RESUMEN
Aberrant RNA splicing in keratinocytes drives inflammatory skin disorders. In the present study, we found that the RNA helicase DDX5 was downregulated in keratinocytes from the inflammatory skin lesions in patients with atopic dermatitis and psoriasis, and that mice with keratinocyte-specific deletion of Ddx5 (Ddx5∆KC) were more susceptible to cutaneous inflammation. Inhibition of DDX5 expression in keratinocytes was induced by the cytokine interleukin (IL)-17D through activation of the CD93-p38 MAPK-AKT-SMAD2/3 signaling pathway and led to pre-messenger RNA splicing events that favored the production of membrane-bound, intact IL-36 receptor (IL-36R) at the expense of soluble IL-36R (sIL-36R) and to the selective amplification of IL-36R-mediated inflammatory responses and cutaneous inflammation. Restoration of sIL-36R in Ddx5∆KC mice with experimental atopic dermatitis or psoriasis suppressed skin inflammation and alleviated the disease phenotypes. These findings indicate that IL-17D modulation of DDX5 expression controls inflammation in keratinocytes during inflammatory skin diseases.
Asunto(s)
Dermatitis Atópica , Interleucina-27 , Psoriasis , Ratones , Animales , Interleucina-27/metabolismo , Dermatitis Atópica/genética , Dermatitis Atópica/patología , Queratinocitos/metabolismo , Piel/patología , Psoriasis/genética , Psoriasis/patología , Inflamación/metabolismoRESUMEN
The mismatch repair (MMR) deficiency of cancer cells drives mutagenesis and offers a useful biomarker for immunotherapy. However, many MMR-deficient (MMR-d) tumors do not respond to immunotherapy, highlighting the need for alternative approaches to target MMR-d cancer cells. Here, we show that inhibition of the ATR kinase preferentially kills MMR-d cancer cells. Mechanistically, ATR inhibitor (ATRi) imposes synthetic lethality on MMR-d cells by inducing DNA damage in a replication- and MUS81 nuclease-dependent manner. The DNA damage induced by ATRi is colocalized with both MSH2 and PCNA, suggesting that it arises from DNA structures recognized by MMR proteins during replication. In syngeneic mouse models, ATRi effectively reduces the growth of MMR-d tumors. Interestingly, the antitumor effects of ATRi are partially due to CD8+ T cells. In MMR-d cells, ATRi stimulates the accumulation of nascent DNA fragments in the cytoplasm, activating the cGAS-mediated interferon response. The combination of ATRi and anti-PD-1 antibody reduces the growth of MMR-d tumors more efficiently than ATRi or anti-PD-1 alone, showing the ability of ATRi to augment the immunotherapy of MMR-d tumors. Thus, ATRi selectively targets MMR-d tumor cells by inducing synthetic lethality and enhancing antitumor immunity, providing a promising strategy to complement and augment MMR deficiency-guided immunotherapy.
Asunto(s)
Linfocitos T CD8-positivos , Reparación de la Incompatibilidad de ADN , Animales , Ratones , Reparación de la Incompatibilidad de ADN/genética , Mutaciones Letales Sintéticas , ADN , InmunoterapiaRESUMEN
Alternative lengthening of telomeres (ALT), a telomerase-independent process maintaining telomeres, is mediated by break-induced replication (BIR). RAD52 promotes ALT by facilitating D-loop formation, but ALT also occurs through a RAD52-independent BIR pathway. Here, we show that the telomere non-coding RNA TERRA forms dynamic telomeric R-loops and contributes to ALT activity in RAD52 knockout cells. TERRA forms R-loops in vitro and at telomeres in a RAD51AP1-dependent manner. The formation of R-loops by TERRA increases G-quadruplexes (G4s) at telomeres. G4 stabilization enhances ALT even when TERRA is depleted, suggesting that G4s act downstream of R-loops to promote BIR. In vitro, the telomeric R-loops assembled by TERRA and RAD51AP1 generate G4s, which persist after R-loop resolution and allow formation of telomeric D-loops without RAD52. Thus, the dynamic telomeric R-loops formed by TERRA and RAD51AP1 enable the RAD52-independent ALT pathway, and G4s orchestrate an R- to D-loop switch at telomeres to stimulate BIR.
