DTX3L ubiquitin ligase ubiquitinates single-stranded nucleic acids.

Ubiquitination typically involves covalent linking of ubiquitin (Ub) to a lysine residue on a protein substrate. Recently, new facets of this process have emerged, including Ub modification of non-proteinaceous substrates like ADP-ribose by the DELTEX E3 ligase family. Here, we show that the DELTEX family member DTX3L expands this non-proteinaceous substrate repertoire to include single-stranded DNA and RNA. Although the N-terminal region of DTX3L contains single-stranded nucleic acid binding domains and motifs, the minimal catalytically competent fragment comprises the C-terminal RING and DTC domains (RD). DTX3L-RD catalyses ubiquitination of the 3'-end of single-stranded DNA and RNA, as well as double-stranded DNA with a 3' overhang of two or more nucleotides. This modification is reversibly cleaved by deubiquitinases. NMR and biochemical analyses reveal that the DTC domain binds single-stranded DNA and facilitates the catalysis of Ub transfer from RING-bound E2-conjugated Ub. Our study unveils the direct ubiquitination of nucleic acids by DTX3L, laying the groundwork for understanding its functional implications.

Ubiquitylation of nucleic acids by DELTEX ubiquitin E3 ligase DTX3L.

The recent discovery of non-proteinaceous ubiquitylation substrates broadened our understanding of this modification beyond conventional protein targets. However, the existence of additional types of substrates remains elusive. Here, we present evidence that nucleic acids can also be directly ubiquitylated via ester bond formation. DTX3L, a member of the DELTEX family E3 ubiquitin ligases, ubiquitylates DNA and RNA in vitro and that this activity is shared with DTX3, but not with the other DELTEX family members DTX1, DTX2 and DTX4. DTX3L shows preference for the 3'-terminal adenosine over other nucleotides. In addition, we demonstrate that ubiquitylation of nucleic acids is reversible by DUBs such as USP2, JOSD1 and SARS-CoV-2 PLpro. Overall, our study proposes reversible ubiquitylation of nucleic acids in vitro and discusses its potential functional implications.

l-Carnitine relieves cachexia-related skeletal muscle fibrosis by inducing deltex E3 ubiquitin ligase 3L to negatively regulate the Runx2/COL1A1 axis.

BACKGROUND: Cancer cachexia-induced skeletal muscle fibrosis (SMF) impairs muscle regeneration, alters the muscle structure and function, reduces the efficacy of anticancer drugs, diminishes the patient's quality of life and shortens overall survival. RUNX family transcription factor 2 (Runx2), a transcription factor, and collagen type I alpha 1 chain (COL1A1), the principal constituent of SMF, have been linked previously, with Runx2 shown to directly modulate COL1A1 mRNA levels. l-Carnitine, a marker of cancer cachexia, can alleviate fibrosis in liver and kidney models; however, its role in cancer cachexia-associated fibrosis and the involvement of Runx2 in the process remain unexplored. METHODS: Female C57 mice (48 weeks old) were inoculated subcutaneously with MC38 cells to establish a cancer cachexia model. A 5 mg/kg dose of l-carnitine or an equivalent volume of water was administered for 14 days via oral gavage, followed by assessments of muscle function (grip strength) and fibrosis. To elucidate the interplay between the deltex E3 ubiquitin ligase 3L(DTX3L)/Runx2/COL1A1 axis and fibrosis in transforming growth factor beta 1-stimulated NIH/3T3 cells, a suite of molecular techniques, including quantitative real-time PCR, western blot analysis, co-immunoprecipitation, molecular docking, immunofluorescence and Duolink assays, were used. The relevance of the DTX3L/Runx2/COL1A1 axis in the gastrocnemius was also explored in the in vivo model. RESULTS: l-Carnitine supplementation reduced cancer cachexia-induced declines in grip strength (>88.2%, P < 0.05) and the collagen fibre area within the gastrocnemius (>57.9%, P < 0.05). At the 5 mg/kg dose, l-carnitine also suppressed COL1A1 and alpha-smooth muscle actin (α-SMA) protein expression, which are markers of SMF and myofibroblasts. Analyses of the TRRUST database indicated that Runx2 regulates both COL1A1 and COL1A2. In vitro, l-carnitine diminished Runx2 protein levels and promoted its ubiquitination. Overexpression of Runx2 abolished the effects of l-carnitine on COL1A1 and α-SMA. Co-immunoprecipitation, molecular docking, immunofluorescence and Duolink assays confirmed an interaction between DTX3L and Runx2, with l-carnitine enhancing this interaction to promote Runx2 ubiquitination. l-Carnitine supplementation restored DTX3L levels to those observed under non-cachectic conditions, both in vitro and in vivo. Knockdown of DTX3L abolished the effects of l-carnitine on Runx2, COL1A1 and α-SMA in vitro. The expression of DTX3L was negatively correlated with the levels of Runx2 and COL1A1 in untreated NIH/3T3 cells. CONCLUSIONS: This study revealed a previously unrecognized link between Runx2 and DTX3L in SMF and demonstrated that l-carnitine exerted a significant therapeutic impact on cancer cachexia-associated SMF, potentially through the upregulation of DTX3L.

