RESUMO
Glioblastoma (GBM) is a deadly and the most common primary brain tumor in adults. Due to their regulation of a high number of mRNA transcripts, microRNAs (miRNAs) are key molecules in the control of biological processes and are thereby promising therapeutic targets for GBM patients. In this regard, we recently reported miRNAs as strong modulators of GBM aggressiveness. Here, using an integrative and comprehensive analysis of the TCGA database and the transcriptome of GBM biopsies, we identified three critical and clinically relevant miRNAs for GBM, miR-17-3p, miR-222, and miR-340. In addition, we showed that the combinatorial modulation of three of these miRNAs efficiently inhibited several biological processes in patient-derived GBM cells of all these three GBM subtypes (Mesenchymal, Proneural, Classical), induced cell death, and delayed tumor growth in a mouse tumor model. Finally, in a doxycycline-inducible model, we observed a significant inhibition of GBM stem cell viability and a significant delay of orthotopic tumor growth. Collectively, our results reveal, for the first time, the potential of miR-17-3p, miR-222 and miR-340 multi-targeting as a promising therapeutic strategy for GBM patients.
Assuntos
Glioblastoma , MicroRNAs , Adulto , Humanos , Animais , Camundongos , MicroRNAs/genética , Glioblastoma/genética , Agressão , Biópsia , Morte Celular , Modelos Animais de DoençasRESUMO
BACKGROUND: An influenza A(H1N1)pdm09 infection was diagnosed in a hematopoietic stem cell transplant recipient during conditioning regimen. He was treated with oral oseltamivir, later combined with intravenous zanamivir. The H275Y neuraminidase (NA) mutation was first detected, and an E119D NA mutation was identified during zanamivir therapy. METHODS: Recombinant wild-type (WT) E119D and E119D/H275Y A(H1N1)pdm09 NA variants were generated by reverse genetics. Susceptibility to NA inhibitors (NAIs) was evaluated with a fluorometric assay using the 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) substrate. Susceptibility to favipiravir (T-705) was assessed using plaque reduction assays. The NA affinity and velocity values were determined with NA enzymatic studies. RESULTS: We identified an influenza A(H1N1)pdm09 E119D mutant that exhibited a marked increase in the 50% inhibitory concentrations against all tested NAIs (827-, 25-, 286-, and 702-fold for zanamivir, oseltamivir, peramivir, and laninamivir, respectively). The double E119D/H275Y mutation further increased oseltamivir and peramivir 50% inhibitory concentrations by 790- and >5000-fold, respectively, compared with the WT. The mutant viruses remained susceptible to favipiravir. The NA affinity and velocity values of the E119D variant decreased by 8.1-fold and 4.5-fold, respectively, compared with the WT. CONCLUSIONS: The actual emergence of a single NA mutation conferring pan-NAI resistance in the clinical setting reinforces the pressing need to develop new anti-influenza strategies.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/genética , Neuraminidase/antagonistas & inibidores , Neuraminidase/genética , Transplante de Células-Tronco , Farmacorresistência Viral/genética , Humanos , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Mutação/fisiologia , Cavidade Nasal/virologia , Neuraminidase/química , Neuraminidase/metabolismo , Zanamivir/farmacologiaRESUMO
Glioblastoma is a deadly malignant brain tumor and one of the most incurable forms of cancer in need of new therapeutic targets. As some cancers are known to be caused by a virus, the discovery of viruses could open the possibility to treat, and perhaps prevent, such a disease. Although an association with viruses such as cytomegalovirus or Simian virus 40 has been strongly suggested, involvement of these and other viruses in the initiation and/or propagation of glioblastoma remains vague, controversial and warrants elucidation. To exhaustively address the association of virus and glioblastoma, we developed and validated a robust metagenomic approach to analyze patient biopsies via high-throughput sequencing, a sensitive tool for virus screening. In addition to traditional clinical diagnostics, glioblastoma biopsies were deep-sequenced and analyzed with a multistage computational pipeline to identify known or potentially discover unknown viruses. In contrast to the studies reporting the presence of viral signatures in glioblastoma, no common or recurring active viruses were detected, despite finding an antiviral-like type I interferon response in some specimens. Our findings highlight a discrete and non-specific viral signature and uncharacterized short RNA sequences in glioblastoma. This study provides new insights into glioblastoma pathogenesis and defines a general methodology that can be used for high-resolution virus screening and discovery in human cancers.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/virologia , Citomegalovirus/imunologia , Glioblastoma/genética , Glioblastoma/virologia , Interferon Tipo I/imunologia , Anticorpos Antivirais/sangue , Neoplasias Encefálicas/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , MetagenômicaRESUMO
Enterovirus 71 (EV71) is one of the most virulent enteroviruses, but the specific molecular features that enhance its ability to disseminate in humans remain unknown. We analyzed the genomic features of EV71 in an immunocompromised host with disseminated disease according to the different sites of infection. Comparison of five full-length genomes sequenced directly from respiratory, gastrointestinal, nervous system, and blood specimens revealed three nucleotide changes that occurred within a five-day period: a non-conservative amino acid change in VP1 located within the BC loop (L97R), a region considered as an immunogenic site and possibly important in poliovirus host adaptation; a conservative amino acid substitution in protein 2B (A38V); and a silent mutation in protein 3D (L175). Infectious clones were constructed using both BrCr (lineage A) and the clinical strain (lineage C) backgrounds containing either one or both non-synonymous mutations. In vitro cell tropism and competition assays revealed that the VP197 Leu to Arg substitution within the BC loop conferred a replicative advantage in SH-SY5Y cells of neuroblastoma origin. Interestingly, this mutation was frequently associated in vitro with a second non-conservative mutation (E167G or E167A) in the VP1 EF loop in neuroblastoma cells. Comparative models of these EV71 VP1 variants were built to determine how the substitutions might affect VP1 structure and/or interactions with host cells and suggest that, while no significant structural changes were observed, the substitutions may alter interactions with host cell receptors. Taken together, our results show that the VP1 BC loop region of EV71 plays a critical role in cell tropism independent of EV71 lineage and, thus, may have contributed to dissemination and neurotropism in the immunocompromised patient.
