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We evaluate the effect of rotating inspections carried out by China's central government in 2016 to 2017 in response to the country's air pollution crisis on the environmental performance of targeted cities and coal power plants. Using a staggered difference-in-differences (DID) design, we find that during one-month inspections concentrations of sulfur dioxide (SO2) at coal power plants in targeted cities are on average lower by 25 to 52% compared to not-yet-inspected cities but revert by 54 to 62% on average once scrutiny ends. Following inspections, SO2 pollution increases more quickly at state-owned plants accountable to the central government, compared to state-owned plants accountable to the local (city or below) government. Our results suggest that for most plants SO2 concentration changes during inspections may have been due primarily to the operation of end-of-pipe SO2 removal devices, while following inspections local state-owned plants may have reduced output.
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As an output effector of the Hippo signaling pathway, the TEAD transcription factor and co-activator YAP play crucial functions in promoting cell proliferation and organ size. The tumor suppressor NF2 has been shown to activate LATS1/2 kinases and interplay with the Hippo pathway to suppress the YAP-TEAD complex. However, whether and how NF2 could directly regulate TEAD remains unknown. We identified a direct link and physical interaction between NF2 and TEAD4. NF2 interacted with TEAD4 through its FERM domain and C-terminal tail and decreased the protein stability of TEAD4 independently of LATS1/2 and YAP. Furthermore, NF2 inhibited TEAD4 palmitoylation and induced the cytoplasmic translocation of TEAD4, resulting in ubiquitination and dysfunction of TEAD4. Moreover, the interaction with TEAD4 is required for NF2 function to suppress cell proliferation. These findings reveal an unanticipated role of NF2 as a binding partner and inhibitor of the transcription factor TEAD, shedding light on an alternative mechanism of how NF2 functions as a tumor suppressor through the Hippo signaling cascade.
Assuntos
Via de Sinalização Hippo , Neurofibromina 2 , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Fatores de Transcrição de Domínio TEA , Humanos , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Células HEK293 , Lipoilação , Neurofibromina 2/metabolismo , Neurofibromina 2/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Estabilidade Proteica , Fatores de Transcrição de Domínio TEA/metabolismo , Proteínas Supressoras de Tumor , UbiquitinaçãoRESUMO
Exposure to particulate matter (PM) from agricultural environments has been extensively reported to cause respiratory health concerns in both animals and agricultural workers. Furthermore, PM from agricultural environments, containing fungal spores, has emerged as a significant threat to public health and the environment. Despite its potential toxicity, the impact of fungal spores present in PM from agricultural environments on the lung microbiome and metabolic profile is not well understood. To address this gap in knowledge, we developed a mice model of immunodeficiency using cyclophosphamide and subsequently exposed the mice to fungal spores via the trachea. By utilizing metabolomics techniques and 16 S rRNA sequencing, we conducted a comprehensive investigation into the alterations in the lung microbiome and metabolic profile of mice exposed to fungal spores. Our study uncovered significant modifications in both the lung microbiome and metabolic profile post-exposure to fungal spores. Additionally, fungal spore exposure elicited noticeable changes in α and ß diversity, with these microorganisms being closely associated with inflammatory factors. Employing non-targeted metabolomics analysis via GC-TOF-MS, a total of 215 metabolites were identified, among which 42 exhibited significant differences. These metabolites are linked to various metabolic pathways, with amino sugar and nucleotide sugar metabolism, as well as galactose metabolism, standing out as the most notable pathways. Cysteine and methionine metabolism, along with glycine, serine and threonine metabolism, emerged as particularly crucial pathways. Moreover, these metabolites demonstrated a strong correlation with inflammatory factors and exhibited significant associations with microbial production. Overall, our findings suggest that disruptions to the microbiome and metabolome may hold substantial relevance in the mechanism underlying fungal spore-induced lung damage in mice.
