RESUMEN
Senataxin is an evolutionarily conserved DNA/RNA helicase, whose dysfunctions are linked to neurodegeneration and cancer. A main activity of this protein is the removal of R-loops, which are nucleic acid structures capable to promote DNA damage and replication stress. Here we found that Senataxin deficiency causes the release of damaged DNA into extranuclear bodies, called micronuclei, triggering the massive recruitment of cGAS, the apical sensor of the innate immunity pathway, and the downstream stimulation of interferon genes. Such cGAS-positive micronuclei are characterized by defective membrane envelope and are particularly abundant in cycling cells lacking Senataxin, but not after exposure to a DNA breaking agent or in absence of the tumor suppressor BRCA1 protein, a partner of Senataxin in R-loop removal. Micronuclei with a discontinuous membrane are normally cleared by autophagy, a process that we show is impaired in Senataxin-deficient cells. The formation of Senataxin-dependent inflamed micronuclei is promoted by the persistence of nuclear R-loops stimulated by the DSIF transcription elongation complex and the engagement of EXO1 nuclease activity on nuclear DNA. Coherently, high levels of EXO1 result in poor prognosis in a subset of tumors lacking Senataxin expression. Hence, R-loop homeostasis impairment, together with autophagy failure and unscheduled EXO1 activity, elicits innate immune response through micronuclei formation in cells lacking Senataxin.
Asunto(s)
Autofagia , Daño del ADN , ADN Helicasas , Inflamación , Enzimas Multifuncionales , Nucleotidiltransferasas , Estructuras R-Loop , ARN Helicasas , Humanos , Autofagia/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/deficiencia , ADN Helicasas/metabolismo , ADN Helicasas/genética , ADN Helicasas/deficiencia , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/deficiencia , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Inmunidad Innata , Inflamación/patología , Inflamación/metabolismo , Inflamación/genética , Enzimas Multifuncionales/metabolismo , Enzimas Multifuncionales/genética , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Fosfoproteínas , ARN Helicasas/metabolismo , ARN Helicasas/genéticaRESUMEN
Highly sensitive detection of low-frequency EGFR-L858R mutation is particularly important in guiding targeted therapy of nonsmall-cell lung carcinoma (NSCLC). To this end, a ligase chain reaction (LCR)-based electrochemical biosensor (e-LCR) with an inverted sandwich-type architecture was provided by combining a cooperation of lambda exonuclease-RecJf exonuclease (λ-RecJf exo). In this work, by designing a knife-like DNA substrate (an overhang ssDNA part referred to the "knife arm") and introducing the λ-RecJf exo, the unreacted DNA probes in the LCR were specially degraded while only the ligated products were preserved, after which the ligated knife-like DNA products were hybridized with capture probes on the gold electrode surface through the "knife arms", forming the inverted sandwich-type DNA structure and bringing the methylene blue-label close to the electrode surface to engender the electrical signal. Finally, the sensitivity of the e-LCR could be improved by 3 orders of magnitude with the help of the λ-RecJf exo, and due to the mutation recognizing in the ligation site of the employed ligase, this method could detect EGFR-L858R mutation down to 0.01%, along with a linear range of 1 fM-10 pM and a limit detection of 0.8 fM. Further, the developed method could distinguish between L858R positive and negative mutations in cultured cell samples, tumor tissue samples, and plasma samples, whose accuracy was verified by the droplet digital PCR, holding a huge potential in liquid biopsy for precisely guiding individualized-treatment of NSCLC patients with advantages of high sensitivity, low cost, and adaptability to point-of-care testing.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Técnicas Electroquímicas , Receptores ErbB , Exodesoxirribonucleasas , Neoplasias Pulmonares , Mutación , Receptores ErbB/genética , Humanos , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Técnicas Biosensibles , Reacción en Cadena de la Ligasa , Límite de Detección , Proteínas ViralesRESUMEN
Activation of the cGAS-STING pathway plays a key role in the innate immune response to cancer through Type-1 Interferon (IFN) production and T cell priming. Accumulation of cytosolic double-stranded DNA (dsDNA) within tumor cells and dying cells is recognized by the DNA sensor cyclic GMP-AMP synthase (cGAS) to create the secondary messenger cGAMP, which in turn activates STING (STimulator of INterferon Genes), resulting in the subsequent expression of IFN-related genes. This process is regulated by Three-prime Repair EXonuclease 1 (TREX1), a 3' â 5' exonuclease that degrades cytosolic dsDNA, thereby dampening activation of the cGAS-STING pathway, which in turn diminishes immunostimulatory IFN secretion. Here, we characterize the activity of VB-85680, a potent small-molecule inhibitor of TREX1. We first demonstrate that VB-85680 inhibits TREX1 exonuclease activity in vitro in lysates from both human and mouse cell lines. We then show that treatment of intact cells with VB-85680 results in activation of downstream STING signaling, and activation of IFN-stimulated genes (ISGs). THP1-Dual™ cells cultured under low-serum conditions exhibited an enhanced ISG response when treated with VB-85680 in combination with exogenous DNA. Collectively, these findings suggest the potential of a TREX1 exonuclease inhibitor to work in combination with agents that generate cytosolic DNA to enhance the acquisition of the anti-tumor immunity widely associated with STING pathway activation.