Asunto(s)
ARN Largo no Codificante , Telomerasa , Homeostasis del Telómero , Telómero/genética , Telómero/metabolismo , Telomerasa/genética , Telomerasa/metabolismo , Estructuras R-Loop/genética , Reparación del ADNRESUMEN
Alternative lengthening of telomeres (ALT) is mediated by break-induced replication (BIR), but how BIR is regulated at telomeres is poorly understood. Here, we show that telomeric BIR is a self-perpetuating process. By tethering PML-IV to telomeres, we induced telomere clustering in ALT-associated PML bodies (APBs) and a POLD3-dependent ATR response at telomeres, showing that BIR generates replication stress. Ablation of BLM helicase activity in APBs abolishes telomere synthesis but causes multiple chromosome bridges between telomeres, revealing a function of BLM in processing inter-telomere BIR intermediates. Interestingly, the accumulation of BLM in APBs requires its own helicase activity and POLD3, suggesting that BIR triggers a feedforward loop to further recruit BLM. Enhancing BIR induces PIAS4-mediated TRF2 SUMOylation, and PIAS4 loss deprives APBs of repair proteins and compromises ALT telomere synthesis. Thus, a BLM-driven and PIAS4-mediated feedforward loop operates in APBs to perpetuate BIR, providing a critical mechanism to extend ALT telomeres.
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Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Retroalimentación Fisiológica , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas Inhibidoras de STAT Activados/genética , ARN Helicasas/genética , Homeostasis del Telómero , Telómero/química , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Línea Celular , Línea Celular Tumoral , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/antagonistas & inhibidores , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Cuerpos de Inclusión Intranucleares/genética , Cuerpos de Inclusión Intranucleares/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/antagonistas & inhibidores , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/metabolismo , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Transducción de Señal , Sumoilación , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/genéticaRESUMEN
R loops arising during transcription induce genomic instability, but how cells respond to the R loop-associated genomic stress is still poorly understood. Here, we show that cells harboring high levels of R loops rely on the ATR kinase for survival. In response to aberrant R loop accumulation, the ataxia telangiectasia and Rad3-related (ATR)-Chk1 pathway is activated by R loop-induced reversed replication forks. In contrast to the activation of ATR by replication inhibitors, R loop-induced ATR activation requires the MUS81 endonuclease. ATR protects the genome from R loops by suppressing transcription-replication collisions, promoting replication fork recovery, and enforcing a G2/M cell-cycle arrest. Furthermore, ATR prevents excessive cleavage of reversed forks by MUS81, revealing a MUS81-triggered and ATR-mediated feedback loop that fine-tunes MUS81 activity at replication forks. These results suggest that ATR is a key sensor and suppressor of R loop-induced genomic instability, uncovering a signaling circuitry that safeguards the genome against R loops.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Estructuras R-Loop/genética , Proteínas de la Ataxia Telangiectasia Mutada/fisiología , Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Daño del ADN , Reparación del ADN , Replicación del ADN/genética , Replicación del ADN/fisiología , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Inestabilidad Genómica/fisiología , Células HeLa , Humanos , Fosforilación , Proteínas Quinasas/metabolismo , Transducción de SeñalRESUMEN
Homologous recombination (HR) repairs DNA double-strand breaks (DSBs) in the S and G2 phases of the cell cycle1-3. Several HR proteins are preferentially recruited to DSBs at transcriptionally active loci4-10, but how transcription promotes HR is poorly understood. Here we develop an assay to assess the effect of local transcription on HR. Using this assay, we find that transcription stimulates HR to a substantial extent. Tethering RNA transcripts to the vicinity of DSBs recapitulates the effects of local transcription, which suggests that transcription enhances HR through RNA transcripts. Tethered RNA transcripts stimulate HR in a sequence- and orientation-dependent manner, indicating that they function by forming DNA-RNA hybrids. In contrast to most HR proteins, RAD51-associated protein 1 (RAD51AP1) only promotes HR when local transcription is active. RAD51AP1 drives the formation of R-loops in vitro and is required for tethered RNAs to stimulate HR in cells. Notably, RAD51AP1 is necessary for the DSB-induced formation of DNA-RNA hybrids in donor DNA, linking R-loops to D-loops. In vitro, RAD51AP1-generated R-loops enhance the RAD51-mediated formation of D-loops locally and give rise to intermediates that we term 'DR-loops', which contain both DNA-DNA and DNA-RNA hybrids and favour RAD51 function. Thus, at DSBs in transcribed regions, RAD51AP1 promotes the invasion of RNA transcripts into donor DNA, and stimulates HR through the formation of DR-loops.
