RESUMO
Recent evidence suggests that animal microRNAs (miRNAs) can target coding sequences (CDSs); however, the pathophysiological importance of such targeting remains unknown. Here, we show that a somatic heterozygous missense mutation (c.402C>G; p.C134W) in FOXL2, a feature shared by virtually all adult-type granulosa cell tumors (AGCTs), introduces a target site for miR-1236, which causes haploinsufficiency of the tumor-suppressor FOXL2. This miR-1236-mediated selective degradation of the variant FOXL2 mRNA is preferentially conducted by a distinct miRNA-loaded RNA-induced silencing complex (miRISC) directed by the Argonaute3 (AGO3) and DHX9 proteins. In both patients and a mouse model of AGCT, abundance of the inversely regulated variant FOXL2 with miR-1236 levels is highly correlated with malignant features of AGCT. Our study provides a molecular basis for understanding the conserved FOXL2 CDS mutation-mediated etiology of AGCT, revealing the existence of a previously unidentified mechanism of miRNA-targeting disease-associated mutations in the CDS by forming a non-canonical miRISC.
Assuntos
Proteína Forkhead Box L2/genética , Proteína Forkhead Box L2/metabolismo , Tumor de Células da Granulosa/genética , MicroRNAs/metabolismo , Mutação , Fases de Leitura Aberta , Desequilíbrio Alélico , Animais , Apoptose , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Morte Celular/fisiologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Tumor de Células da Granulosa/patologia , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , MicroRNAs/genética , Mutação de Sentido Incorreto , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/metabolismo , TranscriptomaRESUMO
Although both the p53 and forkhead box (FOX) family proteins are key transcription factors associated with cancer progression, their direct relationship is unknown. Here, we found that FOX family proteins bind to the non-canonical homotypic cluster of the p53 promoter region (TP53). Analysis of crystal structures of FOX proteins (FOXL2 and FOXA1) bound to the p53 homotypic cluster indicated that they interact with a 2:1 stoichiometry accommodated by FOX-induced DNA allostery. In particular, FOX proteins exhibited distinct dimerization patterns in recognition of the same p53-DNA; dimer formation of FOXA1 involved protein-protein interaction, but FOXL2 did not. Biochemical and biological functional analyses confirmed the cooperative binding of FOX proteins to the TP53 promoter for the transcriptional activation of TP53. In addition, up-regulation of TP53 was necessary for FOX proteins to exhibit anti-proliferative activity in cancer cells. These analyses reveal the presence of a discrete characteristic within FOX family proteins in which FOX proteins regulate the transcription activity of the p53 tumor suppressor via cooperative binding to the TP53 promoter in alternative dimer configurations.
Assuntos
Proteína Forkhead Box L2/metabolismo , Fatores de Transcrição Forkhead , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Proteína Supressora de Tumor p53/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Regiões Promotoras Genéticas , Proteína Supressora de Tumor p53/metabolismoRESUMO
The selective monitoring of G-quadruplex (G4) structures in living cells is important to elucidate their functions and reveal their value as diagnostic or therapeutic targets. Here we report a fluorogenic probe (CV2) able to selectively light-up parallel G4 DNA over antiparallel topologies. CV2 was constructed by conjugating the excimer-forming CV dye with a peptide sequence (l-Arg-l-Gly-glutaric acid) that specifically recognizes G4s. CV2 forms self-assembled, red excimer-emitting nanoaggregates in aqueous media, but specific binding to G4s triggers its disassembly into rigidified monomeric dyes, leading to a dramatic fluorescence enhancement. Moreover, selective permeation of CV2 stains G4s in mitochondria over the nucleus. CV2 was employed for tracking the folding and unfolding of G4s in living cells, and for monitoring mitochondrial DNA (mtDNA) damage. These properties make CV2 appealing to investigate the possible roles of mtDNA G4s in diseases that involve mitochondrial dysfunction.
