RESUMEN
Dynamic interactions between transcription factors govern changes in gene expression that mediate changes in cell state accompanying injury response and regeneration. Transcription factors frequently function as obligate dimers whose activity is often modulated by post-translational modifications. These critical and often transient interactions are not easily detected by traditional methods to investigate protein-protein interactions. This chapter discusses the design and validation of a fusion protein involving a transcription factor tethered to a proximity labeling ligase, APEX2. In this technique, proteins are biotinylated within a small radius of the transcription factor of interest, regardless of time of interaction. Here we discuss the validations required to ensure proper functioning of the transcription factor proximity labeling tool and the sample preparation of biotinylated proteins for mass spectrometry analysis of putative protein interactors.
Asunto(s)
Biotinilación , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Mapeo de Interacción de Proteínas , Factores de Transcripción , Mapeo de Interacción de Proteínas/métodos , Humanos , Factores de Transcripción/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Unión Proteica , Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional , Endonucleasas , Enzimas MultifuncionalesRESUMEN
Templated DNA repair that occurs during homologous recombination and replication stress relies on RAD51. RAD51 activity is positively regulated by BRCA2 and the RAD51 paralogs. The Shu complex is a RAD51 paralog-containing complex consisting of SWSAP1, SWS1, and SPIDR. We demonstrate that SWSAP1-SWS1 binds RAD51, maintains RAD51 filament stability, and enables strand exchange. Using single-molecule confocal fluorescence microscopy combined with optical tweezers, we show that SWSAP1-SWS1 decorates RAD51 filaments proficient for homologous recombination. We also find SWSAP1-SWS1 enhances RPA diffusion on ssDNA. Importantly, we show human sgSWSAP1 and sgSWS1 knockout cells are sensitive to pharmacological inhibition of PARP and APE1. Lastly, we identify cancer variants in SWSAP1 that alter Shu complex formation. Together, we show that SWSAP1-SWS1 stimulates RAD51-dependent high-fidelity repair and may be an important new cancer therapeutic target.
Asunto(s)
ADN de Cadena Simple , Recombinasa Rad51 , Proteína de Replicación A , Recombinasa Rad51/metabolismo , Recombinasa Rad51/genética , Humanos , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/genética , Proteína de Replicación A/metabolismo , Proteína de Replicación A/genética , Reparación del ADN , Unión Proteica , Recombinación Homóloga , Imagen Individual de Molécula , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genéticaRESUMEN
Mitochondrial dysfunction and genomic instability are key hallmarks of aging. The aim of this study was to evaluate whether maintenance of physical capacities at very old age is associated with key hallmarks of aging. To investigate this, we measured mitochondrial bioenergetics, mitochondrial DNA (mtDNA) copy number and DNA repair capacity in peripheral blood mononuclear cells from centenarians. In addition, circulating levels of NAD+/NADH, brain-derived neurotrophic factor (BDNF) and carbonylated proteins were measured in plasma and these parameters were correlated to physical capacities. Centenarians without physical disabilities had lower mitochondrial respiration values including ATP production, reserve capacity, maximal respiration and non-mitochondrial oxygen-consumption rate and had higher mtDNA copy number than centenarians with moderate and severe disabilities (p < 0.05). In centenarian females, grip strength had a positive association with mtDNA copy number (p < 0.05), and a borderline positive trend for activity of the central DNA repair enzyme, APE 1 (p = 0.075), while a negative trend was found with circulating protein carbonylation (p = 0.07) in the entire cohort. Lastly, a trend was observed for a negative association between BDNF and activity of daily living disability score (p = 0.06). Our results suggest that mechanisms involved in maintaining mitochondrial function and genomic stability may be associated with maintenance of physical function in centenarians.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo , Reparación del ADN , ADN Mitocondrial , Mitocondrias , Humanos , Femenino , Reparación del ADN/genética , ADN Mitocondrial/genética , Masculino , Anciano de 80 o más Años , Mitocondrias/metabolismo , Mitocondrias/genética , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/sangre , Factor Neurotrófico Derivado del Encéfalo/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Variaciones en el Número de Copia de ADN , Biomarcadores/sangre , Leucocitos Mononucleares/metabolismo , Metabolismo Energético/genética , Envejecimiento/genética , NAD/metabolismo , NAD/sangre , Carbonilación Proteica , Fuerza de la Mano , Consumo de Oxígeno/genéticaRESUMEN