Assuntos
Metaboloma , Microbiota , Animais , Camundongos , Esporos Fúngicos , Metabolômica , Agricultura , Material ParticuladoRESUMO
Targeting subunits of BAF/PBAF chromatin remodeling complexes has been proposed as an approach to exploit cancer vulnerabilities. Here, we develop proteolysis targeting chimera (PROTAC) degraders of the BAF ATPase subunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL. High-resolution ternary complex crystal structures and biophysical investigation guided rational and efficient optimization toward ACBI1, a potent and cooperative degrader of SMARCA2, SMARCA4 and PBRM1. ACBI1 induced anti-proliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancer cells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify a successful biophysics- and structure-based PROTAC design approach to degrade high profile drug targets, and pave the way toward new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Estrutura Molecular , Proteínas Nucleares/metabolismoRESUMO
In the version of this article originally published, several lines of text in the last paragraph of the right column on page 1 of the PDF were transposed into the bottom paragraph of the left column. The affected text of the left column should read "The ATP-dependent activities of the BAF (SWI/SNF) chromatin remodeling complexes affect the positioning of nucleosomes on DNA and thereby many cellular processes related to chromatin structure, including transcription, DNA repair and decatenation of chromosomes during mitosis12,13." The affected text of the right column should read "SMARCA2/4BD inhibitors are thus precluded from use for the treatment of SMARCA4 mutant cancers but could provide attractive ligands for PROTAC conjugation. Small molecules binding to other bromodomains have been successfully converted into PROTACs by conjugating them with structures capable of binding to the E3 ligases von Hippel-Lindau (VHL) or cereblon5,6,10,11,25,26,27." The errors have been corrected in the PDF version of the paper.
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The kinetochore is a proteinaceous complex that is essential for proper chromosome segregation. As a core member of the inner kinetochore, defects of each subunit in the CENP-H/I/K complex cause dysfunction of kinetochore that leads to chromosome mis-segregation and cell death. However, how the CENP-H/I/K complex assembles and promotes kinetochore function are poorly understood. We here determined the crystal structures of CENP-I N-terminus alone from Chaetomium thermophilum and its complex with CENP-H/K from Thielavia terrestris, and verified the identified interactions. The structures and biochemical analyses show that CENP-H and CENP-K form a heterodimer through both N- and C-terminal interactions. CENP-I integrates into the CENP-H/K complex by binding to the C-terminus of CENP-H, leading to formation of the ternary complex in which CENP-H is sandwiched between CENP-K and CENP-I. Our sequence comparisons and mutational analyses showed that this architecture of the CENP-H/I/K complex is conserved in human. Mutating the binding interfaces of CENP-H for either CENP-K or CENP-I significantly reduced their localizations at centromeres and induced massive chromosome alignment defects during mitosis, suggesting that the identified interactions are critical for CENP-H/I/K complex assembly at the centromere and kinetochore function. Altogether, our findings unveil the evolutionarily conserved assembly mechanism of the CENP-H/I/K complex that is critical for proper chromosome alignment.
Assuntos
Proteína Centromérica A/química , Segregação de Cromossomos/genética , Evolução Molecular , Homologia Estrutural de Proteína , Sequência de Aminoácidos , Centrômero/genética , Proteína Centromérica A/genética , Chaetomium/química , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Cromossomos/genética , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Cinetocoros/química , Mitose/genética , Conformação Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Cisplatin is one of the most effective antitumor agents, but its clinical use is highly limited by its severe side effects, especially nephrotoxicity. Recently, the active form of gasdermin D (GSDMD), termed GSDMD-N, was identified to mediate pyroptotic inflammatory cell death in several diseases. However, the role of the GSDMD-N fragment in cisplatin-induced acute kidney injury (AKI) remains unclear. In the present study, we found that pyroptosis was induced by cisplatin in both mouse kidney tissues and renal tubular epithelial cells, accompanied by increased expression of the GSDMD-N fragment. In GSDMD knockout mice with cisplatin-induced AKI, we found that cisplatin-induced loss of renal function, renal tubular injury, and inflammation was significantly attenuated compared with wild-type mice. Furthermore, the GSDMD-N fragment was overexpressed by an established rapid plasmid tail vein injection approach to evaluate the role of this cleaved form of GSDMD in AKI. As expected, mice with GSDMD-N fragment overexpression in the kidney were more susceptible to cisplatin-induced AKI than control mice, as evidenced by further elevated serum levels of blood urea nitrogen and creatinine, aggravated renal pathology, increased expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1, and enhanced renal inflammatory cytokine secretion, which indicates a pathogenic role of GSDMD-N in cisplatin-induced AKI by triggering cell pyroptosis. Similar results were also observed in renal tubular epithelial cells overexpressing the GSDMD-N fragment. Thus these findings suggested that the activation of GSDMD contributes to cisplatin-induced AKI, possibly through triggering pyroptosis.
