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.

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.

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.

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.

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 insights into the molecular mechanism of TRPT1 for nucleic acid ADP-ribosylation.

Nucleic acid ADP-ribosylation has been established as a novel modification found in a wide diversity of prokaryotic and eukaryotic organisms. tRNA 2'-phosphotransferase 1 (TRPT1/TPT1/KptA) possesses ADP-ribosyltransferase (ART) activity and is able to ADP-ribosylate nucleic acids. However, the underlying molecular mechanism remains elusive. Here, we determined crystal structures of TRPT1s in complex with NAD+ from Homo sapiens, Mus musculus and Saccharomyces cerevisiae. Our results revealed that the eukaryotic TRPT1s adopt common mechanisms for both NAD+ and nucleic acid substrate binding. The conserved SGR motif induces a significant conformational change in the donor loop upon NAD+ binding to facilitate the catalytic reaction of ART. Moreover, the nucleic acid-binding residue redundancy provides structural flexibility to accommodate different nucleic acid substrates. Mutational assays revealed that TRPT1s employ different catalytic and nucleic acid-binding residues to perform nucleic acid ADP-ribosylation and RNA 2'-phosphotransferase activities. Finally, cellular assays revealed that the mammalian TRPT1 is able to promote endocervical HeLa cell survival and proliferation. Together, our results provide structural and biochemical insights into the molecular mechanism of TRPT1 for nucleic acid ADP-ribosylation.

Molecular basis for the reversible ADP-ribosylation of guanosine bases.

Modification of nucleic acids by ADP-ribosylation is catalyzed by various ADP-ribosyltransferases, including the DarT enzyme. The latter is part of the bacterial toxin-antitoxin (TA) system DarTG, which was shown to provide control of DNA replication and bacterial growth as well as protection against bacteriophages. Two subfamilies have been identified, DarTG1 and DarTG2, which are distinguished by their associated antitoxins. While DarTG2 catalyzes reversible ADP-ribosylation of thymidine bases employing a macrodomain as antitoxin, the DNA ADP-ribosylation activity of DarTG1 and the biochemical function of its antitoxin, a NADAR domain, are as yet unknown. Using structural and biochemical approaches, we show that DarT1-NADAR is a TA system for reversible ADP-ribosylation of guanosine bases. DarT1 evolved the ability to link ADP-ribose to the guanine amino group, which is specifically hydrolyzed by NADAR. We show that guanine de-ADP-ribosylation is also conserved among eukaryotic and non-DarT-associated NADAR members, indicating a wide distribution of reversible guanine modifications beyond DarTG systems.

Induction of PARP7 Creates a Vulnerability for Growth Inhibition by RBN2397 in Prostate Cancer Cells.

The ADP-ribosyltransferase PARP7 modulates protein function by conjugating ADP-ribose to the side chains of acceptor amino acids. PARP7 has been shown to affect gene expression in prostate cancer cells and certain other cell types by mechanisms that include transcription factor ADP-ribosylation. Here, we use a recently developed catalytic inhibitor to PARP7, RBN2397, to study the effects of PARP7 inhibition in androgen receptor (AR)-positive and AR-negative prostate cancer cells. We find that RBN2397 has nanomolar potency for inhibiting androgen-induced ADP-ribosylation of the AR. RBN2397 inhibits the growth of prostate cancer cells in culture when cells are treated with ligands that activate the AR, or the aryl hydrocarbon receptor, and induce PARP7 expression. We show that the growth-inhibitory effects of RBN2397 are distinct from its enhancement of IFN signaling recently shown to promote tumor immunogenicity. RBN2397 treatment also induces trapping of PARP7 in a detergent-resistant fraction within the nucleus, which is reminiscent of how inhibitors such as talazoparib affect PARP1 compartmentalization. Because PARP7 is expressed in AR-negative metastatic tumors and RBN2397 can affect cancer cells through multiple mechanisms, PARP7 may be an actionable target in advanced prostate cancer. Significance: RBN2397 is a potent and selective inhibitor of PARP7 that reduces the growth of prostate cancer cells, including a model for treatment-emergent neuroendocrine prostate cancer. RBN2397 induces PARP7 trapping on chromatin, suggesting its mechanism of action might be similar to clinically used PARP1 inhibitors.