In response to DNA double-strand breaks or oxidative stress, ATM-dependent DNA damage response (DDR) is activated to maintain genome integrity. However, it remains elusive whether and how DNA single-strand breaks (SSBs) activate ATM. Here, we provide direct evidence in Xenopus egg extracts that ATM-mediated DDR is activated by a defined SSB structure. Our mechanistic studies reveal that APE1 promotes the SSB-induced ATM DDR through APE1 exonuclease activity and ATM recruitment to SSB sites. APE1 protein can form oligomers to activate the ATM DDR in Xenopus egg extracts in the absence of DNA and can directly stimulate ATM kinase activity in vitro. Our findings reveal distinct mechanisms of the ATM-dependent DDR activation by SSBs in eukaryotic systems and identify APE1 as a direct activator of ATM kinase.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Roturas del ADN de Cadena Simple , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Transducción de Señal , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Animales , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Humanos , Xenopus laevis , Reparación del ADNRESUMEN
Anaplastic thyroid carcinoma (ATC) is a rare but highly aggressive thyroid cancer with poor prognosis. Killing cancer cells by inducing DNA damage or blockage of DNA repair is a promising strategy for chemotherapy. It is reported that aldehyde-reactive alkoxyamines can capture the AP sites, one of the most common DNA lesions, and inhibit apurinic/apyrimidinic endonuclease 1(APE1)-mediated base excision repair (BER), leading to cell death. Whether this strategy can be employed for ATC treatment is rarely investigated. The aim of this study is to exploit GSH-responsive AP site capture reagent (AP probe-net), which responses to the elevated glutathione (GSH) levels in the tumor micro-environment (TME), releasing reactive alkoxyamine to trap AP sites and block the APE1-mediated BER for targeted anti-tumor activity against ATC. In vitro experiments, including MTT andγ-H2AX assays, demonstrate their selective cytotoxicity towards ATC cells over normal thyroid cells. Flow cytometry analysis suggests that AP probe-net arrests the cell cycle in the G2/M phase and induces apoptosis. Western blotting (WB) results show that the expression of apoptotic protein increased with the increased concentration of AP probe-net. Further in vivo experiments reveal that the AP probe-net has a good therapeutic effect on subcutaneous tumors of the ATC cells. In conclusion, taking advantage of the elevated GSH in TME, our study affords a new strategy for targeted chemotherapy of ATC with high selectivity and reduced adverse effects.
Asunto(s)
Apoptosis , Glutatión , Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Carcinoma Anaplásico de Tiroides/patología , Carcinoma Anaplásico de Tiroides/metabolismo , Humanos , Glutatión/metabolismo , Animales , Ratones , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Línea Celular Tumoral , Apoptosis/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Reparación del ADN/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Daño del ADN/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacosRESUMEN
Largely due to its simplicity, while being more like human cells compared to other experimental models, Dictyostelium continues to be of great use to discover basic molecular mechanisms and signaling pathways underlying evolutionarily conserved biological processes. However, the identification of new protein interactions implicated in signaling pathways can be particularly challenging in Dictyostelium due to its extremely fast signaling kinetics coupled with the dynamic nature of signaling protein interactions. Recently, the proximity labeling method using engineered ascorbic acid peroxidase 2 (APEX2) in mammalian cells was shown to allow the detection of weak and/or transient protein interactions and also to obtain spatial and temporal resolution. Here, we describe a protocol for successfully using the APEX2-proximity labeling method in Dictyostelium. Coupled with the identification of the labeled proteins by mass spectrometry, this method expands Dictyostelium's proteomics toolbox and should be widely useful for identifying interacting partners involved in a variety of biological processes in Dictyostelium.