Asunto(s)
ADN/genética , ADN/metabolismo , Recombinación Homóloga/genética , Estructuras R-Loop/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular , ADN/química , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Genes/genética , Genes Reporteros/genética , Proteínas Fluorescentes Verdes/genética , Humanos , Técnicas In Vitro , ARN Mensajero/química , Proteínas de Unión al ARN/metabolismo , Recombinasa Rad51/metabolismoRESUMEN
Numerous DNA repair and signaling proteins function at DNA damage sites to protect the genome. Here, we show that fusion of the promiscuous biotin ligase BirAR118G with RAD18 leads to localized protein biotinylation at DNA damage sites, allowing identification of ZPET (zinc finger protein proximal to RAD eighteen)/ZNF280C as a potential DNA damage response (DDR) protein. ZPET binds ssDNA and localizes to DNA double-strand breaks (DSBs) and stalled replication forks. In vitro, ZPET inhibits MRE11 binding to ssDNA. In cells, ZPET delays MRE11 binding to chromatin after DSB formation and slows DNA end resection through binding ssDNA. ZPET hinders resection independently of 53BP1 and HELB. Cells lacking ZPET displayed enhanced homologous recombination (HR), accelerated replication forks under stress, and increased resistance to DSBs and PARP inhibition. These results not only reveal ZPET as an HR repressor but also suggest that localized protein biotinylation at DNA damage sites is a useful strategy to identify DDR proteins.
Asunto(s)
Biotinilación/métodos , Daño del ADN , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Recombinación Homóloga/genética , Factores de Transcripción/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Línea Celular , Roturas del ADN de Doble Cadena , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli/genética , Técnicas de Silenciamiento del Gen , Humanos , Proteína Homóloga de MRE11/metabolismo , Unión Proteica , Transporte de Proteínas/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Protein function prediction based on amino acid sequence alone is an extremely challenging but important task, especially in metagenomics/metatranscriptomics field, in which novel proteins have been uncovered exponentially from new microorganisms. Many of them are extremely low homology to known proteins and cannot be annotated with homology-based or information integrative methods. To overcome this problem, we proposed a Homology Independent protein Function annotation method (HiFun) based on a unified deep-learning model by reassembling the sequence as protein language. The robustness of HiFun was evaluated using the benchmark datasets and metrics in the CAFA3 challenge. To navigate the utility of HiFun, we annotated 2 212 663 unknown proteins and discovered novel motifs in the UHGP-50 catalog. We proved that HiFun can extract latent function related structure features which empowers it ability to achieve function annotation for non-homology proteins. HiFun can substantially improve newly proteins annotation and expand our understanding of microorganisms' adaptation in various ecological niches. Moreover, we provided a free and accessible webservice at http://www.unimd.org/HiFun, requiring only protein sequences as input, offering researchers an efficient and practical platform for predicting protein functions.
Asunto(s)
Benchmarking , Lenguaje , Secuencia de Aminoácidos , Metagenómica , Anotación de Secuencia MolecularRESUMEN
RNA modifications regulate a variety of cellular processes including DNA repair.The RNA methyltransferase TRDMT1 generates methyl-5-cytosine (m5C) on messen-ger RNA (mRNA) at DNA double-strand breaks (DSBs) in transcribed regions, pro-moting transcription-coupled homologous recombination (HR). Here, we identifiedthat Fragile X mental retardation protein (FMRP) promotes transcription-coupled HRvia its interaction with both the m5C writer TRDMT1 and the m5C eraser ten-eleventranslocation protein 1 (TET1). TRDMT1, FMRP, and TET1 function in a temporalorder at the transcriptionally active sites of DSBs. FMRP displays a higher affinity forDNA:RNA hybrids containing m5C-modified RNA than for hybrids without modifica-tion and facilitates demethylation of m5C by TET1 in vitro. Loss of either the chroma-tin- or RNA-binding domain of FMRP compromises demethylation of damage-inducedm5C in cells. Importantly, FMRP is required for R-loop resolving in cells. Due to unre-solved R-loop and m5C preventing completion of DSB repair, FMRP depletion or lowexpression leads to delayed repair of DSBs at transcriptionally active sites and sensitizescancer cells to radiation in a BRCA-independent manner. Together, ourfindings presentan m5C reader, FMRP, which acts as a coordinator between the m5C writer and eraserto promote mRNA-dependent repair and cell survival in cancer.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil , Citosina , Desmetilación , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genética , Recombinación Homóloga , Humanos , Oxigenasas de Función Mixta/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , ARN/genética , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The inhibition of cell surface crystal adhesion and an appropriate increase in crystal endocytosis contribute to the inhibition of kidney stone formation. In this study, we investigated the effects of different degrees of carboxymethylation on these processes. An injury model was established by treating human renal proximal tubular epithelial (HK-2) cells with 98.3 ± 8.1 nm calcium oxalate dihydrate (nanoCOD) crystals. The HK-2 cells were protected with carboxy (-COOH) Desmodium styracifolium polysaccharides at 1.17% (DSP0), 7.45% (CDSP1), 12.2% (CDSP2), and 17.7% (CDSP3). Changes in biochemical indexes and effects on nanoCOD adhesion and endocytosis were detected. The protection of HK-2 cells from nanoCOD-induced oxidative damage by carboxymethylated Desmodium styracifolium polysaccharides (CDSPs) is closely related to the protection of subcellular organelles, such as mitochondria. CDSPs can reduce crystal adhesion on the cell surface and maintain appropriate crystal endocytosis, thereby reducing the risk of kidney stone formation. CDSP2 with moderate -COOH content showed the strongest protective activity among the CDSPs.