Asunto(s)
Exodesoxirribonucleasas , Fosfoproteínas , Exodesoxirribonucleasas/metabolismo , Humanos , Fosfoproteínas/metabolismo , Ratones , Animales , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Interferones/metabolismo , Inmunidad Innata/efectos de los fármacosRESUMEN
An ultrasensitive method for the visual detection of microRNAs (miRNAs) in cell lysates using a gold nanorod-based lateral flow nucleic acid biosensor (GN-LFNAB) and exonuclease III (Exo III)-assisted signal amplification. The Exo III-catalyzed target recycling strategy is employed to generate a large number of single-strand DNA products, which can be detected by GN-LFNAB visually. With the implementation of a unique recycling strategy, we have demonstrated that the miRNA in the concentration as low as 0.5 pM can be detected without the need for instrumentation, providing a detection limit that surpasses previous reports. The new biosensor is ultrasensitive and can be applied to the reliable monitoring of miRNAs in cell lysates with high accuracy. The approach offers a simple and rapid tool for cancer diagnosis and clinical biomedicine, thanks to its flexibility, simplicity, cost-effectiveness, and convenience. This new method has the potential to significantly improve the detection and monitoring of cancer biomarkers, ultimately contributing to more effective cancer diagnosis and treatment.
Asunto(s)
Técnicas Biosensibles , Exodesoxirribonucleasas , Oro , Límite de Detección , MicroARNs , Nanotubos , Técnicas de Amplificación de Ácido Nucleico , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Oro/química , Técnicas Biosensibles/métodos , Nanotubos/química , MicroARNs/análisis , Humanos , Técnicas de Amplificación de Ácido Nucleico/métodos , Nanopartículas del Metal/químicaRESUMEN
Carcinoembryonic antigen (CEA), a key colon biomarker, demands a precise detection method for cancer diagnosis and prognosis. This study introduces a novel electrochemical aptasensor using a triblock polyadenine probe for ultra-sensitive detection of CEA. The method leverages Exonuclease III (Exo III)-assisted target recycling and hybridization chain reaction. The triblock polyadenine probe self-assembles on the bare gold electrode through the strong affinity between adenine and gold electrode, blocking CEA diffusion and providing a large immobilization surface. CEA binding to hairpin probe 1 (HP1), followed by the hybridization between HP1 and hairpin probe 2 (HP2), triggers DNA cleavage by Exo III, amplifying the signal via a hybridization chain reaction and producing numerous dsDNA walkers that generates a dramatic electrochemical impedance signal. Under optimized conditions, the aptasensor achieved two ultra-low detection limits: 0.39 agâmL-1 within the concentration range of 5 agâmL-1 to 5 × 106 agâmL-1, and 1.5 agâmL-1 within the concentration range of 5 × 106 agâmL-1 to 1 × 1010 agâmL-1. Its performance in human serum samples meets the practical standards, offering a promising new tool for ultrasensitive tumor marker detection, potentially revolutionizing early cancer diagnosis.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Antígeno Carcinoembrionario , Técnicas Electroquímicas , Exodesoxirribonucleasas , Límite de Detección , Hibridación de Ácido Nucleico , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Antígeno Carcinoembrionario/sangre , Humanos , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Poli A/química , Oro/química , ElectrodosRESUMEN
With their regulated Boolean logic operations in vitro and in vivo, DNA logic circuits have shown great promise for target recognition and disease diagnosis. However, significant obstacles must be overcome to improve their operational efficiency and broaden their range of applications. In this study, we propose an Exo III-powered closed-loop DNA circuit (ECDC) architecture that integrates four highly efficient AND logic gates. The ECDC utilizes Exo III as the sole enzyme-activated actuator, simplifying the circuit design and ensuring optimal performance. Moreover, the use of Exo III enables a self-feedback (autocatalytic) mechanism in the dynamic switching between AND logic gates within this circulating