Asunto(s)
Ascorbato Peroxidasas , Dictyostelium , Proteómica , Dictyostelium/metabolismo , Ascorbato Peroxidasas/metabolismo , Ascorbato Peroxidasas/genética , Proteómica/métodos , Mapeo de Interacción de Proteínas/métodos , Espectrometría de Masas/métodos , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Humanos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Transducción de Señal , Coloración y Etiquetado/métodos , Endonucleasas , Enzimas MultifuncionalesRESUMEN
To achieve highly sensitive and reliable detection of apurinic/apyrimidinic endonuclease 1 (APE1), a critical cancer diagnostic biomarker, we designed a DNA walker-based dual-mode biosensor, utilizing cellular endogenous dual enzymes (APE 1 and Flap endonuclease 1 (FEN 1)) to collaborate in activating and propelling DNA walker motion on DNA-functionalized Au nanoparticles. Incorporating both fluorescence and electrochemical detection modes, this system leverages signal amplification from DNA walker movement and cascade amplification through tandem hybridization chain reactions (HCR), achieving highly sensitive detection of APE 1. In the fluorescence mode, continuous DNA walker movement, initiated by APE1 and driven by FEN1, generates a robust signal response within a concentration range of 0.01-500 U mL-1, presenting a good linearity in the concentration range of 0.01-10 U mL-1, with a detection limit of 0.01 U mL-1. In the electrochemical detection module, the cascade upstream DNA walker and downstream HCR dual signal amplification strategy further enhances the sensitivity of APE1 detection, extending the linear range to 0.01-50 U mL-1 and reducing the detection limit to 0.002 U mL-1. Rigorous validation demonstrates the biosensor's specificity and anti-interference capability against multiple enzymes. Moreover, it effectively distinguishes cancer cells from normal cell lysates, exhibiting excellent stability and consistency in the dual-modes. Overall, our findings underscore the efficacy of the developed dual-mode biosensor for detecting APE1 in serum and cell lysates samples, indicating its potential for clinical applications in disease diagnosis.
Asunto(s)
Técnicas Biosensibles , ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN , Endonucleasas de ADN Solapado , Oro , Límite de Detección , Nanopartículas del Metal , Técnicas Biosensibles/métodos , Humanos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , ADN/química , Endonucleasas de ADN Solapado/química , Endonucleasas de ADN Solapado/metabolismo , Nanopartículas del Metal/química , Oro/química , Técnicas Electroquímicas/métodosRESUMEN
The resistance of osteosarcoma (OS) to ionizing radiation (IR) is an obstacle for effective patient treatment. Apurinic/apyrimidinic endonuclease-reduction/oxidation factor 1 (APE1/Ref-1) is a multifunctional protein with DNA repair and reduction/oxidation (redox) activities. We previously revealed the role of APE1 in OS radioresistance; however, whether the redox activity of APE1 is involved in OS radioresistance is unclear. APE1 regulates the activation of ataxia-telangiectasia mutated (ATM), an initiator of DNA damage response that mediates radioresistance in other cancers. The role of APE1 redox activity and ATM activation in OS radioresistance is unknown. Our study revealed that IR increased APE1 expression and ATM activation in OS cells, and APE1 directly regulated ATM activation by its redox activity. The combined use of an APE1 redox inhibitor and ATM inhibitor effectively sensitized OS cells to IR in vitro and in vivo. Mechanistically, the increased radiosensitization of OS cells by the combined use of the two inhibitors was mediated by increased ferroptosis. Co-treatment with the two inhibitors significantly decreased expression of the common targeted transcription factor P53 compared with single inhibitor treatment. Collectively, APE1 redox activity, ATM activation and their crosstalk play important roles in the resistance of OS to irradiation. Synergetic inhibition of APE1 redox activity and ATM activation sensitized OS cells to IR by inducing ferroptosis, which provides a promising strategy for OS radiotherapy.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Neoplasias Óseas , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Ferroptosis , Osteosarcoma , Oxidación-Reducción , Radiación Ionizante , Osteosarcoma/radioterapia , Osteosarcoma/metabolismo , Osteosarcoma/tratamiento farmacológico , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Humanos , Ferroptosis/efectos de los fármacos , Línea Celular Tumoral , Animales , Neoplasias Óseas/metabolismo , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/radioterapia , Tolerancia a Radiación/efectos de los fármacos , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto , Propionatos , BenzoquinonasRESUMEN