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Oxalato de Calcio , Endocitosis , Cálculos Renales , Polisacáridos , Humanos , Oxalato de Calcio/metabolismo , Adhesión Celular/efectos de los fármacos , Línea Celular , Cristalización , Endocitosis/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Cálculos Renales/prevención & control , Cálculos Renales/tratamiento farmacológico , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/metabolismo , Estrés Oxidativo/efectos de los fármacos , Polisacáridos/farmacología , Polisacáridos/química , Supervivencia Celular/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Calcio/metabolismo , Espacio Intracelular/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacosRESUMEN
MOTIVATION: Antibiotic resistance presents a formidable global challenge to public health and the environment. While considerable endeavors have been dedicated to identify antibiotic resistance genes (ARGs) for assessing the threat of antibiotic resistance, recent extensive investigations using metagenomic and metatranscriptomic approaches have unveiled a noteworthy concern. A significant fraction of proteins defies annotation through conventional sequence similarity-based methods, an issue that extends to ARGs, potentially leading to their under-recognition due to dissimilarities at the sequence level. RESULTS: Herein, we proposed an Artificial Intelligence-powered ARG identification framework using a pretrained large protein language model, enabling ARG identification and resistance category classification simultaneously. The proposed PLM-ARG was developed based on the most comprehensive ARG and related resistance category information (>28K ARGs and associated 29 resistance categories), yielding Matthew's correlation coefficients (MCCs) of 0.983 ± 0.001 by using a 5-fold cross-validation strategy. Furthermore, the PLM-ARG model was verified using an independent validation set and achieved an MCC of 0.838, outperforming other publicly available ARG prediction tools with an improvement range of 51.8%-107.9%. Moreover, the utility of the proposed PLM-ARG model was demonstrated by annotating resistance in the UniProt database and evaluating the impact of ARGs on the Earth's environmental microbiota. AVAILABILITY AND IMPLEMENTATION: PLM-ARG is available for academic purposes at https://github.com/Junwu302/PLM-ARG, and a user-friendly webserver (http://www.unimd.org/PLM-ARG) is also provided.
Asunto(s)
Antibacterianos , Inteligencia Artificial , Antibacterianos/farmacología , Farmacorresistencia Microbiana/genética , Genes Bacterianos , MetagenomaRESUMEN
Energy efficiency and security issues are the main concerns in wireless sensor networks (WSNs) because of limited energy resources and the broadcast nature of wireless communication. Therefore, how to improve the energy efficiency of WSNs while enhancing security performance has attracted widespread attention. In order to solve this problem, this paper proposes a new deep reinforcement learning (DRL)-based strategy, i.e., DeepNR strategy, to enhance the energy efficiency and security performance of WSN. Specifically, the proposed DeepNR strategy approximates the Q-value by designing a deep neural network (DNN) to adaptively learn the state information. It also designs DRL-based multi-level decision-making to learn and optimize the data transmission paths in real time, which eventually achieves accurate prediction and decision-making of the network. To further enhance security performance, the DeepNR strategy includes a defense mechanism that responds to detected attacks in real time to ensure the normal operation of the network. In addition, DeepNR adaptively adjusts its strategy to cope with changing network environments and attack patterns through deep learning models. Experimental results show that the proposed DeepNR outperforms the conventional methods, demonstrating a remarkable 30% improvement in network lifespan, a 25% increase in network data throughput, and a 20% enhancement in security measures.