logic circuit. After validating the signal flow and examining the impact of each AND logic gate on the regulation of the circuit, we demonstrate the intelligent determination of miR-21 using the carefully designed ECDC architecture in vitro. The proposed ECDC exhibits a linear detection range for miR-21 from 0 to 300 nM, with a limit of detection (LOD) of approximately 0.01 nM, surpassing most reported methods. It also shows excellent selectivity for miR-21 detection and holds potential for identifying and imaging live cancer cells. This study presents a practical and efficient strategy for monitoring various nucleic acid-based biomarkers in vitro and in vivo through specific sequence modifications, offering significant potential for early cancer diagnosis, bioanalysis, and prognostic clinical applications.
Asunto(s)
Técnicas Biosensibles , Exodesoxirribonucleasas , Límite de Detección , MicroARNs , Humanos , MicroARNs/análisis , Técnicas Biosensibles/métodos , Técnicas Biosensibles/instrumentación , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , ADN/químicaRESUMEN
DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited. Here, we demonstrate a role for the metallo-ß-lactamase fold 5'-3' exonuclease SNM1A in this vital process. Cells disrupted for SNM1A manifest increased sensitivity to radiation and radiomimetic agents and show defects in DSB damage repair. SNM1A is recruited and is retained at the sites of DSB damage via the concerted action of its three highly conserved PBZ, PIP box and UBZ interaction domains, which mediate interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. SNM1A can resect DNA containing oxidative lesions induced by radiation damage at break termini. The combined results reveal a crucial role for SNM1A to digest chemically modified DNA during the repair of DSBs and imply that the catalytic domain of SNM1A is an attractive target for potentiation of radiotherapy.
Asunto(s)
Roturas del ADN de Doble Cadena , Enzimas Reparadoras del ADN , Reparación del ADN , Exodesoxirribonucleasas , Humanos , Roturas del ADN de Doble Cadena/efectos de la radiación , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , ADN/metabolismo , ADN/genética , Ubiquitinación , Proteínas de Ciclo CelularRESUMEN
BACKGROUND: Usual Interstitial Pneumonia (UIP) a fibrosing pneumonia is associated with idiopathic pulmonary fibrosis, chronic autoimmune disease (AID), or hypersensitivity pneumonia. Oxygen radicals, due to tobacco smoke, can damage DNA and might upregulate PARP1. Cytosolic DNA from dying pneumocytes activate cytosolic GMP-AMP-synthase-stimulator of interferon genes (cGAS-STING) pathway and TREX1. Prolonged inflammation induces senescence, which might be inhibited by phagocytosis, eliminating nuclear debris. We aimed to evaluate activation of cGAS-STING-TREX1 pathway in UIP, and if phagocytosis and anti-phagocytosis might counteract inflammation. METHODS: 44 cases of UIP with IPF or AID were studied for the expression of cGAS, pSTING, TREX1 and PARP1. LAMP1 and Rab7 expression served as phagocytosis markers. CD47 protecting phagocytosis and p16 to identify senescent cells were also studied. RESULTS: Epithelial cells in remodeled areas and macrophages expressed cGAS-pSTING, TREX1; epithelia but not macrophages stained for PARP1. Myofibroblasts, endothelia, and bronchial/bronchiolar epithelial cells were all negative except early myofibroblastic foci expressing cGAS. Type II pneumocytes expressed cGAS and PARP1, but less pSTING. TREX1 although expressed was not activated. Macrophages and many regenerating epithelial cells expressed LAMP1 and Rab7. CD47, the 'don't-eat-me-signal', was expressed by macrophages and epithelial cells including senescence cells within the remodeled areas. CONCLUSIONS: The cGAS-STING pathway is activated in macrophages and epithelial cells within remodeled areas. LikelyTREX1 because not activated cannot sufficiently degrade DNA fragments. PARP1 activation points to smoking-induced oxygen radical release, prolonging inflammation and leading to fibrosis. By expressing CD47 epithelial cells within remodeled areas protect themselves from being eliminated by phagocytosis.