Due to the complexity of regulatory networks of disease-related biomarkers, developing simple, sensitive, and accurate methods has remained challenging for precise diagnosis. Herein, an "AND" logic gates DNA molecular machine (LGDM) was constructed, which was powered by the catalytic hairpin assembly (CHA). It was coupled with dual-emission CdTe quantum dots (QDs)-based cation exchange reaction (CER) for label-free, sensitive, and ratiometric fluorescence detection of APE1 and miRNA biomarkers. Benefiting from synergistic signal amplification strategies and a ratiometric fluorometric output mode, this LGDM enables accurate logic computing with robust and significant output signals from weak inputs. It offers improved sensitivity and selectivity even in cell extracts. Using dual-emission spectra CdTe QDs, with a ratiometric signal output mode, ensured good stability and effectively prevented false-positive signals from intrinsic biological interferences compared to the approach relying on a single signal output mode, which enabled the LGDM to achieve rapid, efficient, and accurate natural drug screening against APE1 inhibitors in vitro and cells. The developed method provides impetus to streamline research related to miRNA and APE1, offering significant promise for widespread application in drug development and clinical analysis.
Asunto(s)
Compuestos de Cadmio , ADN-(Sitio Apurínico o Apirimidínico) Liasa , MicroARNs , Puntos Cuánticos , Telurio , Humanos , MicroARNs/análisis , MicroARNs/antagonistas & inhibidores , Telurio/química , Puntos Cuánticos/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/antagonistas & inhibidores , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Compuestos de Cadmio/química , Espectrometría de Fluorescencia , ADN/química , Fluorescencia , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Colorantes Fluorescentes/química , Evaluación Preclínica de Medicamentos , Computadores MolecularesRESUMEN
Companion diagnostics using biomarkers have gained prominence in guiding radiotherapy. However, biopsy-based techniques fail to account for real-time variations in target response and tumor heterogeneity. Herein, we design an activated afterglow/MRI probe as a companion diagnostics tool for dynamically assessing biomarker apurinic/apyrimidinic endonuclease 1(APE1) during radiotherapy in vivo. We employ ultrabright afterglow nanoparticles and ultrasmall FeMnOx nanoparticles as dual contrast agents, significantly broadening signal change range and enhancing the sensitivity of APE1 imaging (limit of detection: 0.0092 U/mL in afterglow imaging and 0.16 U/mL in MRI). We devise longitudinally and transversely subtraction-enhanced imaging (L&T-SEI) strategy to markedly enhance MRI contrast and signal-to-noise ratio between tumor and normal tissue of living female mice. The combined afterglow and MRI facilitate both anatomical and functional imaging of APE1 activity. This probe enables correlation of afterglow and MRI signals with APE1 expression, radiation dosage, intratumor ROS, and DNA damage, enabling early prediction of radiotherapy outcomes (as early as 3 h), significantly preceding tumor size reduction (6 days). By monitoring APE1 levels, this probe allows for early and sensitive detection of liver organ injury, outperforming histopathological analysis. Furthermore, MRI evaluates APE1 expression in radiation-induced abscopal effects provides insights into underlying mechanisms, and supports the development of treatment protocols.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa , Imagen por Resonancia Magnética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Animales , Imagen por Resonancia Magnética/métodos , Femenino , Ratones , Humanos , Línea Celular Tumoral , Medios de Contraste , Neoplasias/radioterapia , Neoplasias/diagnóstico por imagen , Ratones Desnudos , Nanopartículas/química , Ratones Endogámicos BALB C , Radioterapia Guiada por Imagen/métodosRESUMEN
