RESUMO
Lifespan varies within and across species, but the general principles of its control remain unclear. Here, we conducted multi-tissue RNA-seq analyses across 41 mammalian species, identifying longevity signatures and examining their relationship with transcriptomic biomarkers of aging and established lifespan-extending interventions. An integrative analysis uncovered shared longevity mechanisms within and across species, including downregulated Igf1 and upregulated mitochondrial translation genes, and unique features, such as distinct regulation of the innate immune response and cellular respiration. Signatures of long-lived species were positively correlated with age-related changes and enriched for evolutionarily ancient essential genes, involved in proteolysis and PI3K-Akt signaling. Conversely, lifespan-extending interventions counteracted aging patterns and affected younger, mutable genes enriched for energy metabolism. The identified biomarkers revealed longevity interventions, including KU0063794, which extended mouse lifespan and healthspan. Overall, this study uncovers universal and distinct strategies of lifespan regulation within and across species and provides tools for discovering longevity interventions.
Assuntos
Longevidade , Fosfatidilinositol 3-Quinases , Animais , Camundongos , Longevidade/genética , Fosfatidilinositol 3-Quinases/genética , Envelhecimento/genética , Mamíferos/genética , Perfilação da Expressão GênicaRESUMO
Telomerase maintains chromosome ends from humans to yeasts. Recruitment of yeast telomerase to telomeres occurs through its Ku and Est1 subunits via independent interactions with telomerase RNA (TLC1) and telomeric proteins Sir4 and Cdc13, respectively. However, the structures of the molecules comprising these telomerase-recruiting pathways remain unknown. Here, we report crystal structures of the Ku heterodimer and Est1 complexed with their key binding partners. Two major findings are as follows: (1) Ku specifically binds to telomerase RNA in a distinct, yet related, manner to how it binds DNA; and (2) Est1 employs two separate pockets to bind distinct motifs of Cdc13. The N-terminal Cdc13-binding site of Est1 cooperates with the TLC1-Ku-Sir4 pathway for telomerase recruitment, whereas the C-terminal interface is dispensable for binding Est1 in vitro yet is nevertheless essential for telomere maintenance in vivo. Overall, our results integrate previous models and provide fundamentally valuable structural information regarding telomere biology.
Assuntos
Proteínas de Ligação a DNA/química , Simulação de Acoplamento Molecular , Proteínas de Saccharomyces cerevisiae/química , Telomerase/química , Homeostase do Telômero , Proteínas de Ligação a Telômeros/química , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ligação Proteica , RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Telomerase/genética , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Non-homologous end joining is the major double-strand break repair (DSBR) pathway in mammals. DNA-PK is the hub and organizer of multiple steps in non-homologous end joining (NHEJ). Recent high-resolution structures show how two distinct NHEJ complexes "synapse" two DNA ends. One complex includes a DNA-PK dimer mediated by XLF, whereas a distinct DNA-PK dimer forms via a domain-swap mechanism where the C terminus of Ku80 from one DNA-PK protomer interacts with another DNA-PK protomer in trans. Remarkably, the distance between the two synapsed DNA ends in both dimers is the same (â¼115 Å), which matches the distance observed in the initial description of an NHEJ long-range synaptic complex. Here, a mutational strategy is used to demonstrate distinct cellular function(s) of the two dimers: one promoting fill-in end processing, while the other promotes DNA end resection. Thus, the specific DNA-PK dimer formed (which may be impacted by DNA end structure) dictates the mechanism by which ends will be made ligatable.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Animais , Proteínas de Ligação a DNA/genética , Subunidades Proteicas/metabolismo , Reparo do DNA por Junção de Extremidades , Reparo do DNA , DNA/genética , Proteína Quinase Ativada por DNA/genética , Autoantígeno Ku/genética , Mamíferos/metabolismoRESUMO
Aging is associated with DNA accumulation and increased homeostatic proliferation of circulating T cells. Although these attributes are associated with aging-related autoimmunity, their direct contributions remain unclear. Conventionally, KU complex, the regulatory subunit of DNA-dependent protein kinase (DNA-PK), together with the catalytic subunit of DNA-PK (DNA-PKcs), mediates DNA damage repair in the nucleus. Here, we found KU complex abundantly expressed in the cytoplasm, where it recognized accumulated cytoplasmic DNA in aged human and mouse CD4+ T cells. This process enhanced T cell activation and pathology of experimental autoimmune encephalomyelitis (EAE) in aged mice. Mechanistically, KU-mediated DNA sensing facilitated DNA-PKcs recruitment and phosphorylation of the kinase ZAK. This activated AKT and mTOR pathways, promoting CD4+ T cell proliferation and activation. We developed a specific ZAK inhibitor, which dampened EAE pathology in aged mice. Overall, these findings demonstrate a KU-mediated cytoplasmic DNA-sensing pathway in CD4+ T cells that potentiates aging-related autoimmunity.