Asunto(s)
Antígeno CD47 , Exodesoxirribonucleasas , Proteínas de la Membrana , Nucleotidiltransferasas , Fagocitosis , Transducción de Señal , Humanos , Nucleotidiltransferasas/metabolismo , Transducción de Señal/fisiología , Proteínas de la Membrana/metabolismo , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Antígeno CD47/metabolismo , Inflamación/metabolismo , Persona de Mediana Edad , Masculino , Anciano , Fosfoproteínas/metabolismo , Femenino , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Poli(ADP-Ribosa) Polimerasa-1/metabolismoRESUMEN
R-loops cause genome instability, disrupting normal cellular functions. Histone acetylation, particularly by p300/CBP-associated factor (PCAF), is essential for maintaining genome stability and regulating cellular processes. Understanding how R-loop formation and resolution are regulated is important because dysregulation of these processes can lead to multiple diseases, including cancer. This study explores the role of PCAF in maintaining genome stability, specifically for R-loop resolution. We found that PCAF depletion promotes the generation of R-loop structures, especially during ongoing transcription, thereby compromising genome stability. Mechanistically, we found that PCAF facilitates histone H4K8 acetylation, leading to recruitment of the a double-strand break repair protein (MRE11) and exonuclease 1 (EXO1) to R-loop sites. These in turn recruit Fanconi anemia (FA) proteins, including FANCM and BLM, to resolve the R-loop structure. Our findings suggest that PCAF, histone acetylation, and FA proteins collaborate to resolve R-loops and ensure genome stability. This study therefore provides novel mechanistic insights into the dynamics of R-loops as well as the role of PCAF in preserving genome stability. These results may help develop therapeutic strategies to target diseases associated with genome instability.
R-loops are harmful DNA-RNA hybrid structures that cause genome instability, disrupting normal cell functions. This study explored the role of the protein PCAF in resolving R-loops to maintain genome stability. The researchers found that depleting PCAF leads to increased R-loop formation, especially during transcription, compromising the genome. Mechanistically, PCAF facilitates histone acetylation, recruiting proteins like MRE11, EXO1, FANCM and BLM to R-loop sites. These proteins collaborate to resolve R-loop structures. The findings suggest that PCAF, histone acetylation, and these repair proteins work together to untangle R-loops and preserve genome integrity. Understanding this process provides insights into R-loop dynamics and PCAF's role in genome maintenance, potentially leading to therapeutic strategies for diseases associated with genome instability, such as cancer.
Asunto(s)
Inestabilidad Genómica , Histonas , Estructuras R-Loop , Factores de Transcripción p300-CBP , Acetilación , Histonas/metabolismo , Histonas/genética , Factores de Transcripción p300-CBP/metabolismo , Factores de Transcripción p300-CBP/genética , Humanos , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Reparación del ADN , Enzimas Reparadoras del ADNRESUMEN
Age-related microangiopathy, also known as small vessel disease (SVD), causes damage to the brain, retina, liver, and kidney. Based on the DNA damage theory of aging, we reasoned that genomic instability may underlie an SVD caused by dominant C-terminal variants in TREX1, the most abundant 3'-5' DNA exonuclease in mammals. C-terminal TREX1 variants cause an adult-onset SVD known as retinal vasculopathy with cerebral leukoencephalopathy (RVCL or RVCL-S). In RVCL, an aberrant, C-terminally truncated TREX1 mislocalizes to the nucleus due to deletion of its ER-anchoring domain. Since RVCL pathology mimics that of radiation injury, we reasoned that nuclear TREX1 would cause DNA damage. Here, we show that RVCL-associated TREX1 variants trigger DNA damage in humans, mice, and Drosophila, and that cells expressing RVCL mutant TREX1 are more vulnerable to DNA damage induced by chemotherapy and cytokines that up-regulate TREX1, leading to depletion of TREX1-high cells in RVCL mice. RVCL-associated TREX1 mutants inhibit homology-directed repair (HDR), causing DNA deletions and vulnerablility to PARP inhibitors. In women with RVCL, we observe early-onset breast cancer, similar to patients with BRCA1/2 variants. Our results provide a mechanistic basis linking aberrant TREX1 activity to the DNA damage theory of aging, premature senescence, and microvascular disease.