Assuntos
Corantes Fluorescentes , Quadruplex G , Corantes Fluorescentes/química , DNA Mitocondrial , Mitocôndrias/metabolismoRESUMO
meso-Carboxyl-BODIPY responds to small electronic changes resulting from acyl substitution reactions with a marked change in fluorescence. Herein, the minute changes that accompany the thioester to amide conversion encountered in native chemical ligation (NCL) are exploited in the construction of fluorescent "turn-on" probes. Two fluorogenic probes, 1 a and 4, derived from a meso-thioester-BODIPY scaffold, were designed for the selective detection of cysteine (1 a) and aminopeptidase N (4), respectively. The aromatic (1 a) and aliphatic (4) thioesters of meso-carboxyl-BODIPY are nonfluorescent. However, specific analyte-induced conversion to the meso-amide derivative caused significant spectral changes and a dramatic fluorescence enhancement. Probe 1 a exhibited a large fluorescence "turn-on" response with high selectivity toward cysteine via a tandem NCL reaction. Probe 4 was successfully applied to the monitoring and imaging of endogenous aminopeptidase N in live cancer cells.
Assuntos
Cisteína , Corantes Fluorescentes , Compostos de Boro , Antígenos CD13RESUMO
The adaptor-related protein complex 5 subunit mu 1 (AP5M1) is an evolutionally conserved protein with ubiquitous expression in human tissues. However, the major function of AP5M1 in living organisms is unclear owing to few published studies. Here, we demonstrate that AP5M1 is a potent apoptosis-inducing molecule in cervical cancer cells. We also found that AP5M1 upregulated the level of BAX protein, a key pro-apoptotic B cell lymphoma (BCL)-2 family member regulating mitochondrial apoptotic cell death pathway. Moreover, AP5M1 completely lost its apoptotic activity in BAX-knockout or -knockdown cells, indicative of its functional dependence on BAX. Comparative analysis of cervical tissues from patients with cervical carcinoma and non-cancer control revealed a prominent downregulation in AP5M1 expression with a concomitant downregulation in BAX expression; AP5M1 and BAX mRNA expression levels in cervical tissues exhibited a strong positive correlation (râ¯=â¯0.97). Thus, we identified AP5M1 as a previously unrecognized apoptotic protein that governs BAX expression and revealed the association between AP5M1 and malignancy.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Neoplasias do Colo do Útero/metabolismo , Proteína X Associada a bcl-2/metabolismo , Proliferação de Células , Feminino , Células HeLa , Humanos , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/patologiaRESUMO
The successful implementation of photothermal therapy (PTT) in cancer treatment hinges on the development of highly effective photothermal agents (PTAs). Boron dipyrromethene (BODIPY) dyes, being well known for their high brightness and quantum efficiencies, are the antithesis of PTAs. Nonetheless, a systematic exploration of the photophysics and photothermal characteristics of a series of π-extended BODIPY dyes with high absorptivity in the near-infrared (NIR) region has achieved superior photothermal conversion efficiencies (>90%), in both monomeric state and nanoparticles after encapsulation in a biocompatible polyethyleneglycol 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000]. Optimal PTA candidates combine strong NIR absorption provided by extended donor-acceptor conjugation and an optimization of the electronic and steric effects of meso-substituents to maximize photothermal conversion performance. The PTT-optimized meso-CF3-BODIPY, TCF3PEn exhibits exceptional efficacy in inducing cancer cell apoptosis and in vivo tumor ablation using low-power NIR laser irradiation (0.3 W cm-2, 808 nm) as well as excellent biological safety, underscoring its potential for advancing light-induced cancer therapies.
Assuntos
Compostos de Boro , Terapia Fototérmica , Compostos de Boro/química , Compostos de Boro/farmacologia , Animais , Humanos , Camundongos , Terapia Fototérmica/métodos , Nanopartículas/química , Polietilenoglicóis/química , Linhagem Celular Tumoral , Fototerapia/métodos , FosfatidiletanolaminasRESUMO
DNA methylation is an epigenetic modification that regulates gene expression and plays an essential role in hematopoiesis. UHRF1 and DNMT1 are both crucial for regulating genome-wide maintenance of DNA methylation. Specifically, it is well known that hypermethylation is crucial characteristic of acute myeloid leukemia (AML). However, the mechanism underlying how DNA methylation regulates the differentiation of AML cells, including THP-1 is not fully elucidated. In this study, we report that UHRF1 or DNMT1 depletion enhances the phorbol-12-myristate-13-acetate (PMA)-induced differentiation of THP-1 cells. Transcriptome analysis and genome-wide methylation array results showed that depleting UHRF1 or DNMT1 induced changes that made THP-1 cells highly sensitive to PMA. Furthermore, knockdown of UHRF1 or DNMT1 impeded solid tumor formation in xenograft mouse model. These findings suggest that UHRF1 and DNMT1 play a pivotal role in regulating differentiation and proliferation of THP-1 cells and targeting these proteins may improve the efficiency of differentiation therapy in AML patients.