Gamma radiation coupled ADP-ribosyl transferase activity of Pseudomonas aeruginosa PE24 moiety.

The ADP-ribosyl transferase activity of P. aeruginosa PE24 moiety expressed by E. coli BL21 (DE3) was assessed on nitrobenzylidene aminoguanidine (NBAG) and in vitro cultured cancer cell lines. Gene encoding PE24 was isolated from P. aeruginosa isolates, cloned into pET22b( +) plasmid, and expressed in E. coli BL21 (DE3) under IPTG induction. Genetic recombination was confirmed by colony PCR, the appearance of insert post digestion of engineered construct, and protein electrophoresis using sodium dodecyl-sulfate polyacrylamide gel (SDS-PAGE). The chemical compound NBAG has been used to confirm PE24 extract ADP-ribosyl transferase action through UV spectroscopy, FTIR, c13-NMR, and HPLC before and after low-dose gamma irradiation (5, 10, 15, 24 Gy). The cytotoxicity of PE24 extract alone and in combination with paclitaxel and low-dose gamma radiation (both 5 Gy and one shot 24 Gy) was assessed on adherent cell lines HEPG2, MCF-7, A375, OEC, and Kasumi-1 cell suspension. Expressed PE24 moiety ADP-ribosylated NBAG as revealed by structural changes depicted by FTIR and NMR, and the surge of new peaks at different retention times from NBAG in HPLC chromatograms. Irradiating recombinant PE24 moiety was associated with a reduction in ADP-ribosylating activity. The PE24 extract IC50 values were < 10 µg/ml with an acceptable R2 value on cancer cell lines and acceptable cell viability at 10 µg/ml on normal OEC. Overall, the synergistic effects were observed upon combining PE24 extract with low-dose paclitaxel demonstrated by the reduction in IC50 whereas antagonistic effects and a rise in IC50 values were recorded after irradiation by low-dose gamma rays. KEY POINTS: • Recombinant PE24 moiety was successfully expressed and biochemically analyzed. • Low-dose gamma radiation and metal ions decreased the recombinant PE24 cytotoxic activity. • Synergism was observed upon combining recombinant PE24 with low-dose paclitaxel.

Complementary CRISPR genome-wide genetic screens in PARP10-knockout and overexpressing cells identify synthetic interactions for PARP10-mediated cellular survival.

PARP10 is a mono-ADP-ribosyltransferase with multiple cellular functions, including proliferation, apoptosis, metabolism and DNA repair. PARP10 is overexpressed in a significant proportion of tumors, particularly breast and ovarian cancers. Identifying genetic susceptibilities based on PARP10 expression levels is thus potentially relevant for finding new targets for precision oncology. Here, we performed a series of CRISPR genome-wide loss-of-function screens in isogenic control and PARP10-overexpressing or PARP10-knockout cell lines, to identify genetic determinants of PARP10-mediated cellular survival. We found that PARP10-overexpressing cells rely on multiple DNA repair genes for survival, including ATM, the master regulator of the DNA damage checkpoint. Moreover, we show that PARP10 impacts the recruitment of ATM to nascent DNA upon replication stress. Finally, we identify the CDK2-Cyclin E1 complex as essential for proliferation of PARP10-knockout cells. Our work identifies a network of functionally relevant PARP10 synthetic interactions, and reveals a set of factors which can potentially be targeted in personalized cancer therapy.

Comprehensive methylome sequencing reveals prognostic epigenetic biomarkers for prostate cancer mortality.