Asunto(s)
Daño del ADN , Exodesoxirribonucleasas , Fosfoproteínas , Animales , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratones , Reparación del ADN por Recombinación , Fenotipo , Mutación , Drosophila/genética , Envejecimiento/genética , Envejecimiento/metabolismo , Femenino , Drosophila melanogaster/genética , Masculino , Enfermedades de la Retina , Enfermedades Vasculares , Enfermedades Desmielinizantes del Sistema Nervioso Central HereditariasRESUMEN
Oncogene-induced senescence (OIS) arrests cell proliferation in response to replication stress (RS) induced by oncogenes. OIS depends on the DNA damage response (DDR), but also on the cGAS-STING pathway, which detects cytosolic DNA and induces type I interferons (IFNs). Whether and how RS and IFN responses cooperate to promote OIS remains unknown. Here, we show that the induction of OIS by the H-RASV12 oncogene in immortalized human fibroblasts depends on the MRE11 nuclease. Indeed, treatment with the MRE11 inhibitor Mirin prevented RS, micronuclei formation and IFN response induced by RASV12. Overexpression of the cytosolic nuclease TREX1 also prevented OIS. Conversely, overexpression of a dominant negative mutant of TREX1 or treatment with IFN-ß was sufficient to induce RS and DNA damage, independent of RASV12 induction. These data suggest that the IFN response acts as a positive feedback loop to amplify DDR in OIS through a process regulated by MRE11 and TREX1.
Asunto(s)
Senescencia Celular , Daño del ADN , Replicación del ADN , Exodesoxirribonucleasas , Proteína Homóloga de MRE11 , Fosfoproteínas , Transducción de Señal , Humanos , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Proteína Homóloga de MRE11/metabolismo , Proteína Homóloga de MRE11/genética , Senescencia Celular/genética , Fibroblastos/metabolismo , Interferón beta/metabolismo , Interferón beta/genéticaRESUMEN
Clinical trials have identified ARID1A mutations as enriched among patients who respond favorably to immune checkpoint blockade (ICB) in several solid tumor types independent of microsatellite instability. We show that ARID1A loss in murine models is sufficient to induce anti-tumor immune phenotypes observed in ARID1A mutant human cancers, including increased CD8+ T cell infiltration and cytolytic activity. ARID1A-deficient cancers upregulated an interferon (IFN) gene expression signature, the ARID1A-IFN signature, associated with increased R-loops and cytosolic single-stranded DNA (ssDNA). Overexpression of the R-loop resolving enzyme, RNASEH2B, or cytosolic DNase, TREX1, in ARID1A-deficient cells prevented cytosolic ssDNA accumulation and ARID1A-IFN gene upregulation. Further, the ARID1A-IFN signature and anti-tumor immunity were driven by STING-dependent type I IFN signaling, which was required for improved responsiveness of ARID1A mutant tumors to ICB treatment. These findings define a molecular mechanism underlying anti-tumor immunity in ARID1A mutant cancers.