Relatively low levels of antioxidant enzymes coupled with high oxygen metabolism result in the formation of numerous oxidative DNA damages in the tissues of the central nervous system. Recently, kynurenic acid (KYNA), knowns for its neuroprotective properties, has gained increasing attention in this context. Therefore, our hypothesis assumed that increased KYNA levels in the brain would positively influence mRNA expression of selected enzymes of the base excision repair pathway as well as enhance their efficiency in excising damaged nucleobases in specific areas of the sheep brain. The study was conducted on adult anestrous sheep (n = 18), in which two different doses of KYNA (20 and 100 µg/day) were infused into the third brain ventricle for three days. Molecular and biochemical analysis included the hypothalamus (preoptic and mediol-basal areas), hippocampus (CA3 field) and amygdala (central amygdaloid nucleus), dissected from the brain of sheep euthanized immediately after the last infusion. The results revealed a significant increase P < 0.001) in the relative mRNA abundance of N-methylpurine DNA glycosylase (MPG) following administration of both dose of KYNA across all examined tissues. The transcription of thymine-DNA glycosylase (TDG) increased significantly (P < 0.001) in all tissues in response to the lower KYNA dose compared to the control group. Moreover, 8-oxoguanine (8-oxoG) DNA glycosylase (OGG1) mRNA levels were also higher in both animal groups (P < 0.001). In addition, in the hypothalamus, hippocampus and amygdala, AP endonuclease 1 (APE1) mRNA expression increased under both doses of KYNA. Moreover, the both dose of KYNA significantly stimulated the efficiency of 8-oxoG excision in hypothalamus and amygdala (P < 0.05-0.001). The lower and higher doses of KYNA significantly influenced the effectiveness of εA and εC in all structures (P < 0.01-0.001). In conclusion, the favorable effect of KYNA in the brain may include the protection of genetic material in nerve and glial cells by stimulating the expression and efficiency of BER pathway enzymes.
Asunto(s)
Encéfalo , ADN Glicosilasas , Reparación del ADN , Ácido Quinurénico , Animales , Reparación del ADN/efectos de los fármacos , Ovinos , Ácido Quinurénico/metabolismo , ADN Glicosilasas/metabolismo , ADN Glicosilasas/genética , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Hipotálamo/metabolismo , Hipotálamo/efectos de los fármacos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Daño del ADN/efectos de los fármacos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Femenino , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Reparación por EscisiónRESUMEN
The nucleolus has core functions in ribosome biosynthesis, but also acts as a regulatory hub in a plethora of non-canonical processes, including cellular stress. Upon DNA damage, several DNA repair factors shuttle between the nucleolus and the nucleoplasm. Yet, the molecular mechanisms underlying such spatio-temporal protein dynamics remain to be deciphered. Here, we present a novel imaging platform to investigate nucleolar-nucleoplasmic protein shuttling in living cells. For image acquisition, we used a commercially available automated fluorescence microscope and for image analysis, we developed a KNIME workflow with implementation of machine learning-based tools. We validated the method with different nucleolar proteins, i.e., PARP1, TARG1 and APE1, by monitoring their shuttling dynamics upon oxidative stress. As a paradigm, we analyzed PARP1 shuttling upon H2O2 treatment in combination with a range of pharmacological inhibitors in a novel reporter cell line. These experiments revealed that inhibition of SIRT7 results in a loss of nucleolar PARP1 localization. Finally, we unraveled specific differences in PARP1 shuttling dynamics after co-treatment with H2O2 and different clinical PARP inhibitors. Collectively, this work delineates a highly sensitive and versatile bioimaging platform to investigate swift nucleolar-nucleoplasmic protein shuttling in living cells, which can be employed for pharmacological screening and in-depth mechanistic analyses.