Assuntos
Envelhecimento/imunologia , Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/imunologia , Citoplasma/imunologia , Proteína Quinase Ativada por DNA/imunologia , DNA/imunologia , Inflamação/imunologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/imunologia , Proliferação de Células/fisiologia , Reparo do DNA/imunologia , Células HEK293 , Humanos , Células Jurkat , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Células U937RESUMO
The DNA-dependent protein kinase (DNA-PK) initially protects broken DNA ends but then promotes their processing during non-homologous end joining (NHEJ). Before ligation by NHEJ, DNA hairpin ends generated during V(D)J recombination must be opened by the Artemis nuclease, together with autophosphorylated DNA-PK. Structures of DNA-PK bound to DNA before and after phosphorylation, and in complex with Artemis and a DNA hairpin, reveal an essential functional switch. When bound to open DNA ends in its protection mode, DNA-PK is inhibited for cis-autophosphorylation of the so-called ABCDE cluster but activated for phosphorylation of other targets. In contrast, DNA hairpin ends promote cis-autophosphorylation. Phosphorylation of four Thr residues in ABCDE leads to gross structural rearrangement of DNA-PK, widening the DNA binding groove for Artemis recruitment and hairpin cleavage. Meanwhile, Artemis locks DNA-PK into the kinase-inactive state. Kinase activity and autophosphorylation of DNA-PK are regulated by different DNA ends, feeding forward to coordinate NHEJ events.
Assuntos
Dano ao DNA , Reparo do DNA por Junção de Extremidades , DNA de Neoplasias/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Neoplasias do Colo do Útero/enzimologia , DNA de Neoplasias/genética , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Ativação Enzimática , Feminino , Células HEK293 , Células HeLa , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Conformação de Ácido Nucleico , Fosforilação , Ligação Proteica , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
DNA-dependent protein kinase (DNA-PK), like all phosphatidylinositol 3-kinase-related kinases (PIKKs), is composed of conserved FAT and kinase domains (FATKINs) along with solenoid structures made of HEAT repeats. These kinases are activated in response to cellular stress signals, but the mechanisms governing activation and regulation remain unresolved. For DNA-PK, all existing structures represent inactive states with resolution limited to 4.3 Å at best. Here, we report the cryoelectron microscopy (cryo-EM) structures of DNA-PKcs (DNA-PK catalytic subunit) bound to a DNA end or complexed with Ku70/80 and DNA in both inactive and activated forms at resolutions of 3.7 Å overall and 3.2 Å for FATKINs. These structures reveal the sequential transition of DNA-PK from inactive to activated forms. Most notably, activation of the kinase involves previously unknown stretching and twisting within individual solenoid segments and loosens DNA-end binding. This unprecedented structural plasticity of helical repeats may be a general regulatory mechanism of HEAT-repeat proteins.