Asunto(s)
Linfocitos T CD8-positivos , Proteínas de Unión al ADN , Interferón Tipo I , Proteínas de la Membrana , Neoplasias , Transducción de Señal , Factores de Transcripción , Animales , Humanos , Ratones , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Exodesoxirribonucleasas/metabolismo , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Interferón Tipo I/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Mutación , Neoplasias/inmunología , Neoplasias/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Factores de Transcripción/metabolismo , Masculino , Quimiocinas/genética , Quimiocinas/metabolismoRESUMEN
DNA replication faces challenges from DNA lesions originated from endogenous or exogenous sources of stress, leading to the accumulation of single-stranded DNA (ssDNA) that triggers the activation of the ATR checkpoint response. To complete genome replication in the presence of damaged DNA, cells employ DNA damage tolerance mechanisms that operate not only at stalled replication forks but also at ssDNA gaps originated by repriming of DNA synthesis downstream of lesions. Here, we demonstrate that human cells accumulate post-replicative ssDNA gaps following replicative stress induction. These gaps, initiated by PrimPol repriming and expanded by the long-range resection factors EXO1 and DNA2, constitute the principal origin of the ssDNA signal responsible for ATR activation upon replication stress, in contrast to stalled forks. Strikingly, the loss of EXO1 or DNA2 results in synthetic lethality when combined with BRCA1 deficiency, but not BRCA2. This phenomenon aligns with the observation that BRCA1 alone contributes to the expansion of ssDNA gaps. Remarkably, BRCA1-deficient cells become addicted to the overexpression of EXO1, DNA2 or BLM. This dependence on long-range resection unveils a new vulnerability of BRCA1-mutant tumors, shedding light on potential therapeutic targets for these cancers.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Proteína BRCA1 , ADN Helicasas , Replicación del ADN , ADN de Cadena Simple , Exodesoxirribonucleasas , Humanos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/genética , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Replicación del ADN/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , ADN Helicasas/metabolismo , ADN Helicasas/genética , Supervivencia Celular/genética , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Daño del ADNRESUMEN
The cGAS-STING pathway detects cytosolic DNA and activates a signaling cascade that results in a type I interferon (IFN) response. The endoplasmic reticulum (ER)-associated exonuclease TREX1 suppresses cGAS-STING by eliminating DNA from the cytosol. Mutations that compromise TREX1 function are linked to autoinflammatory disorders, including systemic lupus erythematosus (SLE) and Aicardi-Goutières syndrome (AGS). Despite key roles in regulating cGAS-STING and suppressing excessive inflammation, the impact of many disease-associated TREX1 mutations-particularly those outside of the core catalytic domains-remains poorly understood. Here, we characterize a recessive AGS-linked TREX1 P61Q mutation occurring within the poorly characterized polyproline helix (PPII) motif. In keeping with its position outside of the catalytic core or ER targeting motifs, neither the P61Q mutation, nor aggregate proline-to-alanine PPII mutation, disrupts TREX1 exonuclease activity, subcellular localization, or cGAS-STING regulation in overexpression systems. Introducing targeted mutations into the endogenous TREX1 locus revealed that PPII mutations destabilize the protein, resulting in impaired exonuclease activity and unrestrained cGAS-STING activation. Overall, these results demonstrate that TREX1 PPII mutations, including P61Q, impair proper immune regulation and lead to autoimmune disease through TREX1 destabilization.
Asunto(s)
Enfermedades Autoinmunes del Sistema Nervioso , Exodesoxirribonucleasas , Proteínas de la Membrana , Mutación , Malformaciones del Sistema Nervioso , Nucleotidiltransferasas , Fosfoproteínas , Humanos , Secuencias de Aminoácidos , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Células HEK293 , Lupus Eritematoso Sistémico/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Malformaciones del Sistema Nervioso/genética , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transducción de Señal/genéticaRESUMEN