Asunto(s)
Nucléolo Celular , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Poli(ADP-Ribosa) Polimerasa-1 , Humanos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Nucléolo Celular/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Microscopía Fluorescente/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Estrés Oxidativo , Peróxido de Hidrógeno/farmacología , Núcleo Celular/metabolismo , Sirtuinas/metabolismo , Transporte de Proteínas , Proteínas Nucleares/metabolismo , Células HeLa , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Aprendizaje AutomáticoRESUMEN
Apurinic/apyrimidinic endonuclease 1 (APE1), as a vital base excision repair enzyme, is essential for maintaining genomic integrity and stability, and its abnormal expression is closely associated with malignant tumors. Herein, we constructed an electrochemiluminescence (ECL) biosensor for detecting APE1 activity by combining nanoconfined ECL silver nanoclusters (Ag NCs) with X-shaped DNA recognizer-triggered cascade amplification. Specifically, the Ag NCs were prepared and confined in the glutaraldehyde-cross-linked chitosan hydrogel network using the one-pot method, resulting in a strong ECL response and exceptional stability in comparison with discrete Ag NCs. Furthermore, the self-assembled X-shaped DNA recognizers were designed for APE1 detection, which not only improved reaction kinetics due to the ordered arrangement of recognition sites but also achieved high sensitivity by utilizing the recognizer-triggered cascade amplification of strand displacement amplification (SDA) and DNAzyme catalysis. As expected, this biosensor achieved sensitive ECL detection of APE1 in the range of 1.0 × 10-3 U·µL-1 to 1.0 × 10-10 U·µL-1 with the detection limit of 2.21 × 10-11 U·µL-1, rendering it a desirable approach for biomarker detection.
Asunto(s)
Técnicas Biosensibles , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Técnicas Electroquímicas , Mediciones Luminiscentes , Nanopartículas del Metal , Plata , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/análisis , Plata/química , Humanos , Nanopartículas del Metal/química , Técnicas Electroquímicas/métodos , Mediciones Luminiscentes/métodos , Técnicas Biosensibles/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ADN/química , Límite de Detección , ADN Catalítico/química , ADN Catalítico/metabolismoRESUMEN
Clinical success with poly (ADP-ribose) polymerase inhibitors (PARPi) is impeded by inevitable resistance and associated cytotoxicity. Depletion of Amplified in Liver Cancer 1 (ALC1), a chromatin-remodeling enzyme, can overcome these limitations by hypersensitizing BReast CAncer genes 1/2 (BRCA1/2) mutant cells to PARPi. Here, we demonstrate that PARPi hypersensitivity upon ALC1 loss is reliant on its role in promoting the repair of chromatin buried abasic sites. We show that ALC1 enhances the ability of the abasic site processing enzyme, Apurinic/Apyrimidinic endonuclease 1 (APE1) to cleave nucleosome-occluded abasic sites. However, unrepaired abasic sites in ALC1-deficient cells are readily accessed by APE1 at the nucleosome-free replication forks. APE1 cleavage leads to fork breakage and trapping of PARP1/2 upon PARPi treatment, resulting in hypersensitivity. Collectively, our studies reveal how cells overcome the chromatin barrier to repair abasic lesions and uncover cleavage of abasic sites as a mechanism to overcome limitations of PARPi.