Assuntos
Reparo do DNA por Junção de Extremidades , Proteína Quinase Ativada por DNA/química , Autoantígeno Ku/química , Complexos Multiproteicos/química , Microscopia Crioeletrônica , Proteína Quinase Ativada por DNA/genética , Ativação Enzimática , Células HEK293 , Células HeLa , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestruturaRESUMO
The characteristics of the sleep drivers and the mechanisms through which sleep relieves the cellular homeostatic pressure are unclear. In flies, zebrafish, mice, and humans, DNA damage levels increase during wakefulness and decrease during sleep. Here, we show that 6 h of consolidated sleep is sufficient to reduce DNA damage in the zebrafish dorsal pallium. Induction of DNA damage by neuronal activity and mutagens triggered sleep and DNA repair. The activity of the DNA damage response (DDR) proteins Rad52 and Ku80 increased during sleep, and chromosome dynamics enhanced Rad52 activity. The activity of the DDR initiator poly(ADP-ribose) polymerase 1 (Parp1) increased following sleep deprivation. In both larva zebrafish and adult mice, Parp1 promoted sleep. Inhibition of Parp1 activity reduced sleep-dependent chromosome dynamics and repair. These results demonstrate that DNA damage is a homeostatic driver for sleep, and Parp1 pathways can sense this cellular pressure and facilitate sleep and repair activity.
Assuntos
Comportamento Animal , Encéfalo , Dano ao DNA , Reparo do DNA , Neurônios , Poli(ADP-Ribose) Polimerase-1 , Sono , Proteínas de Peixe-Zebra , Animais , Feminino , Masculino , Animais Geneticamente Modificados , Encéfalo/enzimologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Camundongos Endogâmicos C57BL , Neurônios/enzimologia , Neurônios/patologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/fisiologia , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Fatores de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Several compounds have been used for atherosclerosis treatment, including clinical trials; however, no anti-atherosclerotic drugs based on hemodynamic force-mediated atherogenesis have been discovered. Our previous studies demonstrated that "small mothers against decapentaplegic homolog 1/5" (Smad1/5) is a convergent signaling molecule for chemical [e.g., bone morphogenetic proteins (BMPs)] and mechanical (e.g., disturbed flow) stimulations and hence may serve as a promising hemodynamic-based target for anti-atherosclerosis drug development. The goal of this study was to develop a high-throughput screening (HTS) platform to identify potential compounds that can inhibit disturbed flow- and BMP-induced Smad1/5 activation and atherosclerosis. Through HTS using a Smad1/5 downstream target inhibitor of DNA binding 1 (Id-1) as a luciferase reporter, we demonstrated that KU-55933 and Apicidin suppressed Id-1 expression in AD-293 cells. KU-55933 (10 µM), Apicidin (10 µM), and the combination of half doses of each [1/2(K + A)] inhibited disturbed flow- and BMP4-induced Smad1/5 activation in human vascular endothelial cells (ECs). KU-55933, Apicidin, and 1/2(K + A) treatments caused 50.6%, 47.4%, and 73.3% inhibitions of EC proliferation induced by disturbed flow, respectively, whereas EC inflammation was only suppressed by KU-55933 and 1/2(K + A), but not Apicidin alone. Administrations of KU-55933 and 1/2(K + A) to apolipoprotein E-deficient mice inhibited Smad1/5 activation in ECs in athero-susceptible regions, thereby suppressing endothelial proliferation and inflammation, with the attenuation of atherosclerotic lesions in these mice. A unique drug screening platform has been developed to demonstrate that KU-55933 and its combination with Apicidin are promising therapeutic compounds for atherosclerosis based on hemodynamic considerations.