Assuntos
Injúria Renal Aguda/metabolismo , Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Rim/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/patologia , Animais , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Rim/patologia , Masculino , Camundongos , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Piroptose/fisiologiaRESUMO
OBJECTIVES: Although persistent human papillomavirus (HPV) infection is a major cause of cervical squamous intra-epithelial neoplasia, the relationship between vaginal microbiota and different grades of squamous intra-epithelial neoplasia is not well established. We explored the possible relationship between the vaginal microbiota and the progression of cervical squamous intra-epithelial neoplasia. METHODS: We evaluated 69 women who attended the Obstetrics and Gynecology Hospital of Fudan University. The vaginal bacterial composition of three groups of women was characterized by deep sequencing of bar-coded 16S rRNA gene fragments (V3-4) using Illumina MiSeq. Exclusion criteria were any previous hysterectomy, history of cervical or other lower genital cancer, and/or destructive therapy of the cervix. Women who had autoimmune disorders, who were HIV positive, who received antibiotics within 15 days of sampling, or who had engaged in sexual intercourse or douching within 48 hours prior to sampling were also excluded. P values for age and proportions of organisms were calculated using one-way ANOVA and p values for HPV status and community state types (CSTs) were calculated using a χ2 test. RESULTS: The vaginal bacterial composition of three groups of women, those without an intra-epithelial lesion or malignancy (n=31), those with a low-grade squamous intra-epithelial lesion (LSIL) (n=22), and those with a high-grade squamous intra-epithelial lesion (HSIL) (n=16) were analyzed. Lactobacillus was the most dominant genus overall. Prevotella and Streptococcus were increased in the HSIL group. Cervical disease progression was associated with the prevalence of high-risk HPV infection. Squamous intra-epithelial neoplasia converted the vaginal bacterial community structure from CSTs IV to II. Microbiota diversity was more pronounced in CST types II and IV (p<0.001), especially in type II. We found a significant enrichment in the Peptostreptococcaceae family, Pseudomonadales order, and other types of bacteria in the group of women without intra-epithelial lesions or malignancy compared with women with squamous intra-epithelial neoplasia. We found enrichment in Delftia in the LSIL and HSIL groups compared with the group without an intra-epithelial lesion or malignancy. CONCLUSIONS: Our results show that the vaginal microbiota is directly or indirectly related to the progression of squamous intra-epithelial neoplasia, and Delftia might be a microbiological hallmark of cervical pre-cancerous lesions.
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Microbiota , Displasia do Colo do Útero/microbiologia , Vagina/microbiologia , Adulto , Estudos de Casos e Controles , China , Progressão da Doença , Feminino , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/diagnóstico , Displasia do Colo do Útero/patologiaRESUMO
BACKGROUND: Atherosclerosis involves disorders in lipoprotein metabolism and inflammation. Mitochondrial dysfunction plays a critical role in promoting cell apoptosis, inflammation, and oxidative stress involved in the progress of atherosclerosis. Whereas the direct effect of mitochondrial activity modulation on atherogenesis remains unclear. This study evaluated the effect of a mitochondrial complex inhibitor on atherosclerosis in ApoE-deficient mice as well as the potential mechanisms. METHODS AND RESULTS: We treated ApoE-deficient mice with mitochondrial complex I inhibitor rotenone in the western diet and found that rotenone attenuated early and advanced atherosclerosis with no effect on serum lipoprotein levels. Mechanistic investigation showed that rotenone suppressed primary macrophage foam cell formation possibly by suppressing CD36. In addition, we also found that the inhibitory effect of rotenone on VSMC proliferation and migration possibly by targeting the PI3K/AKT signaling. Consistently, mitochondrial complex III inhibitor azoxystrobin also exhibited similar actions as rotenone in VSMCs but not in macrophages. CONCLUSIONS: Inhibition of mitochondrial activity could significantly attenuate atherosclerosis possibly by modulating CD36-mediated macrophage foam cell formation and PI3K/AKT signaling pathway-associated VSMC activation. Targeting mitochondrial activity might be the potential therapeutic strategy for atherosclerosis.
Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/metabolismo , Aterosclerose/patologia , Células Espumosas/metabolismo , Mitocôndrias/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Animais , Apolipoproteínas E/metabolismo , Antígenos CD36/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Células Espumosas/efeitos dos fármacos , Lipídeos/química , Lipoproteínas LDL/farmacologia , Masculino , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Rotenona/toxicidade , Estrobilurinas/farmacologiaRESUMO
CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single-guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock-in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to E. coli engineering.
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Sistemas CRISPR-Cas , Escherichia coli/genética , Técnicas de Inativação de Genes , Engenharia Genética , Genoma Bacteriano , Francisella/genética , Staphylococcus aureus/genética , Streptococcus pyogenes/genéticaRESUMO
Heterochromatin is a heritable form of gene repression, with critical roles in development and cell identity. Understanding how chromatin factors results in such repression is a fundamental question. Chromatin is assembled and disassembled during transcription, replication and repair by anti-silencing function 1 (Asf1), a highly conserved histone chaperone. Transcription and DNA replication are also affected by histone modifications that modify nucleosome dynamics, such as H2B ubiquitylation (H2Bub). We report here that H2Bub and Asf1 cooperatively promote transcriptional silencing at yeast telomeres and mating loci. Through real time monitoring of HML (Hidden MAT Left) locus silencing, we found that transcriptional repression was slowly initiated and never fully established in mutants lacking both Asf1 and H2Bub. These findings are consistent with impaired HML silencer-binding and spreading of repressor proteins, Sir2 and Sir3. In addition, mutants lacking H2Bub and Asf1 show defects in both nucleosome assembly and higher-order heterochromatin organization at the HML locus. Our findings reveal a novel role for H2Bub and Asf1 in epigenetic silencing at mating loci. Thus, the interplay between H2Hbub and Asf1 may fine-tune nucleosome dynamics and SIR protein recruitment, and represent an ongoing requirement for proper formation and maintenance of heterochromatin.
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Proteínas de Ciclo Celular/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitinação , Proteínas de Ciclo Celular/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Inativação Gênica , Genes Fúngicos Tipo Acasalamento/genética , Heterocromatina/genética , Histonas/genética , Modelos Genéticos , Chaperonas Moleculares/genética , Mutação , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Telômero/genética , Telômero/metabolismoRESUMO
BACKGROUND: Long noncoding RNA HULC is highly up-regulation in human hepatocellular carcinoma (HCC). However, the functions of HULC in hepatocarcinogenesis remains unclear. METHODS: RT-PCR, Western blotting, Chromatin immunoprecipitation (CHIP) assay, RNA Immunoprecipitation (RIP) and tumorignesis test in vitro and in vivo were performed. RESULTS: HULC is negatively associated with expression of PTEN or miR15a in patients of human liver cancer. Moreover, HULC accelerates malignant progression of liver cancer cells in vitro and in vivo. Mechanistically, HULC increasesthe expression of P62 via decreasing mature miR15a. On the other hand, excessive HULC increases the expression of LC3 on the level of transcription and then activates LC3 through Sirt1 (a deacetylase). Notably, HULC enhanced the interplay between LC3 and ATG3. Furthermore, HULC also increases the expression of becline-1(autophagy related gene). Therefore, HULC increases the cellular autophagy by increasing LC3II dependent on Sirt1.Noteworthy, excessive HULC reduces the expression of PTEN, ß-catenin and enhances the expression of SAPK/JUNK, PKM2, CDK2, NOTCH1, C-Jun in liver cancer cells. Of significance, our observations also revealed that HULC inhibited PTEN through ubiquitin-proteasome system mediated by autophagy-P62.Ultimately,HULC activates AKT-PI3K-mTOR pathway through inhibiting PTEN in human liver cancer cells. CONCLUSIONS: This study elucidates a novel mechanism that lncRNA HULC produces a vital function during hepatocarcinogenesis.