Oligomerization mediated by the D2 domain of DTX3L is critical for DTX3L-PARP9 reading function of mono-ADP-ribosylated androgen receptor.

Deltex proteins are a family of E3 ubiquitin ligases that encode C-terminal RING and DTC domains that mediate interactions with E2 ubiquitin-conjugating enzymes and recognize ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N-terminal domain architecture. The N-terminal D1 and D2 domains of DTX3L mediate homo-oligomerization, and the D3 domain interacts with PARP9, a protein that contains tandem macrodomains with ADP-ribose reader function. While DTX3L and PARP9 are known to heterodimerize, and assemble into a high molecular weight oligomeric complex, the nature of the oligomeric structure, including whether this contributes to the ADP-ribose reader function is unknown. Here, we report a crystal structure of the DTX3L N-terminal D2 domain and show that it forms a tetramer with, conveniently, D2 symmetry. We identified two interfaces in the structure: a major, conserved interface with a surface of 973 Å2 and a smaller one of 415 Å2. Using native mass spectrometry, we observed molecular species that correspond to monomers, dimers and tetramers of the D2 domain. Reconstitution of DTX3L knockout cells with a D1-D2 deletion mutant showed the domain is dispensable for DTX3L-PARP9 heterodimer formation, but necessary to assemble an oligomeric complex with efficient reader function for ADP-ribosylated androgen receptor. Our results suggest that homo-oligomerization of DTX3L is important for the DTX3L-PARP9 complex to read mono-ADP-ribosylation on a ligand-regulated transcription factor.

DTX3L Accelerates Pancreatic cancer Progression via FAK/PI3K/AKT Axis.

DTX3L (Deltex E3 ubiquitin ligase 3 L) is an E3 ubiquitin ligase, a member of the deltex family. It is also known as B-lymphoma and BAL-associated protein (BBAP). DTX3L has been proven to play an important role in various tumor development; however, its role in pancreatic cancer remains unknown. So, we analyzed the DTX3L expression in pancreatic cancer based on the TCGA database and verified it in our samples by qRT­PCR and western blot. We identified that DTX3L was highly expressed in pancreatic cancer, and its expression level was significantly negatively correlated with patients' survival. Using CCK8, colony formation, transwell, and wound healing assays, we found that upregulated DTX3L promotes pancreatic cancer cell proliferation, invasion, and migration. Mechanically, DTX3L combined with EGFR (epidermal growth factor receptor) and prevented the ubiquitination degradation of it. Upregulated EGFR activated the FAK/PI3K/Akt pathway and promoted the progression of pancreatic cancer. Moreover, we found that DTX3L can weaken pancreatic cancer cells' sensitivity to chemotherapy using the orthotopic implant tumor model. In conclusion, DTX3L accelerates pancreatic cancer progression by EGFR dependent FAK/PI3K/Akt pathway activation and may become a potential target for pancreatic cancer treatment.

DTX3L mediated ubiquitination of cGAS suppresses antitumor immunity in pancreatic cancer.