Assuntos
Aterosclerose , Células Endoteliais , Morfolinas , Pironas , Humanos , Animais , Camundongos , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Aterosclerose/tratamento farmacológico , Hemodinâmica , InflamaçãoRESUMO
Whether cell types exposed to a high level of environmental insults possess cell type-specific prosurvival mechanisms or enhanced DNA damage repair capacity is not well understood. BRN2 is a tissue-restricted POU domain transcription factor implicated in neural development and several cancers. In melanoma, BRN2 plays a key role in promoting invasion and regulating proliferation. Here we found, surprisingly, that rather than interacting with transcription cofactors, BRN2 is instead associated with DNA damage response proteins and directly binds PARP1 and Ku70/Ku80. Rapid PARP1-dependent BRN2 association with sites of DNA damage facilitates recruitment of Ku80 and reprograms DNA damage repair by promoting Ku-dependent nonhomologous end-joining (NHEJ) at the expense of homologous recombination. BRN2 also suppresses an apoptosis-associated gene expression program to protect against UVB-, chemotherapy- and vemurafenib-induced apoptosis. Remarkably, BRN2 expression also correlates with a high single-nucleotide variation prevalence in human melanomas. By promoting error-prone DNA damage repair via NHEJ and suppressing apoptosis of damaged cells, our results suggest that BRN2 contributes to the generation of melanomas with a high mutation burden. Our findings highlight a novel role for a key transcription factor in reprogramming DNA damage repair and suggest that BRN2 may impact the response to DNA-damaging agents in BRN2-expressing cancers.
Assuntos
Apoptose , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Homeodomínio/metabolismo , Melanoma/genética , Melanoma/fisiopatologia , Mutação/genética , Fatores do Domínio POU/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Homeodomínio/genética , Humanos , Autoantígeno Ku/metabolismo , Fatores do Domínio POU/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ligação Proteica , Domínios Proteicos , Transporte ProteicoRESUMO
Chromosomal deletion rearrangements mediated by repetitive elements often involve repeats separated by several kilobases and sequences that are divergent. While such rearrangements are likely induced by DNA double-strand breaks (DSBs), it has been unclear how the proximity of DSBs relative to repeat sequences affects the frequency of such events. We generated a reporter assay in mouse cells for a deletion rearrangement involving repeats separated by 0.4 Mb. We induced this repeat-mediated deletion (RMD) rearrangement with two DSBs: the 5' DSB that is just downstream from the first repeat and the 3' DSB that is varying distances upstream of the second repeat. Strikingly, we found that increasing the 3' DSB/repeat distance from 3.3 kb to 28.4 kb causes only a modest decrease in rearrangement frequency. We also found that RMDs are suppressed by KU70 and RAD51 and promoted by RAD52, CtIP, and BRCA1. In addition, we found that 1%-3% sequence divergence substantially suppresses these rearrangements in a manner dependent on the mismatch repair factor MSH2, which is dominant over the suppressive role of KU70. We suggest that a DSB far from a repeat can stimulate repeat-mediated rearrangements, but multiple pathways suppress these events.
Assuntos
Quebra Cromossômica , Deleção Cromossômica , Sequências Repetitivas de Ácido Nucleico , Animais , DNA/química , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Autoantígeno Ku/fisiologia , Camundongos , Proteína 2 Homóloga a MutS/fisiologia , Rad51 Recombinase/fisiologia , Proteína Rad52 de Recombinação e Reparo de DNA/fisiologia , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/fisiologiaRESUMO
Deficiency in chromatin assembly factor-1 (CAF-1) in plants through dysfunction of its components, FASCIATA1 and 2 (FAS1, FAS2), leads to the specific and progressive loss of rDNA and telomere repeats in plants. This loss is attributed to defective repair mechanisms for the increased DNA breaks encountered during replication, a consequence of impaired replication-dependent chromatin assembly. In this study, we explore the role of KU70 in these processes. Our findings reveal that, although the rDNA copy number is reduced in ku70 mutants when compared with wild-type plants, it is not markedly affected by diverse KU70 status in fas1 mutants. This is consistent with our previous characterisation of rDNA loss in fas mutants as a consequence part of the single-strand annealing pathway of homology-dependent repair. In stark contrast to rDNA, KU70 dysfunction fully suppresses the loss of telomeres in fas1 plants and converts telomeres to their elongated and heterogeneous state typical for ku70 plants. We conclude that the alternative telomere lengthening pathway, known to be activated in the absence of KU70, overrides progressive telomere loss due to CAF-1 dysfunction.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fator 1 de Modelagem da Cromatina , Proteínas de Ligação a DNA , Homeostase do Telômero , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fator 1 de Modelagem da Cromatina/metabolismo , Fator 1 de Modelagem da Cromatina/genética , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Mutação , Telômero/metabolismo , Telômero/genética , Cromossomos de Plantas/metabolismoRESUMO