Assuntos
DNA (Citosina-5-)-Metiltransferases , Metilação de DNA , Humanos , Animais , Camundongos , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação para Baixo , Células THP-1 , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Diferenciação Celular/genética , Hematopoese , Macrófagos/metabolismoRESUMO
Interferon-inducible guanylate-binding proteins (GBPs) are well-known for mediating host-defense mechanisms against cellular pathogens. Emerging evidence suggests that GBPs are also implicated in tumorigenesis; however, their underlying molecular mechanism is still unknown. In this study, we identified that GBP1 and GBP2 interact with MCL-1, the key prosurvival member of the BCL-2 family, via its BH3 domain. GBPs induce caspase-dependent apoptosis in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cells, where the proapoptotic BCL-2 member, BAK, is an indispensable mediator. In particular, GBP2 completely inhibited the MCL-1-mediated promotion of the survival of CML cells through competitive inhibition, resulting in BAK liberation from MCL-1. Concurrently, GBP2 dramatically upregulates BAK expression via its inhibition of the PI3K/AKT pathway. Moreover, paclitaxel upregulates GBP2 expression, and paclitaxel-induced apoptotic activity was distinctively compromised by knockout of GBP2 in CML cells. Bioinformatics analyses of leukemia databases revealed that transcripts of GBPs were generally downregulated in leukemia patients and that GBPs were favorable prognosis markers. Thus, these findings provide molecular evidence of GBPs as apoptosis-inducing proteins of leukemia cells and suggest that GBPs are attractive targets for the development of chemotherapeutics.
RESUMO
The balance between major DNA double-strand break (DSB) repair pathways is influenced by binding of the Ku complex, a XRCC5/6 heterodimer, to DSB ends, initiating non-homologous end joining (NHEJ) but preventing additional DSB end resection and homologous recombination (HR). However, the key molecular cue for Ku recruitment to DSB sites is unknown. Here, we report that FOXL2, a forkhead family transcriptional factor, directs DSB repair pathway choice by acetylation-dependent binding to Ku. Upon DSB induction, SIRT1 translocates to the nucleus and deacetylates FOXL2 at lysine 124, leading to liberation of XRCC5 and XRCC6 from FOXL2 and formation of the Ku complex. FOXL2 ablation enhances Ku recruitment to DSB sites, imbalances DSB repair kinetics by accelerating NHEJ and inhibiting HR, and thus leads to catastrophic genomic events. Our study unveils the SIRT1-(de)acetylated FOXL2-Ku axis that governs the balance of DSB repair pathways to maintain genome integrity.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteína Forkhead Box L2/metabolismo , Autoantígeno Ku/metabolismo , Acetilação , Linhagem Celular Tumoral , Proteína Forkhead Box L2/genética , Células HEK293 , Recombinação Homóloga , Humanos , Autoantígeno Ku/genética , Mutação , Ligação Proteica/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sirtuína 1/metabolismoRESUMO
Citrus Huanglongbing (HLB) is one of the most devastating citrus diseases worldwide. Sensitive and accurate assays are vital for efficient prevention of the spread of HLB-associated "Candidatus Liberibacter spp". "Candidatus Liberibacter spp" that infect Citrus includes "Candidatus Liberibacter asiaticus" (Las), "Candidatus Liberibacter africanus" (Laf) and "Candidatus Liberibacter americanus" (Lam). Of them, Las is the most widespread species. In this study, a set of nested PCR primer pairs were screened to diagnose Las, and the nested PCR method greatly enhanced the sensitivity to detect Las up to 10 times and 100 times compared to qPCR and conventional PCR, respectively. Totally, 1112 samples from 5 different citrus cultivars in 39 different counties and cities were assayed by nested PCR. The results show that 384 samples were HLB-infected; the highest positive detection rate was 79.7% from the lopsided fruit samples, and the lowest positive detection rate was 16.3% from the apical dieback samples. The results indicate that the designed nested PCR primer pairs can detect Las from different symptomatic tissues, different citrus cultivars and different geographic regions. The set of nested PCR primers designed in the present study will provide a very useful supplementation to the current approaches for Las detection.