Malachite green (MG) has been illicitly employed in aquaculture as a parasiticide, however, its teratogenic and carcinogenic effects pose a significant human health threat. Herein, a dual-mode colorimetric and electrochemical aptasensor was fabricated for MG detection, capitalizing on the robust catalytic and peroxidase-like activity of P-CeO2NR@Mxene and good capture efficiency of a tetrahedral DNA nanostructure (TDN) designed with multiple aptamers (m-TDN). P-CeO2NR@Mxene-modified complementary DNA (cDNA) served as both colorimetric and electrochemical probe. m-TDN was attached to AuE to capture MG and P-CeO2NR@Mxene/cDNA. The superior aptamer and MG binding to cDNA regulated signals and enabled precise MG quantification. The further introduced Exo I enabled aptamer hydrolysis, releasing MG for further binding rounds, allowing target recycling amplification. Under the optimal conditions, the aptasensor reached an impressively low detection limit 95.4 pM in colorimetric mode and 83.6 fM in electrochemical mode. We believe this dual-mode approach holds promise for veterinary drug residue detection.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Colorimetría , Técnicas Electroquímicas , Colorantes de Rosanilina , Aptámeros de Nucleótidos/química , Colorantes de Rosanilina/química , Colorantes de Rosanilina/análisis , Técnicas Biosensibles/instrumentación , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Límite de Detección , Contaminación de Alimentos/análisisRESUMEN
Nowadays, novel and efficient signal amplification strategy in electrochemiluminescence (ECL) platform is urgently needed to enhance the sensitivity of biosensor. In this work, the dual ECL signal enhancement strategy was constructed by the interactions of Pd nanoparticles attached covalent organic frameworks (Pd NPs@COFs) with tris (bipyridine) ruthenium (RuP) and Exonuclease III (Exo.III) cycle reaction. Within this strategy, the COFs composite was generated from the covalent reaction between 2-nitro-1,4-phenylenediamine (NPD) and trialdehyde phloroglucinol (Tp), and then animated by glutamate (Glu) to attach the Pd NPs. Next, the "signal on" ECL biosensor was constructed by the coordination assembly of thiolation capture DNA (cDNA) onto the Pd NPs@COFs modified electrode. After the aptamer recognition of progesterone (P4) with hairpin DNA 1 (HP1), the Exo. III cycle reaction was initiated with HP2 to generate free DNA, which hybridized with cDNA to form double-stranded DNA (dsDNA). For that, the RuP was embedded into the groove of dsDNA and achieved the ultrasensitive detection of P4 with a lower limit of detection (LOD) down to 0.45 pM, as well as the excellent selectivity and stability. This work expands the COFs-based materials application in ECL signal amplification and valuable DNA cyclic reaction in biochemical testing field.
Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas , Exodesoxirribonucleasas , Nanopartículas del Metal , Estructuras Metalorgánicas , Paladio , Progesterona , Nanopartículas del Metal/química , Estructuras Metalorgánicas/química , Paladio/química , Progesterona/análisis , Progesterona/química , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Límite de Detección , Mediciones Luminiscentes/métodos , Humanos , ADN/químicaRESUMEN
MicroRNAs (miRNAs) can serve as biomarkers for early-diagnosis, therapy, and postoperative care of cervical cancer. Sensitive and reliable quantification of miRNA remains a huge challenge due to its low expressing levels and background interference. Herein, we propose a novel exonuclease-III (Exo-III)-propelled DNAzyme cascade for sensitive and high-efficient miRNA analysis. This method involves the engineering of compact DNAzyme hairpin probes, including the H1 probe and H2 probe. The H1 probe is designed with exposed analyte recognition subunits that can specifically recognize target miRNA. This recognition triggers two processes: Exo-iii-assisted target regeneration and successive substrate cleavage catalyzed by DNAzyme. The unique character of Exo-III that catalyzes removal of mononucleotides from the blunt or recessed 3'-OH termini of dsDNA confers the approach with a minimal background signal. The multiple signal cycles provided an abundant signal amplification and consequently, the method exhibited a low limit of detection of 3.12 fM, and a better specificity over several homologous miRNAs. In summary, this powerful Exo-III driven DNAzyme cascaded system offers broader and more adaptable methods for comprehending the activities of miRNA in various biological occurrences.