DNA double-strand break (DSB) repair is essential for maintaining our genomes. Mre11-Rad50-Nbs1 (MRN) and Ku70-Ku80 (Ku) direct distinct DSB repair pathways, but the interplay between these complexes at a DSB remains unclear. Here, we use high-throughput single-molecule microscopy to show that MRN searches for free DNA ends by one-dimensional facilitated diffusion, even on nucleosome-coated DNA. Rad50 binds homoduplex DNA and promotes facilitated diffusion, whereas Mre11 is required for DNA end recognition and nuclease activities. MRN gains access to occluded DNA ends by removing Ku or other DNA adducts via an Mre11-dependent nucleolytic reaction. Next, MRN loads exonuclease 1 (Exo1) onto the free DNA ends to initiate DNA resection. In the presence of replication protein A (RPA), MRN acts as a processivity factor for Exo1, retaining the exonuclease on DNA for long-range resection. Our results provide a mechanism for how MRN promotes homologous recombination on nucleosome-coated DNA.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/enzimologia , Reparo de DNA por Recombinação , Imagem Individual de Molécula , Hidrolases Anidrido Ácido , Proteínas de Ciclo Celular/genética , Adutos de DNA/genética , Adutos de DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Difusão , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Proteína Homóloga a MRE11 , Microscopia de Fluorescência , Proteínas Nucleares/genética , Nucleossomos/genética , Fatores de TempoRESUMO
The budding yeast Mre11-Rad50-Xrs2 (MRX) complex and Sae2 function together in DNA end resection during homologous recombination. Here we show that the Ku complex shields DNA ends from exonucleolytic digestion but facilitates endonucleolytic scission by MRX with a dependence on ATP and Sae2. The incision site is enlarged into a DNA gap via the exonuclease activity of MRX, which is stimulated by Sae2 without ATP being present. RPA renders a partially resected or palindromic DNA structure susceptible to MRX-Sae2, and internal protein blocks also trigger DNA cleavage. We present models for how MRX-Sae2 creates entry sites for the long-range resection machinery.
Assuntos
Reparo do DNA por Junção de Extremidades , Reparo do DNA/fisiologia , Endonucleases/metabolismo , Exonucleases/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Clivagem do DNA , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Ativação Enzimática/genética , Exodesoxirribonucleases/metabolismo , Complexos Multiproteicos/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The yeast Mre11-Rad50-Xrs2 (MRX) complex and Sae2 function together to initiate DNA end resection, an essential early step in homology-dependent repair of DNA double-strand breaks (DSBs). In this issue of Genes & Development, Wang and colleagues (pp. 2331-2336) and Reginato and colleagues (pp. 2325-2330) report that a variety of physiological protein blocks, including Ku, RPA, and nucleosomes, stimulate MRX-Sae2 endonuclease cleavage in vitro. These studies have important implications for how cells deal with a range of barriers to end resection and highlight the crucial role of Sae2 in activating MRX cleavage at the correct cell cycle stage.
Assuntos
Endodesoxirribonucleases/genética , Proteínas de Saccharomyces cerevisiae/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/genética , Exodesoxirribonucleases/genética , Saccharomyces cerevisiae/genéticaRESUMO
DNA double-strand break repair by homologous recombination is initiated by DNA end resection, which is commenced by the Mre11-Rad50-Xrs2 complex and Sae2 in yeast. Here we report that the nonhomologous end joining factor Ku limits the exonuclease activity of Mre11 and promotes its endonuclease to cleave 5'-terminated DNA strands at break sites. Following initial endonucleolytic cleavage past the obstacle, Exo1 specifically extends the resection track, leading to the generation of long 3' overhangs that are required for homologous recombination. These experiments provide mechanistic insights into how short-range and long-range DNA end resection enzymes overcome obstacles near broken DNA ends to initiate recombination.