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Carcinoma Hepatocelular/patologia , Proteínas de Transporte/genética , Neoplasias Hepáticas/patologia , MicroRNAs/genética , RNA Longo não Codificante/genética , Animais , Autofagia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Transplante de Neoplasias , Proteínas de Ligação a RNA/genética , Transdução de SinaisRESUMO
Candida albicans is a major fungal opportunistic pathogen for humans. In the treatment of C. albicans, azole drugs target the sterol 14α-demethylase (CYP51) encoded by ERG11 gene. Most studies have focused on the fact that the ERG11 mutant results in drug resistance, but its mechanism of action as a drug target has not been described yet. Our results showed that deletion of ERG11 reduced filamentous and invasive growth, and impaired hyphal elongation in sensing serum. Lack of ERG11 increased susceptibility to H2O2 and was defective in clearing reactive oxygen species. ERG11 may affect oxidative stress adaptation by specifically downregulating CAT1 expression. In addition, C. albicans cells lacking ERG11 were more efficiently killed by macrophages and became avirulent in vivo. This study is the first to indicate that ERG11 plays an essential role in hyphal elongation, oxidative stress adaptation and virulence in C. albicans. We speculated that azole drugs not only inhibit the growth of C. albicans, but also assist the host immune system in clearing the fungal organism. The new understanding of mechanisms of action of antifungal drugs should facilitate the development of treatment strategies for resistant fungal infections.
Assuntos
Candida albicans/fisiologia , Candida albicans/patogenicidade , Proteínas Fúngicas/metabolismo , Hifas/crescimento & desenvolvimento , Estresse Oxidativo , Esterol 14-Desmetilase/metabolismo , Animais , Candida albicans/enzimologia , Candida albicans/crescimento & desenvolvimento , Candidíase/microbiologia , Feminino , Proteínas Fúngicas/genética , Deleção de Genes , Peróxido de Hidrogênio/farmacologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Estresse Oxidativo/efeitos dos fármacos , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esterol 14-Desmetilase/genética , Análise de Sobrevida , VirulênciaRESUMO
BACKGROUND: Core fucosylation (addition of fucose in α-1,6-linkage to core N-acetylglucosamine of N-glycans) catalyzed by fucosyltransferase 8 (FUT8) is critical for signaling receptors involved in many physiological and pathological processes such as cell growth, adhesion, and tumor metastasis. Transforming growth factor-ß (TGF-ß)-induced epithelial-mesenchymal transition (EMT) regulates the invasion and metastasis of breast tumors. However, whether receptor core fucosylation affects TGF-ß signaling during breast cancer progression remains largely unknown. METHOD: In this study, gene expression profiling and western blot were used to validate the EMT-associated expression of FUT8. Lentivirus-mediated gain-of-function study, short hairpin RNA (shRNA) or CRISPR/Cas9-mediated loss-of-function studies and pharmacological inhibition of FUT8 were used to elucidate the molecular function of FUT8 during TGF-ß-induced EMT in breast carcinoma cells. In addition, lectin blot, luciferase assay, and in vitro ligand binding assay were employed to demonstrate the involvement of FUT8 in the TGF-ß1 signaling pathway. The role of FUT8 in breast cancer migration, invasion, and metastasis was confirmed using an in vitro transwell assay and mammary fat pad xenograft in vivo tumor model. RESULTS: Gene expression profiling analysis revealed that FUT8 is upregulated in TGF-ß-induced EMT; the process was associated with the migratory and invasive abilities of several breast carcinoma cell lines. Gain-of-function and loss-of-function studies demonstrated that FUT8 overexpression stimulated the EMT process, whereas FUT8 knockdown suppressed the invasiveness of highly aggressive breast carcinoma cells. Furthermore, TGF-ß receptor complexes might be core fucosylated by FUT8 to facilitate TGF-ß binding and enhance downstream signaling. Importantly, FUT8 inhibition suppressed the invasive ability of highly metastatic breast cancer cells and impaired their lung metastasis. CONCLUSIONS: Our results reveal a positive feedback mechanism of FUT8-mediated receptor core fucosylation that promotes TGF-ß signaling and EMT, thus stimulating breast cancer cell invasion and metastasis.