BACKGROUND: Prostate cancer is a clinically heterogeneous disease with a subset of patients rapidly progressing to lethal-metastatic prostate cancer. Current clinicopathological measures are imperfect predictors of disease progression. Epigenetic changes are amongst the earliest molecular changes in tumourigenesis. To find new prognostic biomarkers to enable earlier intervention and improved outcomes, we performed methylome sequencing of DNA from patients with localised prostate cancer and long-term clinical follow-up. METHODS: We used whole-genome bisulphite sequencing (WGBS) to comprehensively map and compare DNA methylation of radical prostatectomy tissue between patients with lethal disease (n = 7) and non-lethal (n = 8) disease (median follow-up 19.5 years). Validation of differentially methylated regions (DMRs) was performed in an independent cohort (n = 185, median follow-up 15 years) using targeted multiplex bisulphite sequencing of candidate regions. Survival was assessed via univariable and multivariable analyses including clinicopathological measures (log-rank and Cox regression models). RESULTS: WGBS data analysis identified cancer-specific methylation patterns including CpG island hypermethylation, and hypomethylation of repetitive elements, with increasing disease risk. We identified 1420 DMRs associated with prostate cancer-specific mortality (PCSM), which showed enrichment for gene sets downregulated in prostate cancer and de novo methylated in cancer. Through comparison with public prostate cancer datasets, we refined the DMRs to develop an 18-gene prognostic panel. Applying this panel to an independent cohort, we found significant associations between PCSM and hypermethylation at EPHB3, PARP6, TBX1, MARCH6 and a regulatory element within CACNA2D4. Strikingly in a multivariable model, inclusion of CACNA2D4 methylation was a better predictor of PCSM versus grade alone (Harrell's C-index: 0.779 vs. 0.684). CONCLUSIONS: Our study provides detailed methylome maps of non-lethal and lethal prostate cancer and identifies novel genic regions that distinguish these patient groups. Inclusion of our DNA methylation biomarkers with existing clinicopathological measures improves prognostic models of prostate cancer mortality, and holds promise for clinical application.

A novel risk stratification model based on the Children's Hepatic Tumours International Collaboration-Hepatoblastoma Stratification and deoxyribonucleic acid methylation analysis for hepatoblastoma.

INTRODUCTION: Hepatoblastoma (HB) is the most common paediatric liver tumour, and epigenetic aberrations may be important in HB development. Recently, the Children's Hepatic Tumors International Collaboration-Hepatoblastoma Stratification (CHIC-HS) developed risk stratification based on clinicopathological factors. This study aimed to construct a more accurate model by integrating CHIC-HS with molecular factors based on DNA methylation. METHODS: HB tumour specimens (N = 132) from patients treated with the Japanese Pediatric Liver Tumors Group-2 protocol were collected and subjected to methylation analysis by bisulfite pyrosequencing. Associations between methylation status and clinicopathological factors, overall survival (OS), and event-free survival (EFS) were retrospectively analysed. We investigated the effectiveness of the evaluation of methylation status in each CHIC-HS risk group and generated a new risk stratification model. RESULTS: Most specimens (82%) were from post-chemotherapy tissue. Hypermethylation in ≥2 of the four genes (RASSF1A, PARP6, OCIAD2, and MST1R) was significantly associated with poorer OS and EFS. Multivariate analysis indicated that ≥2 methylated genes was an independent prognostic factor (hazard ratios of 6.014 and 3.684 for OS and EFS, respectively). Two or more methylated genes was also associated with poorer OS in the CHIC-very low (VL)-/low (L)-risk and CHIC-intermediate (I) risk groups (3-year OS rates were 83% vs. 98% and 50% vs. 95%, respectively). The 3-year OS rates of the VL/L, I, and high-risk groups in the new stratification model were 98%, 90%, and 62% (vs. CHIC-HS [96%, 82%, and 65%, respectively]), optimising CHIC-HS. CONCLUSIONS: Our proposed stratification system considers individual risk in HB and may improve patient clinical management.

The zinc-binding motif in tankyrases is required for the structural integrity of the catalytic ADP-ribosyltransferase domain.

Tankyrases are ADP-ribosylating enzymes that regulate many physiological processes in the cell and are considered promising drug targets for cancer and fibrotic diseases. The catalytic ADP-ribosyltransferase domain of tankyrases contains a unique zinc-binding motif of unknown function. Recently, this motif was suggested to be involved in the catalytic activity of tankyrases. In this work, we set out to study the effect of the zinc-binding motif on the activity, stability and structure of human tankyrases. We generated mutants of human tankyrase (TNKS) 1 and TNKS2, abolishing the zinc-binding capabilities, and characterized the proteins biochemically and biophysically in vitro. We further generated a crystal structure of TNKS2, in which the zinc ion was oxidatively removed. Our work shows that the zinc-binding motif in tankyrases is a crucial structural element which is particularly important for the structural integrity of the acceptor site. While mutation of the motif rendered TNKS1 inactive, probably due to introduction of major structural defects, the TNKS2 mutant remained active and displayed an altered activity profile compared to the wild-type.