Assuntos
Reparo do DNA por Junção de Extremidades , Endonucleases/metabolismo , Exonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Animais , Clivagem do DNA , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Ativação Enzimática/genética , Exodesoxirribonucleases/metabolismo , Complexos Multiproteicos/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Células Sf9RESUMO
Radiotherapy is one of the mainstay treatments for hepatocellular carcinoma (HCC). However, a substantial number of patients with HCC develop radioresistance and eventually suffer from tumor progression or relapse, which is a major impediment to the use of radiotherapy. Therefore, elucidating the mechanisms underlying radioresistance and identifying novel therapeutic targets to improve patient prognosis are important in HCC management. In this study, using in vitro and in vivo models, laser microirradiation and live cell imaging methods, and coimmunoprecipitation assays, we report that a DNA repair enhancer, human positive cofactor 4 (PC4), promotes nonhomologous end joining-based DNA repair and renders HCC cells resistant to radiation. Mechanistically, PC4 interacts with poly (ADP-ribose) polymerase 1 and directs Ku complex PARylation, resulting in the successful recruitment of the Ku complex to damaged chromatin and increasing the efficiency of nonhomologous end joining repair. Clinically, PC4 is highly expressed in tumor tissues and is correlated with poor prognosis in patients with HCC. Taken together, our data suggest that PC4 is a DNA repair driver that can be targeted to radiosensitize HCC cells.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/radioterapia , Dano ao DNA , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Neoplasias Hepáticas/genética , Recidiva Local de Neoplasia , Poli ADP Ribosilação , Tolerância a RadiaçãoRESUMO
Komagataella phaffii (Pichia pastoris) is a methylotrophic yeast that is favored by industry and academia mainly for expression of heterologous proteins. However, its full potential as a host for bio-production of valuable compounds cannot be fully exploited as genetic tools are lagging behind those that are available for baker's yeast. The emergence of CRISPR-Cas9 technology has significantly improved the efficiency of gene manipulations of K. phaffii, but improvements in gene-editing methods are desirable to further accelerate engineering of this yeast. In this study, we have developed a versatile vector-based CRISPR-Cas9 method and showed that it works efficiently at different genetic loci using linear DNA fragments with very short targeting sequences including single-stranded oligonucleotides. Notably, we performed site-specific point mutations and full gene deletions using short (90 nt) single-stranded oligonucleotides at very high efficiencies. Lastly, we present a strategy for transient inactivation of non-homologous end-joining (NHEJ) pathway, where KU70 gene is disrupted by a visual marker (uidA gene). This system enables precise CRISPR-Cas9 based editing (including multiplexing) and facilitates simple reversion to NHEJ-proficient genotype. In conclusion, the tools presented in this study can be applied for easy and efficient engineering of K. phaffii strains and are compatible with high-throughput automated workflows.
RESUMO
Cerebral ischemic stroke is a cerebrovascular disease, which is related to DNA damage. Many researches have shown that Ku70 is a key regulator for DNA damage. Here, we aimed to explore Ku70 roles in cerebral ischemic stroke and its potential molecular mechanism. In our study, neural stem cells (NSCs) were induced by oxygen-glucose deprivation/reoxygenation (OGD/R) for constructing cerebral ischemic stroke cell model. CCK8 assay, Brdu/GFP staining, flow cytometry and TUNEL staining were performed to examine cell proliferation, cell cycle and apoptosis, respectively. Relative mRNA and protein levels were detected by quantitative real-time PCR and western blot analysis, respectively. Ku70 positive cells were examined by immunofluorescence staining. Comet assay was employed to determine DNA damage. Animal experiments were performed to assess the effect of transplanting NSCs and Ku70-overexpressed NSCs on neurological deficits, infarct volume, brain edema and bloodâbrain barrier (BBB) integrity in middle cerebral artery occlusion (MCAO) model. Our data found that Ku70 expression was decreased in NSCs after OGD/R. Overexpression of Ku70 reduced DNA damage and apoptosis of OGD/R-induced NSCs. Knockdown of Ku70 promoted the activity of ATM/p53. Moreover, KU60019 (ATM-specific inhibitor) reversed the promoting effects of Ku70 silencing on DNA damage and apoptosis in OGD/R-induced NSCs. In animal experiments, transplantation of NSCs-overexpressed Ku70 enhanced cell survival, improved motor function, reduced infarct volume, relieved brain edema and alleviated BBB dysfunction in MCAO mice models. In conclusion, Ku70 overexpression repressed the DNA damage and apoptosis in OGD/R-induced NSCs by regulating ATM/p53 pathway, and transplantation of NSCs-overexpressed Ku70 played neuroprotective effects in MCAO mice models.