RESUMEN
Petanin, an acylated anthocyanin from the Solanaceae family, shows potential in tyrosinase inhibitory activity and anti-melanogenic effects; however, its mechanism remains unclear. Therefore, to investigate the underlying mechanism of petanin's anti-melanogenic effects, the enzyme activity, protein expression and mRNA transcription of melanogenic and related signaling pathways in zebrafish using network pharmacology, molecular docking and molecular dynamics simulation were combined for analysis. The results showed that petanin could inhibit tyrosinase activity and melanogenesis, change the distribution and arrangement of melanocytes and the structure of melanosomes, reduce the activities of catalase (CAT) and peroxidase (POD) and enhance the activity of glutathione reductase (GR). It also up-regulated JNK phosphorylation, inhibited ERK/RSK phosphorylation and down-regulated CREB/MITF-related protein expression and mRNA transcription. These results were consistent with the predictions provided through network pharmacology and molecular docking. Thus, petanin could inhibit the activity of tyrosinase and the expression of tyrosinase by inhibiting and negatively regulating the tyrosinase-related signaling pathway ERK/CREB/MITF through p-JNK. In conclusion, petanin is a good tyrosinase inhibitor and anti-melanin natural compound with significant market prospects in melanogenesis-related diseases and skin whitening cosmetics.
Asunto(s)
Melaninas , Simulación del Acoplamiento Molecular , Pez Cebra , Animales , Pez Cebra/metabolismo , Melaninas/metabolismo , Melaninas/biosíntesis , Fosforilación , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Monofenol Monooxigenasa/metabolismo , Monofenol Monooxigenasa/antagonistas & inhibidores , Factor de Transcripción Asociado a Microftalmía/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Melanocitos/metabolismo , Melanocitos/efectos de los fármacosRESUMEN
To explore the composition of anthocyanins and expand their biological activities, anthocyanins were systematically isolated and purified from tubers of Solanum tuberosum L., and their tyrosinase inhibitory activity was investigated. In this study, two new anthocyanin degradation compounds, norpetanin (9) and 4-O-(p-coumaryl) rhamnose (10), along with 17 known anthocyanins and their derivatives, were isolated and purified from an acid-ethanolic extract of fresh purple potato tubers. Their structures were elucidated via 1D and 2D NMR and HR-ESI-MS and compared with those reported in the literature. The extracts were evaluated for anthocyanins and their derivatives using a tyrosinase inhibitor screening kit and molecular docking technology, and the results showed that petanin, norpetanin, 4-O-(p-coumaryl) rhamnose, and lyciruthephenylpropanoid D/E possessed tyrosinase inhibitory activity, with 50% inhibiting concentration (IC50) values of 122.37 ± 8.03, 115.53 ± 7.51, 335.03 ± 12.99, and 156.27 ± 11.22 µM (Mean ± SEM, n = 3), respectively. Furthermore, petanin was validated against melanogenesis in zebrafish; it was found that it could significantly inhibit melanin pigmentation (p < 0.001), and the inhibition rate of melanin was 17% compared with the normal group. This finding may provide potential treatments for diseases with abnormal melanin production, and high-quality raw materials for whitening cosmetics.
Asunto(s)
Antocianinas , Solanum tuberosum , Animales , Antocianinas/farmacología , Monofenol Monooxigenasa , Melaninas , Simulación del Acoplamiento Molecular , Ramnosa , Pez CebraRESUMEN
This corrects the article DOI: 10.1038/nature21671.