Expression of the mono-ADP-ribosyltransferase ART1 by tumor cells mediates immune resistance in non-small cell lung cancer.

Most patients with non-small cell lung cancer (NSCLC) do not achieve durable clinical responses from immune checkpoint inhibitors, suggesting the existence of additional resistance mechanisms. Nicotinamide adenine dinucleotide (NAD)-induced cell death (NICD) of P2X7 receptor (P2X7R)-expressing T cells regulates immune homeostasis in inflamed tissues. This process is mediated by mono-adenosine 5'-diphosphate (ADP)-ribosyltransferases (ARTs). We found an association between membranous expression of ART1 on tumor cells and reduced CD8 T cell infiltration. Specifically, we observed a reduction in the P2X7R+ CD8 T cell subset in human lung adenocarcinomas. In vitro, P2X7R+ CD8 T cells were susceptible to ART1-mediated ADP-ribosylation and NICD, which was exacerbated upon blockade of the NAD+-degrading ADP-ribosyl cyclase CD38. Last, in murine NSCLC and melanoma models, we demonstrate that genetic and antibody-mediated ART1 inhibition slowed tumor growth in a CD8 T cell-dependent manner. This was associated with increased infiltration of activated P2X7R+CD8 T cells into tumors. In conclusion, we describe ART1-mediated NICD as a mechanism of immune resistance in NSCLC and provide preclinical evidence that antibody-mediated targeting of ART1 can improve tumor control, supporting pursuit of this approach in clinical studies.

Generation of Single-Domain Antibody-Based Recombinant Immunotoxins.

The discovery of single-domain antibodies has opened new avenues for drug development. Single-domain antibodies, also known as nanobodies, can access buried epitopes that are inaccessible to conventional antibodies. These antigen-binding domains have a high level of solubility and stability, which makes them well suited for therapeutic development. This chapter will discuss the design, production, and testing of single-domain antibody-based recombinant immunotoxins. Recombinant immunotoxins are chimeric proteins that combine the specificity of an antibody with the ribosomal-inhibitory domain of a bacterial toxin. Immunotoxins using the Pseudomonas exotoxin domain have been well studied in clinical trials. Recently, an anti-CD22 immunotoxin was granted marketing approval for use in patients with relapsed or refractory hairy cell leukemia. This supports the idea that treatment with recombinant immunotoxins can be explored for cancers that have not responded to standard therapies.

Reduced Colonic Mucosal Injury in 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Poly ADP-Ribose Polymerase (TIPARP/PARP7)-Deficient Mice.

Poly-ADP-ribose polymerases (PARPs) are important regulators of the immune system, including TCDD-inducible poly-ADP-ribose polymerase (TIPARP), also known as poly-ADP-ribose polymerase 7 (PARP7). PARP7 negatively regulates aryl hydrocarbon receptor (AHR) and type I interferon (IFN-I) signaling, both of which have been implicated in intestinal homeostasis and immunity. Since the loss of PARP7 expression increases AHR and IFN-I signaling, we used a murine dextran sulfate sodium (DSS)-induced colitis model to investigate the effect of PARP7 loss on DSS-induced intestinal inflammation. DSS-exposed Parp7-/- mice had less body weight loss, lower disease index scores, and reduced expression of several inflammation genes, including interleukin IL-6, C-x-c motif chemokine ligand 1 (Cxcl1), and lipocalin-2, when compared with wild-type mice. However, no significant difference was observed between genotypes in the colonic expression of the AHR target gene cytochrome P450 1A1 (Cyp1a1). Moreover, no significant differences in microbial composition were observed between the genotypes. Our findings demonstrate that the absence of PARP7 protein results in an impaired immune response to colonic inflammation and suggests that PARP7 may participate in the recruitment of immune cells to the inflammation site, which may be due to its role in IFN-I signaling rather than AHR signaling.

ADP-ribosyltransferases, an update on function and nomenclature.

ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD+ onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.

Ribosylation of the CD8αß heterodimer permits binding of the nonclassical major histocompatibility molecule, H2-Q10.