Assuntos
Enterovirus Humano A/fisiologia , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/virologia , Neurônios/virologia , Proteínas Estruturais Virais/genética , Ligação Viral , Adulto , Substituição de Aminoácidos , Animais , Líquido da Lavagem Broncoalveolar/virologia , Células CACO-2 , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , DNA Viral/genética , Enterovirus Humano A/genética , Enterovirus Humano A/imunologia , Fezes/virologia , Humanos , Hospedeiro Imunocomprometido , Masculino , Mutação , Neuroblastoma , Células Vero , Proteínas Estruturais Virais/sangue , Proteínas Estruturais Virais/líquido cefalorraquidiano , Proteínas Estruturais Virais/imunologiaRESUMO
The p53 tumour suppressor gene, the most frequently mutated gene in human cancer, encodes a transcription factor that contains sequence-specific DNA binding and homo-tetramerization domains. Interestingly, the affinities of p53 for specific and non-specific DNA sites differ by only one order of magnitude, making it hard to understand how this protein recognizes its specific DNA targets in vivo. We describe here the structure of a p53 polypeptide containing both the DNA binding and oligomerization domains in complex with DNA. The structure reveals that sequence-specific DNA binding proceeds via an induced fit mechanism that involves a conformational switch in loop L1 of the p53 DNA binding domain. Analysis of loop L1 mutants demonstrated that the conformational switch allows DNA binding off-rates to be regulated independently of affinities. These results may explain the universal prevalence of conformational switching in sequence-specific DNA binding proteins and suggest that proteins like p53 rely more on differences in binding off-rates, than on differences in affinities, to recognize their specific DNA sites.
Assuntos
DNA/metabolismo , Conformação Proteica , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Análise Mutacional de DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Polarização de Fluorescência , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação ProteicaRESUMO
Microfluidics is a promising technology for the rapid identification of protein crystallization conditions. However, most of the existing systems utilize silicone elastomers as the chip material which, despite its many benefits, is highly permeable to water vapour. This limits the time available for protein crystallization to less than a week. Here, the use of a cyclic olefin homopolymer-based microfluidics system for protein crystallization and in situ X-ray diffraction is described. Liquid handling in this system is performed in 2 mm thin transparent cards which contain 500 chambers, each with a volume of 320 nl. Microbatch, vapour-diffusion and free-interface diffusion protocols for protein crystallization were implemented and crystals were obtained of a number of proteins, including chicken lysozyme, bovine trypsin, a human p53 protein containing both the DNA-binding and oligomerization domains bound to DNA and a functionally important domain of Arabidopsis Morpheus' molecule 1 (MOM1). The latter two polypeptides have not been crystallized previously. For X-ray diffraction analysis, either the cards were opened to allow mounting of the crystals on loops or the crystals were exposed to X-rays in situ. For lysozyme, an entire X-ray diffraction data set at 1.5 A resolution was collected without removing the crystal from the card. Thus, cyclic olefin homopolymer-based microfluidics systems have the potential to further automate protein crystallization and structural genomics efforts.
Assuntos
Cicloparafinas/química , Microfluídica , Polímeros/química , ATPases Associadas a Diversas Atividades Celulares , Animais , Arabidopsis , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Bovinos , Galinhas , Cristalização , Cicloparafinas/metabolismo , Genômica , Humanos , Microfluídica/instrumentação , Microfluídica/métodos , Muramidase/química , Muramidase/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Polímeros/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Tripsina/química , Tripsina/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Difração de Raios XRESUMO
The mechanisms by which eukaryotic cells sense DNA double-strand breaks (DSBs) in order to initiate checkpoint responses are poorly understood. 53BP1 is a conserved checkpoint protein with properties of a DNA DSB sensor. Here, we solved the structure of the domain of 53BP1 that recruits it to sites of DSBs. This domain consists of two tandem tudor folds with a deep pocket at their interface formed by residues conserved in the budding yeast Rad9 and fission yeast Rhp9/Crb2 orthologues. In vitro, the 53BP1 tandem tudor domain bound histone H3 methylated on Lys 79 using residues that form the walls of the pocket; these residues were also required for recruitment of 53BP1 to DSBs. Suppression of DOT1L, the enzyme that methylates Lys 79 of histone H3, also inhibited recruitment of 53BP1 to DSBs. Because methylation of histone H3 Lys 79 was unaltered in response to DNA damage, we propose that 53BP1 senses DSBs indirectly through changes in higher-order chromatin structure that expose the 53BP1 binding site.