Asunto(s)
ADN Catalítico , Exodesoxirribonucleasas , MicroARNs , Neoplasias del Cuello Uterino , MicroARNs/análisis , MicroARNs/genética , MicroARNs/metabolismo , ADN Catalítico/metabolismo , ADN Catalítico/química , ADN Catalítico/genética , Humanos , Exodesoxirribonucleasas/metabolismo , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/genética , Femenino , Límite de Detección , Técnicas Biosensibles/métodosRESUMEN
The DNA exonuclease three-prime repair exonuclease 1 (TREX1) is critical for preventing autoimmunity in mice and humans by degrading endogenous cytosolic DNA, which otherwise triggers activation of the innate cGAS/STING pathway leading to the production of type I IFNs. As tumor cells are prone to aberrant cytosolic DNA accumulation, we hypothesized that they are critically dependent on TREX1 activity to limit their immunogenicity. Here, we show that in tumor cells, TREX1 restricts spontaneous activation of the cGAS/STING pathway, and the subsequent induction of a type I IFN response. As a result, TREX1 deficiency compromised in vivo tumor growth in mice. This delay in tumor growth depended on a functional immune system, systemic type I IFN signaling, and tumor-intrinsic cGAS expression. Mechanistically, we show that tumor TREX1 loss drove activation of CD8+ T cells and NK cells, prevented CD8+ T-cell exhaustion, and remodeled an immunosuppressive myeloid compartment. Consequently, TREX1 deficiency combined with T-cell-directed immune checkpoint blockade. Collectively, we conclude that TREX1 is essential to limit tumor immunogenicity, and that targeting this innate immune checkpoint remodels the tumor microenvironment and enhances antitumor immunity by itself and in combination with T-cell-targeted therapies. See related article by Toufektchan et al., p. 673.
Asunto(s)
Exodesoxirribonucleasas , Inmunidad Innata , Proteínas de la Membrana , Nucleotidiltransferasas , Fosfoproteínas , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Animales , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Ratones , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Humanos , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/genética , Interferón Tipo I/metabolismo , Ratones Noqueados , Ratones Endogámicos C57BL , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Transducción de Señal , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéuticoRESUMEN
Werner (WRN) syndrome protein is a multifunctional enzyme with helicase, ATPase, and exonuclease activities that are necessary for numerous DNA-related transactions in the human cell. Recent studies identified WRN as a synthetic lethal target in cancers. In this study, a series of new N-arylquinazoline-4-amine analogs were designed and synthesized based on structure optimization of quinazoline. The structures of the thirty-two newly synthesized compounds were confirmed by 1H NMR, 13C NMR and ESI-MS. The anticancer activity in vitro against chronic myeloid leukemia cells (K562), non-small cell lung cancer cells (A549), human prostate cancer cells (PC3), and cervical cancer cells (HeLa) of the target compounds was evaluated. Among them, the inhibition ratio of compounds 17d, 18a, 18b, 11 and 23a against four cancer cells at 5 µM concentration were more than 50 %. The IC50 values of compounds 18a and 18b were 0.3 ± 0.01 µM and 0.05 ± 0.02 µM in K562 cells respectively, compared with HeLa and A549 cells, 18a and 18b were more sensitive to K562 cells. In addition, the PC3 cells with WRN overexpression (PC3-WRN) was constructed, 18a and 18b and 23a were more sensitive to PC3-WRN cells compared with the control group cells (PC3-NC). Then, the cell viability of the novel WRN inhibitors were further assessed by colony formation assay. Compared with PC3-NC cells, 18b and 23a had obvious inhibitory effect on PC3-WRN cell at 1000 nM. In summary, these results indicated that the compounds 18b and 23a could be WRN protein inhibitor with potent anticancer properties in vitro.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , RecQ Helicasas , Exodesoxirribonucleasas/metabolismo , Células HeLaRESUMEN
Chromosomal instability is a hallmark of human cancer that is associated with aggressive disease characteristics. Chromosome mis-segregations help fuel natural selection, but they risk provoking a cGAS-STING immune response through the accumulation of cytosolic DNA. The mechanisms of how tumors benefit from chromosomal instability while mitigating associated risks, such as enhanced immune surveillance, are poorly understood. Here, we identify cGAS-STING-dependent upregulation of the nuclease TREX1 as an adaptive, negative feedback mechanism that promotes immune evasion through digestion of cytosolic DNA. TREX1 loss diminishes tumor growth, prolongs survival of host animals, increases tumor immune infiltration, and potentiates response to immune checkpoint blockade selectively in tumors capable of mounting a type I IFN response downstream of STING. Together, these data demonstrate that TREX1 induction shields chromosomally unstable tumors from immune surveillance by dampening type I IFN production and suggest that TREX1 inhibitors might be used to selectively target tumors that have retained the inherent ability to mount an IFN response downstream of STING. See related article by Lim et al., p. 663.