The CD8αß heterodimer plays a crucial role in the stabilization between major histocompatibility complex class I molecules (MHC-I) and the T cell receptor (TCR). The interaction between CD8 and MHC-I can be regulated by posttranslational modifications, which are proposed to play an important role in the development of CD8 T cells. One modification that has been proposed to control CD8 coreceptor function is ribosylation. Utilizing NAD+, the ecto-enzyme adenosine diphosphate (ADP) ribosyl transferase 2.2 (ART2.2) catalyzes the addition of ADP-ribosyl groups onto arginine residues of CD8α or ß chains and alters the interaction between the MHC and TCR complexes. To date, only interactions between modified CD8 and classical MHC-I (MHC-Ia), have been investigated and the interaction with non-classical MHC (MHC-Ib) has not been explored. Here, we show that ADP-ribosylation of CD8 facilitates the binding of the liver-restricted nonclassical MHC, H2-Q10, independent of the associated TCR or presented peptide, and propose that this highly regulated binding imposes an additional inhibitory leash on the activation of CD8-expressing cells in the presence of NAD+. These findings highlight additional important roles for nonclassical MHC-I in the regulation of immune responses.

Identification of the Mouse T Cell ADP-Ribosylome Uncovers ARTC2.2 Mediated Regulation of CD73 by ADP-Ribosylation.

Mouse T cells express the ecto-ADP-ribosyltransferase ARTC2.2, which can transfer the ADP-ribose group of extracellular nicotinamide adenine dinucleotide (NAD+) to arginine residues of various cell surface proteins thereby influencing their function. Several targets of ARTC2.2, such as P2X7, CD8a and CD25 have been identified, however a comprehensive mouse T cell surface ADP-ribosylome analysis is currently missing. Using the Af1521 macrodomain-based enrichment of ADP-ribosylated peptides and mass spectrometry, we identified 93 ADP-ribsoylated peptides corresponding to 67 distinct T cell proteins, including known targets such as CD8a and CD25 but also previously unknown targets such as CD73. We evaluated the impact of ADP-ribosylation on the capability of CD73 to generate adenosine from adenosine monophosphate. Our results show that extracellular NAD+ reduces the enzymatic activity of CD73 HEK cells co-transfected with CD73/ARTC2.2. Importantly, NAD+ significantly reduced CD73 activity on WT CD8 T cells compared to ARTC2ko CD8 T cells or WT CD8 T cells treated with an ARTC2.2-blocking nanobody. Our study provides a comprehensive list of T cell membrane proteins that serve as targets for ADP-ribosylation by ARTC2.2 and whose function may be therefore affected by ADP-ribosylation.

Identification of the role of mono-ADP-ribosylation in colorectal cancer by integrated transcriptome analysis.

Our previous study clarified the carcinogenic properties of arginine-specific mono-ADP-ribosyltransferase 1 (ART1), which results in a critical post-translational modification that changes the structure and function of proteins and is widely involved in important processes. This study provides, for the first time, a comprehensive transcriptomic analysis of colorectal cancer cells with ART1 silencing by Illumina RNA-Seq and related verification experiments. Lentiviral infection was used to construct a CT-26 cell line with stable knockdown of the ART1 gene, and a whole transcriptome sequencing technique was performed to identify differentially expressed genes (DEGs). GO and KEGG classification/enrichment analyses and verification experiments were performed to determine the role of ART1 in the progression of colorectal cancer. A total of 5552 DEGs, GO functions and KEGG pathways with the highest enrichment, various SNPs, and diverse splicing patterns were identified. Importantly, knockdown of ART1 affected the splicing of certain key genes related to tumor cell growth and downregulated the expression of the key gene PTBP1 for alternative splicing. The overall attenuation of the endoplasmic reticulum unfolded protein response (UPR) signaling pathway caused by the inhibition of mono-ADP-ribosylation of GRP78 could disrupt UPR signaling, leading to the occurrence of apoptosis to impede tumorigenesis. ART1, which is clustered in organelles, may promote the development of colorectal cancer by participating in a variety of new mechanisms, including endoplasmic reticulum stress regulation and alternative splicing, and may be a good clinical drug target for targeted therapy of CRC.