Asunto(s)
Proteína BRCA1 , Proteína BRCA2 , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Humanos , Línea Celular Tumoral , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/deficiencia , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/deficiencia , Reparación del ADN/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Femenino , Cromatina/metabolismo , Mutación , Daño del ADN/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Replicación del ADN/efectos de los fármacos , Nucleosomas/metabolismo , ADN Helicasas , Proteínas de Unión al ADNRESUMEN
A fluorogenic aptamer (FA)-based hybridization chain reaction (HCR) could provide a sensitive and label-free signal amplification method for imaging molecules in living cells. However, existing FA-HCR methods usually face some problems, such as a complicated design and significant background leakage, which greatly limit their application. Herein, we developed an FA-centered HCR (FAC-HCR) method based on a remote toehold-mediated strand displacement reaction. Compared to traditional HCRs mediated by four hairpin probes (HPs) and two HPs, the FAC-HCR displayed significantly decreased background leakage and improved sensitivity. Furthermore, the FAC-HCR was used to test a non-nucleic acid target, apurinic/apyrimidinic endonuclease 1 (APE1), an important BER-involved endonuclease. The fluorescence analysis results confirmed that FAC-HCR can reach a detection limit of 0.1174 U/mL. By using the two HPs for FAC-HCR with polyetherimide-based nanoparticles, the activity of APE1 in living cells can be imaged. In summary, this study could provide a new idea to design an FA-based HCR and improve the performance of HCRs in live cell imaging.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Hibridación de Ácido Nucleico , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Humanos , Colorantes FluorescentesRESUMEN
BACKGROUND: Non-small cell lung cancer (NSCLC) represents a highly immunogenic malignancy. Immunologic tolerance facilitated by myeloid-derived suppressor cells (MDSCs) is implicated in primary or secondary resistance mechanisms in NSCLC. The potential role of APE1 in regulating NSCLC metastasis by targeting MDSCs remains uncertain. METHODS: This study utilized a plasmid, Plxpsp-mGM-CSF, to induce elevated granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in A549 cells. Tumor transplantation experiments involved A549, A549+GM-CSF, and A549+GM-CSF-siAPE1 cell lines. Evaluation encompassed MDSCs, Treg cells, IgG, CD3, and CD8 levels. RESULTS: Notably, lung cancer tissues and cells displayed markedly reduced APE1 expression. siAPE1 transfection significantly curtailed tumor growth compared to the A549+GM-CSF group. APE1 knockdown orchestrated immune system modulation in lung tumor mice, characterized by diminished MDSCs but augmented Treg cells, IgG, CD3, and CD8. Additionally, APE1 knockdown led to reduced levels of pro-MDSC cytokines (HGF, CCL5, IL-6, CCL12) and a concurrent upregulation of the anti-MDSC cytokine IL-1ra. Furthermore, APE1 knockdown impeded cell viability in both A549 and H1650 cells. CONCLUSIONS: Transplantation of A549-GM-CSF amplified MDSC levels, fostering accelerated tumor growth, while mitigating MDSC levels through APE1 knockdown hindered tumor progression and alleviated inflammatory infiltration in lung cancer tissues. Strategies targeting the APE1/MDSC axis offer a promising approach for lung cancer prevention and treatment, presenting novel insights for NSCLC management.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Neoplasias Pulmonares , Células Supresoras de Origen Mieloide , Células Supresoras de Origen Mieloide/inmunología , Células Supresoras de Origen Mieloide/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Animales , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/metabolismo , Humanos , Ratones , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Células A549 , Técnicas de Silenciamiento del Gen , Metástasis de la Neoplasia , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Masculino , FemeninoRESUMEN
CTC1-STN1-TEN1 (CST) is a single-stranded DNA binding protein vital for telomere length maintenance with additional genome-wide roles in DNA replication and repair. While CST was previously shown to function in double-strand break repair and promote replication restart, it is currently unclear whether it has specialized roles in other DNA repair pathways. Proper and efficient repair of DNA is critical to protecting genome integrity. Telomeres and other G-rich regions are strongly predisposed to oxidative DNA damage in the form of 8-oxoguanines, which are typically repaired by the base-excision repair (BER) pathway. Moreover, recent studies suggest that CST functions in the repair of oxidative DNA lesions. Therefore, we tested whether CST interacts with and regulates BER protein activity. Here, we show that CST robustly stimulates proteins involved in BER, including OGG1, Pol ß, APE1, and LIGI, on both telomeric and non-telomeric DNA substrates. Biochemical reconstitution of the pathway indicates that CST stimulates BER. Finally, knockout of STN1 or CTC1 leads to increased levels of 8-oxoguanine, suggesting defective BER in the absence of CST. Combined, our results define an undiscovered function of CST in BER, where it acts as a stimulatory factor to promote efficient genome-wide oxidative repair.