RESUMEN
Cell proliferation and survival require the faithful maintenance and propagation of genetic information, which are threatened by the ubiquitous sources of DNA damage present intracellularly and in the external environment. A system of DNA repair, called the DNA damage response, detects and repairs damaged DNA and prevents cell division until the repair is complete. Here we report that methylation at the 6 position of adenosine (m6A) in RNA is rapidly (within 2 min) and transiently induced at DNA damage sites in response to ultraviolet irradiation. This modification occurs on numerous poly(A)+ transcripts and is regulated by the methyltransferase METTL3 (methyltransferase-like 3) and the demethylase FTO (fat mass and obesity-associated protein). In the absence of METTL3 catalytic activity, cells showed delayed repair of ultraviolet-induced cyclobutane pyrimidine adducts and elevated sensitivity to ultraviolet, demonstrating the importance of m6A in the ultraviolet-responsive DNA damage response. Multiple DNA polymerases are involved in the ultraviolet response, some of which resynthesize DNA after the lesion has been excised by the nucleotide excision repair pathway, while others participate in trans-lesion synthesis to allow replication past damaged lesions in S phase. DNA polymerase κ (Pol κ), which has been implicated in both nucleotide excision repair and trans-lesion synthesis, required the catalytic activity of METTL3 for immediate localization to ultraviolet-induced DNA damage sites. Importantly, Pol κ overexpression qualitatively suppressed the cyclobutane pyrimidine removal defect associated with METTL3 loss. Thus, we have uncovered a novel function for RNA m6A modification in the ultraviolet-induced DNA damage response, and our findings collectively support a model in which m6A RNA serves as a beacon for the selective, rapid recruitment of Pol κ to damage sites to facilitate repair and cell survival.
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Daño del ADN/efectos de la radiación , Metilación , ARN/química , ARN/metabolismo , Rayos Ultravioleta , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Animales , Biocatálisis/efectos de la radiación , Línea Celular , Supervivencia Celular/efectos de la radiación , Reparación del ADN/efectos de la radiación , Replicación del ADN/efectos de la radiación , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Metilación/efectos de la radiación , Metiltransferasas/deficiencia , Metiltransferasas/metabolismo , Ratones , Poli A/metabolismo , ARN/efectos de la radiación , Fase S/efectos de la radiaciónRESUMEN
SLX4, a coordinator of multiple DNA structure-specific endonucleases, is important for several DNA repair pathways. Noncovalent interactions of SLX4 with ubiquitin are required for localizing SLX4 to DNA interstrand crosslinks (ICLs), yet how SLX4 is targeted to other functional contexts remains unclear. Here, we show that SLX4 binds SUMO-2/3 chains via SUMO-interacting motifs (SIMs). The SIMs of SLX4 are dispensable for ICL repair but important for processing CPT-induced replication intermediates, suppressing fragile site instability, and localizing SLX4 to ALT telomeres. The localization of SLX4 to laser-induced DNA damage also requires the SIMs, as well as DNA end resection, UBC9, and MDC1. Furthermore, the SUMO binding of SLX4 enhances its interaction with specific DNA-damage sensors or telomere-binding proteins, including RPA, MRE11-RAD50-NBS1, and TRF2. Thus, the interactions of SLX4 with SUMO and ubiquitin increase its affinity for factors recognizing different DNA lesions or telomeres, helping to direct the SLX4 complex in distinct functional contexts.
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Genoma , Recombinasas/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Daño del ADN , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Recombinasas/genética , Alineación de Secuencia , Transducción de Señal , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Telómero/efectos de la radiación , Ubiquitina/genética , Enzimas Ubiquitina-Conjugadoras/genética , Ubiquitinas/genética , Rayos UltravioletaRESUMEN
Integration analysis of multi-omics data provides a comprehensive landscape for understanding biological systems and mechanisms. The abundance of high-quality multi-omics data (genomics, transcriptomics, methylomics and phenomics) for the model organism Arabidopsis thaliana enables scientists to study the genetic mechanism of many biological processes. However, no resource is available to provide comprehensive and systematic multi-omics associations for Arabidopsis. Here, we developed an Arabidopsis thaliana Multi-omics Association Database (AtMAD, http://www.megabionet.org/atmad), a public repository for large-scale measurements of associations between genome, transcriptome, methylome, pathway and phenotype in Arabidopsis, designed for facilitating identification of eQTL, emQTL, Pathway-mQTL, Phenotype-pathway, GWAS, TWAS and EWAS. Candidate variants/methylations/genes were identified in AtMAD for specific phenotypes or biological processes, many of them are supported by experimental evidence. Based on the multi-omics association strategy, we have identified 11 796 cis-eQTLs and 10 119 trans-eQTLs. Among them, 68 837 environment-eQTL associations and 149 622 GWAS-eQTL associations were identified and stored in AtMAD. For expression-methylation quantitative trait loci (emQTL), we identified 265 776 emQTLs and 122 344 pathway-mQTLs. For TWAS and EWAS, we obtained 62 754 significant phenotype-gene associations and 3 993 379 significant phenotype-methylation associations, respectively. Overall, the multi-omics associated network in AtMAD will provide new insights into exploring biological mechanisms of plants at multi-omics levels.