Assuntos
Neoplasias da Mama/genética , Fucosiltransferases/genética , Invasividade Neoplásica/genética , Fator de Crescimento Transformador beta1/genética , Neoplasias da Mama/patologia , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Fucose/genética , Fucose/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Lentivirus/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica , Fosforilação , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de Sinais/genéticaRESUMO
Metabolic engineering often necessitates chromosomal integration of multiple genes but integration of large genes into Escherichia coli remains difficult. CRISPR/Cas9 is an RNA-guided system which enables site-specific induction of double strand break (DSB) and programmable genome editing. Here, we hypothesized that CRISPR/Cas9-triggered DSB could enhance homologous recombination and augment integration of large DNA into E. coli chromosome. We demonstrated that CRISPR/Cas9 system was able to trigger DSB in >98% of cells, leading to subsequent cell death, and identified that mutagenic SOS response played roles in the cell survival. By optimizing experimental conditions and combining the λ-Red proteins and linear dsDNA, CRISPR/Cas9-induced DSB enabled homologous recombination of the donor DNA and replacement of lacZ gene in the MG1655 strain at efficiencies up to 99%, and allowed high fidelity, scarless integration of 2.4, 3.9, 5.4, and 7.0 kb DNA at efficiencies approaching 91%, 92%, 71%, and 61%, respectively. The CRISPR/Cas9-assisted gene integration also functioned in different E. coli strains including BL21 (DE3) and W albeit at different efficiencies. Taken together, our methodology facilitated precise integration of dsDNA as large as 7 kb into E. coli with efficiencies exceeding 60%, thus significantly ameliorating the editing efficiency and overcoming the size limit of integration using the commonly adopted recombineering approach. Biotechnol. Bioeng. 2017;114: 172-183. © 2016 Wiley Periodicals, Inc.
Assuntos
Sistemas CRISPR-Cas/genética , DNA/genética , Escherichia coli/genética , Edição de Genes/métodos , Engenharia Metabólica/métodos , Sobrevivência Celular , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Plasmídeos/genética , Resposta SOS em Genética/genéticaRESUMO
Long noncoding RNA CUDR plays an important role during tumorigenesis. Herein, we demonstrate that SET1A cooperates with CUDR to accelerate hepatocarcinogenesis and promote malignant transformation of hepatocyte-like stem cells. Mechanistically, CUDR enhances the phosphorylation of RB1, C-myc expression, and the interplay between the SET1A and pRB1. Notably, CUDR acts as a sponge cushion that shows a link between SET1A and pRB1, producing a activated pRB1-SET1A complex. On the other hand, the pRB1-SET1A complex may carry methyls(me) to occupy the position of H3K4, resulting in specific tri-methylation of forth lysine of histone H3 (H3K4me3). Thereby, the H3K4me3 loads on the TRF2 promoter region which causes the TRF2 overexpression. Ultimately, the excessive TRF2 binds to telomere repeat DNA, prolonging the telomere length. These findings provide the first demonstration that SET1A cooperates with CUDR to play a positive potential role during hepatocarcinogenesis and hepatocyte-like stem cells' malignant transformation epigenetically.