Asunto(s)
Daño del ADN , Reparación del ADN , Proteínas de Unión a Telómeros , Humanos , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Unión a Telómeros/genética , Telómero/metabolismo , Telómero/genética , ADN Glicosilasas/metabolismo , ADN Glicosilasas/genética , Estrés Oxidativo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Guanina/análogos & derivados , Guanina/metabolismo , ADN Polimerasa beta/metabolismo , ADN Polimerasa beta/genética , Reparación por EscisiónRESUMEN
Due to the potential off-tumor signal leakage and limited biomarker content, there is an urgent need for stimulus-responsive and amplification-based tumor molecular imaging strategies. Therefore, two tetrahedral framework DNA (tFNA-Hs), tFNA-H1AP, and tFNA-H2, were rationally engineered to form a polymeric tFNA network, termed an intelligent DNA network, in an AND-gated manner. The intelligent DNA network was designed for tumor-specific molecular imaging by leveraging the elevated expression of apurinic/apyrimidinic endonuclease 1 (APE1) in tumor cytoplasm instead of normal cells and the high expression of miRNA-21 in tumor cytoplasm. The activation of tFNA-H1AP can be achieved through specific recognition and cleavage by APE1, targeting the apurinic/apyrimidinic site (AP site) modified within the stem region of hairpin 1 (H1AP). Subsequently, miRNA-21 facilitates the hybridization of activated H1AP on tFNA-H1AP with hairpin 2 (H2) on tFNA-H2, triggering a catalytic hairpin assembly (CHA) reaction that opens the H1AP at the vertices of tFNA-H1AP to bind with H2 at the vertices of tFNA-H2 and generate fluorescence signals. Upon completion of hybridization, miRNA-21 is released, initiating the subsequent cycle of the CHA reaction. The AND-gated intelligent DNA network can achieve specific tumor molecular imaging in vivo and also enables risk stratification of neuroblastoma patients.
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ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN , MicroARNs , Humanos , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , MicroARNs/metabolismo , MicroARNs/análisis , ADN/química , ADN/metabolismo , Imagen Molecular/métodos , Animales , Imagen ÓpticaRESUMEN
MicroRNA (miRNA) and apurinic/apyrimidinic endonuclease 1 (APE1) have been reported to be closely associated with cancers, making them potential crucial biomarkers and therapeutic targets. However, focusing on the detection of a single target is not conducive to the diagnosis and prognosis assessment of diseases. In this study, an AND logic-gate-based dual-locking hairpin-mediated catalytic hairpin assembly (DL-CHA) was developed for sensitive and specific detection of microRNA and APE1. By addition of a lock to each of the hairpins, with APE1 and microRNA serving as keys, fluorescence signals could only be detected in the presence of simultaneous stimulation by APE1 and miRNA-224. This indicated that the biosensor could operate as an AND logic gate. DL-CHA exhibited advantages such as a low background, rapid response, and high logic capability. Therefore, the biosensor serves as a novel approach to cancer diagnosis with significant potential applications.
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Técnicas Biosensibles , ADN-(Sitio Apurínico o Apirimidínico) Liasa , MicroARNs , MicroARNs/análisis , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Humanos , Técnicas Biosensibles/métodos , Lógica , Límite de DetecciónRESUMEN
APE2 plays important roles in the maintenance of genomic and epigenomic stability including DNA repair and DNA damage response. Accumulating evidence has suggested that APE2 is upregulated in multiple cancers at the protein and mRNA levels and that APE2 upregulation is correlative with higher and lower overall survival of cancer patients depending on tumor type. However, it remains unknown how APE2 protein abundance is maintained and regulated in cells. Here, we provide the first evidence of APE2 regulation via the posttranslational modification ubiquitin. APE2 is poly-ubiquitinated via K48-linked chains and degraded via the ubiquitin-proteasome system where K371 is the key residue within APE2 responsible for its ubiquitination and degradation. We further characterize MKRN3 as the E3 ubiquitin ligase for APE2 ubiquitination in cells and in vitro. In summary, this study offers the first definition of the APE2 proteostasis network and lays the foundation for future studies pertaining to the posttranslational modification regulation and functions of APE2 in genome integrity and cancer etiology/treatment.