C3bot was added apically to the skin model, IL-6 abundance was reduced, but no further effects on the psoriasis-like phenotype of the epidermis were observed. In summary, C3bot inhibits the cytokine-induced expression of IL-6 and thus may have an impact on the pro-inflammatory immune response in the psoriasis-like phenotype.

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.

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.

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.

Novel Anti-Mesothelin Nanobodies and Recombinant Immunotoxins with Pseudomonas Exotoxin Catalytic Domain for Cancer Therapeutics.

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli, achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC50 values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.

High Norepinephrine State Induces Growth of Colorectal Cancer Cells via ADP-Ribosyltransferase 1 in Type 2 Diabetes Mellitus.

BACKGROUND: Patients with type 2 diabetes mellitus have a higher susceptibility for colorectal cancer and poorer prognosis, but the mechanism is still unknown. Here, we investigated the effect of ADP-ribosyltransferase 1 (ART1) on the growth of colorectal cancer in an animal model of diabetes with high norepinephrine status, as well as the potential mechanism. METHODS: We evaluated the size and weight of transplanted CT26 cell tumors with different ART1 expression levels in a mouse model of diabetes, as well as the survival time. CCK8 and flow cytometry were used to evaluate the growth of CT26 cells in vitro. Western blot was performed to analyze differentially expressed proteins in the ART1-modulated pathway. RESULTS: High levels of norepinephrine and ART1 favored the proliferation of CT26 cells in vitro and in vivo. Moreover, inhibition of norepinephrine-dependent proliferation was observed in ART1-silenced CT26 cells compared to those with normal ART1 expression. Following reduction of the serum norepinephrine level by surgery, the size and weight of transplanted CT26 cell tumors was significantly reduced compared to non-operated and sham-operated mice. Furthermore, the expression of ART1, mTOR, STAT3, and p-AKT protein in the tumor tissue of diabetic mice was higher than in non-diabetic mice. Following reduction of the norepinephrine level by renal denervation (RD), expression of the proliferation-related proteins mTOR, STAT3, p-AKT protein decreased, but no change was seen for ART1 expression. At the same concentration of norepinephrine, ART1 induced the expression of p-AKT, mTOR, STAT3, CyclinD1 and c-myc in CT26 cells in vitro. CONCLUSIONS: We conclude that faster growth of colorectal cancer in high norepinephrine conditions requires the expression of ART1, and that high ART1 expression may be a novel target for the treatment of diabetes-associated colorectal cancer.

Cellular Uptake and Cytotoxicity of Clostridium perfringens Iota-Toxin.

Clostridium perfringens iota-toxin is composed of two separate proteins: a binding protein (Ib) that recognizes a host cell receptor and promotes the cellular uptake of a catalytic protein and (Ia) possessing ADP-ribosyltransferase activity that induces actin cytoskeleton disorganization. Ib exhibits the overall structure of bacterial pore-forming toxins (PFTs). Lipolysis-stimulated lipoprotein receptor (LSR) is defined as a host cell receptor for Ib. The binding of Ib to LSR causes an oligomer formation of Ib in lipid rafts of plasma membranes, mediating the entry of Ia into the cytoplasm. Ia induces actin cytoskeleton disruption via the ADP-ribosylation of G-actin and causes cell rounding and death. The binding protein alone disrupts the cell membrane and induces cytotoxicity in sensitive cells. Host cells permeabilized by the pore formation of Ib are repaired by a Ca2+-dependent plasma repair pathway. This review shows that the cellular uptake of iota-toxin utilizes a pathway of plasma membrane repair and that Ib alone induces cytotoxicity.

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.

Structural and biochemical analysis of the PARP1-homology region of PARP4/vault PARP.

PARP4 is an ADP-ribosyltransferase that resides within the vault ribonucleoprotein organelle. Our knowledge of PARP4 structure and biochemistry is limited relative to other PARPs. PARP4 shares a region of homology with PARP1, an ADP-ribosyltransferase that produces poly(ADP-ribose) from NAD+ in response to binding DNA breaks. The PARP1-homology region of PARP4 includes a BRCT fold, a WGR domain, and the catalytic (CAT) domain. Here, we have determined X-ray structures of the PARP4 catalytic domain and performed biochemical analysis that together indicate an active site that is open to NAD+ interaction, in contrast to the closed conformation of the PARP1 catalytic domain that blocks access to substrate NAD+. We have also determined crystal structures of the minimal ADP-ribosyltransferase fold of PARP4 that illustrate active site alterations that restrict PARP4 to mono(ADP-ribose) rather than poly(ADP-ribose) modifications. We demonstrate that PARP4 interacts with vault RNA, and that the BRCT is primarily responsible for the interaction. However, the interaction does not lead to stimulation of mono(ADP-ribosylation) activity. The BRCT-WGR-CAT of PARP4 has lower activity than the CAT alone, suggesting that the BRCT and WGR domains regulate catalytic output. Our study provides first insights into PARP4 structure and regulation and expands understanding of PARP structural biochemistry.

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.