Assuntos
Transformação Celular Neoplásica/genética , Epigênese Genética , Histona-Lisina N-Metiltransferase/genética , Neoplasias Hepáticas/genética , RNA Longo não Codificante/genética , Células-Tronco/patologia , Animais , Transformação Celular Neoplásica/patologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Fosforilação , Regiões Promotoras Genéticas , Proteína do Retinoblastoma/metabolismo , Regulação para CimaRESUMO
The influence of mono-ubiquitylation of histone H2B (H2Bub) on transcription via nucleosome reassembly has been widely documented. Recently, it has also been shown that H2Bub promotes recovery from replication stress; however, the underling molecular mechanism remains unclear. Here, we show that H2B ubiquitylation coordinates activation of the intra-S replication checkpoint and chromatin re-assembly, in order to limit fork progression and DNA damage in the presence of replication stress. In particular, we show that the absence of H2Bub affects replication dynamics (enhanced fork progression and reduced origin firing), leading to γH2A accumulation and increased hydroxyurea sensitivity. Further genetic analysis indicates a role for H2Bub in transducing Rad53 phosphorylation. Concomitantly, we found that a change in replication dynamics is not due to a change in dNTP level, but is mediated by reduced Rad53 activation and destabilization of the RecQ helicase Sgs1 at the fork. Furthermore, we demonstrate that H2Bub facilitates the dissociation of the histone chaperone Asf1 from Rad53, and nucleosome reassembly behind the fork is compromised in cells lacking H2Bub. Taken together, these results indicate that the regulation of H2B ubiquitylation is a key event in the maintenance of genome stability, through coordination of intra-S checkpoint activation, chromatin assembly and replication fork progression.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Montagem e Desmontagem da Cromatina , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , Replicação do DNA , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Hidroxiureia/farmacologia , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Nucleossomos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , UbiquitinaçãoRESUMO
Cyanobacteria hold promise as a cell factory for producing biofuels and bio-derived chemicals, but genome engineering of cyanobacteria such as Synechococcus elongatus PCC 7942 poses challenges because of their oligoploidy nature and long-term instability of the introduced gene. CRISPR-Cas9 is a newly developed RNA-guided genome editing system, yet its application for cyanobacteria engineering has yet to be reported. Here we demonstrated that CRISPR-Cas9 system can effectively trigger programmable double strand break (DSB) at the chromosome of PCC 7942 and provoke cell death. With the co-transformation of template plasmid harboring the gene cassette and flanking homology arms, CRISPR-Cas9-mediated DSB enabled precise gene integration, ameliorated the homologous recombination efficiency and allowed the use of lower amount of template DNA and shorter homology arms. The CRISPR-Cas9-induced cell death imposed selective pressure and enhanced the chance of concomitant integration of gene cassettes into all chromosomes of PCC 7942, hence accelerating the process of obtaining homogeneous and stable recombinant strains. We further explored the feasibility of engineering cyanobacteria by CRISPR-Cas9-assisted simultaneous glgc knock-out and gltA/ppc knock-in, which improved the succinate titer to 435.0±35.0µg/L, an ≈11-fold increase when compared with that of the wild-type cells. These data altogether justify the use of CRISPR-Cas9 for genome engineering and manipulation of metabolic pathways in cyanobacteria.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genoma Bacteriano/genética , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Ácido Succínico/metabolismo , Synechococcus/fisiologia , Apoptose/genética , Vias Biossintéticas/genética , Melhoramento Genético/métodos , Ácido Succínico/isolamento & purificação , Synechococcus/citologiaRESUMO
BACKGROUND: Cyanobacterium Synechococcus elongatus PCC 7942 holds promise for biochemical conversion, but gene deletion in PCC 7942 is time-consuming and may be lethal to cells. CRISPR interference (CRISPRi) is an emerging technology that exploits the catalytically inactive Cas9 (dCas9) and single guide RNA (sgRNA) to repress sequence-specific genes without the need of gene knockout, and is repurposed to rewire metabolic networks in various procaryotic cells. RESULTS: To employ CRISPRi for the manipulation of gene network in PCC 7942, we integrated the cassettes expressing enhanced yellow fluorescent protein (EYFP), dCas9 and sgRNA targeting different regions on eyfp into the PCC 7942 chromosome. Co-expression of dCas9 and sgRNA conferred effective and stable suppression of EYFP production at efficiencies exceeding 99%, without impairing cell growth. We next integrated the dCas9 and sgRNA targeting endogenous genes essential for glycogen accumulation (glgc) and succinate conversion to fumarate (sdhA and sdhB). Transcription levels of glgc, sdhA and sdhB were effectively suppressed with efficiencies depending on the sgRNA binding site. Targeted suppression of glgc reduced the expression to 6.2%, attenuated the glycogen accumulation to 4.8% and significantly enhanced the succinate titer. Targeting sdhA or sdhB also effectively downregulated the gene expression and enhanced the succinate titer ≈12.5-fold to ≈0.58-0.63 mg/L. CONCLUSIONS: These data demonstrated that CRISPRi-mediated gene suppression allowed for re-directing the cellular carbon flow, thus paving a new avenue to rationally fine-tune the metabolic pathways in PCC 7942 for the production of biotechnological products.