Asunto(s)
Arabidopsis/genética , Biología Computacional/métodos , Bases de Datos Genéticas , Estudios de Asociación Genética , Genoma de Planta , Arabidopsis/metabolismo , Minería de Datos , Redes Reguladoras de Genes , Interacción Gen-Ambiente , Estudio de Asociación del Genoma Completo , Genotipo , Internet , Metaboloma , Anotación de Secuencia Molecular , Fenotipo , Programas Informáticos , TranscriptomaRESUMEN
Objective: To explore and analyze the efficacy of luteolin on the proliferation and apoptosis of cerebral glioma and its possible mechanism, providing a theoretical basis for luteolin in glioma treatment. Methods: The cell line of Human cerebral glioma U87 was utilized for our current research. The study group were added with 0, 20, 40, and 80 µmol/L luteolin, respectively. Luteolin's Inhibitory function in the proliferation of U87 cells was detected by CCK-8, the Transwell chamber test was used to measure cell migration and invasion, while flow cytometry was used to assess apoptosis and Western Blot to gauge the expression of JNK/STAT3 pathway proteins. Results: In comparison with the control group, the intervention of various doses of luteolin could effectively inhibit glioma cell proliferation, the inhibitory rate uplifted remarkably with an increase of dose as well as intervention time (95% CI. 92.160-107.494, P < .05), which presented a significant time and dose dependence. The apoptosis rate of the low-dose, medium-dose, and high-dose categories was higher than that of the comparison group after 2 days of luteolin intervention (P < .05), and the apoptosis rate appeared to increase with the increase in intervention dose (P < .05). After 2 days of luteolin intervention, there were significantly more migratory and invasive cells in 3 categories than that in the control group (P < .05), and the number increased as the luteolin intervention dose was raised (P < .05). Bax and Cyt-c expressions dropped after 2 days of luteolin treatment when compared with the control set (P < .05), and the expression of Bax and Cyt-c fell considerably with increasing luteolin dose. Bcl-2, Caspase-3 and PARP expressions were significantly elevated in comparison to the control group (P < .05), and the increase was more obvious with the rise of the luteolin dose. Conclusion: Luteolin has a good inhibiting function on the proliferation of glioma cells, inhibiting cellular invasion and migration and promoting the apoptosis of glioma cells and the expression of apoptosis-related proteins, which has laid a good foundation for the application of luteolin to treat glioma cells.
RESUMEN
Doxorubicin (Dox) is one of the most frequently prescribed anti-cancer drugs. However, treatment with Dox is limited due to cumulative cardiotoxicity. 3-O-ß-d-Sophorosylkaempferol-7-O-{3-O-[2(E)-2,6-dimethyl-6-hydroxyocta-2,7-dienoyl]}-α-L-rhamnoside (F-A), kaempferol 3-sophoroside 7-rhamnoside (F-B), and hippophanone (F-C) were successfully obtained by purification and separation of seabuckthorn seed residue in our previous research. This study was undertaken to investigate the protective effect of three flavonoids against Dox-induced H9c2 cell apoptosis. Cell proliferation was detected by MTT assay. 2',7'-Dichlorofluorescein diacetate (DCFH-DA) was used to determine the production of intracellular reactive oxygen species (ROS). ATP content was measured using an assay kit. Transmission electron microscopy (TEM) was used to observe changes in mitochondrial ultrastructure. The expression levels of proteins (p-JNK, JNK, p-Akt, Akt, p-P38, P38, p-ERK, ERK, p-Src, Src, Sab, IRE1α, Mfn1, Mfn2, and cleaved caspase-3) were evaluated by Western blot. Molecular docking was performed using AutoDock Vina. The three flavonoids could significantly relieve Dox-induced cardiac injury and inhibit cardiomyocyte apoptosis. The mechanisms were mainly related to the stability of mitochondrial structure and function maintained by suppressing the production of intracellular ROS, p-JNK and cleaved caspase-3, and increasing ATP contents and protein expression of mitochondrial mitofusin (Mfn1, Mfn2), Sab and p-Src. Pretreatment with flavonoids from Hippophae rhamnoides Linn. can reduce Dox-induced H9c2 cell apoptosis based on the 'JNK-Sab-Ros' signal pathway.