The global incidence of pancreatic cancer is associated with a high mortality rate and one of the lowest survival rates among all types of cancer. The clinical management modalities for pancreatic cancer encompass surgical intervention, chemotherapy, radiation therapy, targeted therapy, immunotherapy, or a combination thereof. Nevertheless, the diagnosis of pancreatic cancer often occurs at an advanced stage, thereby restricting treatment options and diminishing the prospects of achieving a cure. The cGAS-STING pathway has emerged as a potential target for antitumor therapy due to its role in promoting immune responses against cancer cells. Activation of the cGAS-STING pathway in tumor cells can lead to the production of pro-inflammatory cytokines and type I interferons, which can enhance the recruitment and activation of immune cells to the tumor microenvironment. The cGAS protein was detected in only a half of tumor tissues in pancreatic cancer patients and the underlying mechanism is still elusive. In this study, we have identified the E3 ligase DTX3L as a key regulator of cGAS-STING signaling in pancreatic cancer cells by mediating the ubiquitination and degradation of cGAS. The expression levels of DTX3L were found to be upregulated in pancreatic tumor tissues and correlated with a poor prognosis for patients with pancreatic cancer. Silencing of DTX3L resulted in enhanced activation of the cGAS-STING signaling pathway and improved antitumor immunity for pancreatic cancer, suggesting that targeting the DTX3L-cGAS axis could hold promise for the treatment of this disease.

Discovery of DTX3L inhibitors through a homogeneous FRET-based assay that monitors formation and removal of poly-ubiquitin chains.

Ubiquitination is a reversible protein post-translational modification in which consequent enzymatic activity results in the covalent linking of ubiquitin to a target protein. Once ubiquitinated, a protein can undergo multiple rounds of ubiquitination on multiple sites or form poly-ubiquitin chains. Ubiquitination regulates various cellular processes, and dysregulation of ubiquitination has been associated with more than one type of cancer. Therefore, efforts have been carried out to identify modulators of the ubiquitination cascade. Herein, we present the development of a FRET-based assay that allows us to monitor ubiquitination activity of DTX3L, a RING-type E3 ubiquitin ligase. Our method shows a good signal window with a robust average Z' factor of 0.76 on 384-well microplates, indicating a good assay for screening inhibitors in a high-throughput setting. From a validatory screening experiment, we have identified the first molecules that inhibit DTX3L with potencies in the low micromolar range. We also demonstrate that the method can be expanded to study deubiquitinases, such as USP28, that reduce FRET due to hydrolysis of fluorescent poly-ubiquitin chains.

DTX3L Enhances Type I Interferon Antiviral Response by Promoting the Ubiquitination and Phosphorylation of TBK1.

Studies already revealed that some E3 ubiquitin ligases participated in the immune response after viral infection by regulating the type I interferon (IFN) pathway. Here, we demonstrated that type I interferon signaling enhanced the translocation of ETS1 to the nucleus and the promoter activity of E3 ubiquitin ligase DTX3L (deltex E3 ubiquitin ligase 3L) after virus infection and thus increased the expression of DTX3L. Further experiments suggested that DTX3L ubiquitinated TBK1 at K30 and K401 sites on K63-linked ubiquitination pathway. DTX3L was also necessary for mediating the phosphorylation of TBK1 through binding with the tyrosine kinase SRC: both together enhanced the activation of TBK1. Therefore, DTX3L, being an important positive-feedback regulator of type I interferon, exerted a key role in antiviral response. IMPORTANCE Our present study evaluated DTX3L as an antiviral molecule by promoting IFN production and establishing an IFN-ß-ETS1-DTX3L-TBK1 positive-feedback loop as a novel immunomodulatory step to enhance interferon signaling and inhibit respiratory syncytial virus (RSV) infection. Our finding enriches and complements the biological function of DTX3L and provides a new strategy to protect against lung diseases such as bronchiolitis and pneumonia that develop with RSV.

DTX3L induced NLRP3 ubiquitination inhibit R28 cell pyroptosis in OGD/R injury.

Ischemia/reperfusion (I/R) injury is one of the most common etiologies in many diseases. Retinal I/R leads to cytokine storm, resulting in tissue damage and cell death. Pyroptosis, a novel type of regulated cell death, occurs after cellular I/R injury. In this study, we established an oxygen glucose deprivation (OGD/R) cellular model (R28) to simulate retinal I/R injury. We conducted an LDH assay, and EthD-III and PI staining procedures to confirm pyroptosis. Mass spectrometry and bioinformatics analysis were used to identify the possible proteins interacting with NLRP3. Co-IP and various molecular biology techniques were used to investigate the possible modes regulating NLRP3 by DTX3L. EthD-III, PI staining and LDH assays demonstrated pyroptosis induced by OGD/R injury, mediated via NLRP3 pathway. Mass spectrometry and bioinformatics analysis screened out three candidate proteins interacting with NLRP3, and further Co-IP experiment indicated that DTX-3L may interact with NLRP3 to regulate its protein levels after injury. Co-IP experiments and various molecular biology methods demonstrated that DTX3L ubiquitinates NLRP3 resulting in pyroptosis after R28 OGD/R injury. Further, NLRP3 LRR and DTX3L RING domains interact with each other. Our study demonstrated that DTX3L may ubiquitinate NLRP3 to regulate OGD/R-induced pyroptosis globally in R28 cells.