Assuntos
Edema Encefálico , Isquemia Encefálica , AVC Isquêmico , Células-Tronco Neurais , Traumatismo por Reperfusão , Acidente Vascular Cerebral , Camundongos , Animais , Edema Encefálico/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Isquemia Encefálica/metabolismo , Acidente Vascular Cerebral/metabolismo , Células-Tronco Neurais/metabolismo , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/metabolismo , Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , AVC Isquêmico/metabolismo , ApoptoseRESUMO
COVID-19 pandemic has generated high demand for natural rubber gloves (NR) leading to crucial issues of rubber waste and waste management such as burning, dumping, stockpiling, discarding waste in landfills. Hence, rubber biodegradation by microorganisms is an alternative solution to the problem. The biodegradation method is environmentally friendly but normally extremely slow. Numerous microorganisms can degrade NR as a source of carbon and energy. In this study, Rhodococcus pyridinivorans KU1 was isolated from the consortium CK from previous study. The 40% rubber weight loss was detected after incubated for 2 months. The bacterial colonization and cavities on the surface of rubber were identified using a scanning electron microscope (SEM). The result demonstrated the critical degradation of the rubber surface, indicating that bacteria can degrade rubber and use it as their sole carbon source. The result of whole-genome sequencing (WGS) revealed a gene that is 99.9% identical to lcp which is responsible for poly (cis-1,4-isoprene) degradation. The results from Meta16S rRNA sequencing showed that the microbial communities were slightly shifted during the 2-month degradation, depending on the presence of monomers or oligomers appeared during the degradation process. The majority of species were soil bacteria such as phylum Proteobacteria, Actinobacteria, and Firmicutes. Members of Pseudoxanthomonas seemed to be the dominant degraders throughout the degradation.
Assuntos
Rhodococcus , Borracha , Humanos , Pandemias , Firmicutes , CarbonoRESUMO
Spleen tyrosine kinase (Syk) plays a crucial role as a target for allergy treatment due to its involvement in immunoreceptor signaling. The purpose of this study was to identify natural inhibitors of Syk and assess their effects on the IgE-mediated allergic response in mast cells and ICR mice. A list of eight compounds was selected based on pharmacophore and molecular docking, showing potential inhibitory effects through virtual screening. Among these compounds, sophoraflavanone G (SFG) was found to inhibit Syk activity in an enzymatic assay, with an IC50 value of 2.2 µM. To investigate the conformational dynamics of the SYK-SFG system, we performed molecular dynamics simulations. The stability of the binding between SFG and Syk was evaluated using root mean square deviation (RMSD) and root mean square fluctuation (RMSF). In RBL-2H3 cells, SFG demonstrated a dose-dependent suppression of IgE/BSA-induced mast cell degranulation, with no significant cytotoxicity observed at concentrations below 10.0 µM within 24 h. Furthermore, SFG reduced the production of TNF-α and IL-4 in RBL-2H3 cells. Mechanistic investigations revealed that SFG inhibited downstream signaling proteins, including phospholipase Cγ1 (PLCγ1), as well as mitogen-activated protein kinases (AKT, Erk1/2, p38, and JNK), in mast cells in a dose-dependent manner. Passive cutaneous anaphylaxis (PCA) experiments demonstrated that SFG could reduce ear swelling, mast cell degranulation, and the expression of COX-2 and IL-4. Overall, our findings identify naturally occurring SFG as a direct inhibitor of Syk that effectively suppresses mast cell degranulation both in vitro and in vivo.