The SARS-CoV-2 Nsp3 macrodomain reverses PARP9/DTX3L-dependent ADP-ribosylation induced by interferon signaling.

SARS-CoV-2 nonstructural protein 3 (Nsp3) contains a macrodomain that is essential for coronavirus pathogenesis and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the reversal of protein ADP-ribosylation, a posttranslational modification catalyzed by host poly(ADP-ribose) polymerases (PARPs). However, the main cellular targets of the coronavirus macrodomain that mediate this effect are currently unknown. Here, we use a robust immunofluorescence-based assay to show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this assay can be used to screen for on-target and cell-active macrodomain inhibitors. This IFN-induced ADP-ribosylation is dependent on PARP9 and its binding partner DTX3L, but surprisingly the expression of the Nsp3 macrodomain or the deletion of either PARP9 or DTX3L does not impair IFN signaling or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyze this end product of IFN signaling, rather than to suppress the IFN response itself.

Differential MicroRNA Expression Involved in Endometrial Receptivity of Goats.

Endometrial receptivity represents one of the leading factors affecting the successful implantation of embryos during early pregnancy. However, the mechanism of microRNAs (miRNAs) to establish goat endometrial receptivity remains unclear. This study was intended to identify potential miRNAs and regulatory mechanisms associated with establishing endometrial receptivity through integrating bioinformatics analysis and experimental verification. MiRNA expression profiles were obtained by high-throughput sequencing, resulting in the detection of 33 differentially expressed miRNAs (DEMs), followed by their validation through quantitative RT-PCR. Furthermore, 10 potential transcription factors (TFs) and 1316 target genes of these DEMs were obtained, and the TF-miRNA and miRNA-mRNA interaction networks were constructed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that these miRNAs were significantly linked to establishing endometrial receptivity. Moreover, the fluorescence in situ hybridization (FISH) analysis, dual-luciferase report assay, and immunohistochemistry (IHC) analysis corroborated that chi-miR-483 could directly bind to deltex E3 ubiquitin ligase 3L (DTX3L) to reduce its expression level. In conclusion, our findings contribute to a better understanding of molecular mechanisms regulating the endometrial receptivity of goats, and they provide a reference for improving embryo implantation efficiency.

DTX3L/ARTD9 contributes to inflammation of fibroblast-like synoviocytes by increasing STAT1 translocation.

Deltex-3-like (DTX3L), an E3 ligase, which is also known as B-lymphoma and BAL-associated protein (BBAP), is a member of the Deltex (DTX) family and was originally identified as a binding partner of diphtheria-toxin-like ADP-ribosyltransferase-9 (ARTD9). The present study found that DTX3L and ARTD9 were upregulated in synovial tissues obtained from rheumatoid arthritis (RA) patients compared with those from the controls. Healthy synovial tissues were obtained by arthroscopic biopsy from patients with meniscus injury (n = 10 samples) without a history of RA in the Orthopedic Department of the Affiliated Hospital of Nantong University. FLSs were isolated from RA patients who underwent total knee arthroplasty. We performed dual immunofluorescence staining on DTX3L and ARTD9, and these data strongly demonstrated that DTX3L and ARTD9 were colocalized with fibroblast-like synoviocytes (FLSs) in patients with RA. Furthermore, Western blot assays were performed to confirm that the expression levels of DTX3L and ARTD9 in the FLSs increased in a time-dependent manner and peaked at 24 h after TNF-α stimulation. Further, the inhibition of endogenous DTX3L and ARTD9 expression by RNA interference significantly suppressed the TNF-α-induced MMP-9 and IL-6 expression, as shown by Western blots. In contrast, overexpressing DTX3L and ARTD9 increased the MMP-9 and IL-6 mRNA levels in the TNF-α-stimulated FLSs. Moreover, DTX3L and ARTD9 associated with STAT1 under TNF-α-stimulated conditions to modulate STAT1 nuclear localization and transcriptional activity in an immunofluorescence staining assay. Collectively, our findings provide evidence that DTX3L and ARTD9 contribute to the production of inflammatory cytokines in FLSs from RA patients and may play a key role in the inflammatory process of RA via the STAT1 signal transduction pathway.

N6-methyladenosine methyltransferase METTL3 affects the phenotype of cerebral arteriovenous malformation via modulating Notch signaling pathway.

BACKGROUND: Cerebral arteriovenous malformation (AVM) is a serious life-threatening congenital cerebrovascular disease. Specific anatomical features, such as nidus size, location, and venous drainage, have been validated to affect treatment outcomes. Until recently, molecular biomarkers and corresponding molecular mechanism related to anatomical features and treatment outcomes remain unknown. METHODS: RNA N6-methyladenosine (m6A) Methyltransferase METTL3 was identified as a differentially expressed gene in groups with different lesion sizes by analyzing the transcriptome sequencing (RNA-seq) data. Tube formation and wound healing assays were performed to investigate the effect of METTL3 on angiogenesis. In addition, Methylated RNA Immunoprecipitation Sequencing technology (MeRIP-seq) was performed to screen downstream targets of METTL3 in endothelial cells and to fully clarify the specific underlying molecular mechanisms affecting the phenotype of cerebral AVM. RESULTS: In the current study, we found that the expression level of METTL3 was reduced in the larger pathological tissues of cerebral AVMs. Moreover, knockdown of METTL3 significantly affected angiogenesis of the human endothelial cells. Mechanistically, down-regulation of METTL3 reduced the level of heterodimeric Notch E3 ubiquitin ligase formed by DTX1 and DTX3L, thereby continuously activating the Notch signaling pathway. Ultimately, the up-regulated downstream genes of Notch signaling pathway dramatically affected the angiogenesis of endothelial cells. In addition, we demonstrated that blocking Notch pathway with DAPT could restore the phenotype of METTL3 deficient endothelial cells. CONCLUSIONS: Our findings revealed the mechanism by which m6A modification regulated the angiogenesis and might provide potential biomarkers to predict the outcome of treatment, as well as provide suitable pharmacological targets for preventing the formation and progression of cerebral AVM.

LIPG signaling promotes tumor initiation and metastasis of human basal-like triple-negative breast cancer.

Current understanding of aggressive human basal-like triple-negative breast cancer (TNBC) remains incomplete. In this study, we show endothelial lipase (LIPG) is aberrantly overexpressed in basal-like TNBCs. We demonstrate that LIPG is required for in vivo tumorigenicity and metastasis of TNBC cells. LIPG possesses a lipase-dependent function that supports cancer cell proliferation and a lipase-independent function that promotes invasiveness, stemness and basal/epithelial-mesenchymal transition features of TNBC. Mechanistically, LIPG executes its oncogenic function through its involvement in interferon-related DTX3L-ISG15 signaling, which regulates protein function and stability by ISGylation. We show that DTX3L, an E3-ubiquitin ligase, is required for maintaining LIPG protein levels in TNBC cells by inhibiting proteasome-mediated LIPG degradation. Inactivation of LIPG impairs DTX3L-ISG15 signaling, indicating the existence of DTX3L-LIPG-ISG15 signaling. We further reveal LIPG-ISG15 signaling is lipase-independent. We demonstrate that DTX3L-LIPG-ISG15 signaling is essential for malignancies of TNBC cells. Targeting this pathway provides a novel strategy for basal-like TNBC therapy.

Effects of DTX3L on the cell proliferation, adhesion, and drug resistance of multiple myeloma cells.

Cell adhesion-mediated drug resistance is an important factor that influences the effects of chemotherapy in multiple myeloma. DTX3L, a ubiquitin ligase, plays a key role in cell-cycle-related process. Here, we found that the expression of DTX3L gradually increased during the proliferation of myeloma cells, which resulted in arrest of the cell cycle in the G1 phase and promoted the adherence of myeloma cells to fibronectin or bone marrow stromal cells. In addition, silencing of DTX3L improved sensitivity to chemotherapy drugs in multiple myeloma cell lines adherent to bone marrow stromal cells and increased the expression of caspase-3 and poly-adenosine diphosphate-ribose polymerase, two markers of apoptosis. Finally, we also found that DTX3L expression was regulated by focal adhesion kinase. Taken together, the results of this study show that DTX3L plays an important role in the proliferation and cell adhesion-mediated drug resistance of multiple myeloma cells and as such may play a key role in the development of multiple myeloma.