RESUMO
Idiopathic pulmonary fibrosis is a lethal lung fibrotic disease, associated with aging with a mean survival of 2-5 years and no curative treatment. The GSE4 peptide is able to rescue cells from senescence, DNA and oxidative damage, inflammation, and induces telomerase activity. Here, we investigated the protective effect of GSE4 expression in vitro in rat alveolar epithelial cells (AECs), and in vivo in a bleomycin model of lung fibrosis. Bleomycin-injured rat AECs, expressing GSE4 or treated with GSE4-PLGA/PEI nanoparticles showed an increase of telomerase activity, decreased DNA damage, and decreased expression of IL6 and cleaved-caspase 3. In addition, these cells showed an inhibition in expression of fibrotic markers induced by TGF-ß such as collagen-I and III among others. Furthermore, treatment with GSE4-PLGA/PEI nanoparticles in a rat model of bleomycin-induced fibrosis, increased telomerase activity and decreased DNA damage in proSP-C cells. Both in preventive and therapeutic protocols GSE4-PLGA/PEI nanoparticles prevented and attenuated lung damage monitored by SPECT-CT and inhibited collagen deposition. Lungs of rats treated with bleomycin and GSE4-PLGA/PEI nanoparticles showed reduced expression of α-SMA and pro-inflammatory cytokines, increased number of pro-SPC-multicellular structures and increased DNA synthesis in proSP-C cells, indicating therapeutic efficacy of GSE4-nanoparticles in experimental lung fibrosis and a possible curative treatment for lung fibrotic patients.
Assuntos
Apoptose/efeitos dos fármacos , Bleomicina/farmacologia , Dano ao DNA/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Nanopartículas/uso terapêutico , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Colágeno/efeitos dos fármacos , Colágeno/metabolismo , Humanos , Pulmão/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/farmacologiaRESUMO
BACKGROUND: Telomeres are nucleoprotein structures present at the terminal region of the chromosomes. Mutations in genes coding for proteins involved in telomere maintenance are causative of a number of disorders known as telomeropathies. The genetic origin of these diseases is heterogeneous and has not been determined for a significant proportion of patients. METHODS: This article describes the genetic characterization of a cohort of patients. Telomere length was determined by Southern blot and quantitative PCR. Nucleotide variants were analyzed either by high-resolution melting analysis and Sanger sequencing of selected exons or by massive sequencing of a panel of genes. RESULTS: Forty-seven patients with telomere length below the 10% of normal population, affected with three telomeropathies: dyskeratosis congenita (4), aplastic anemia (22) or pulmonary fibrosis (21) were analyzed. Eighteen of these patients presented known pathogenic or novel possibly pathogenic variants in the telomere-related genes TERT, TERC, RTEL1, CTC1 and ACD. In addition, the analyses of a panel of 188 genes related to haematological disorders indicated that a relevant proportion of the patients (up to 35%) presented rare variants in genes related to DNA repair or in genes coding for proteins involved in the resolution of complex DNA structures, that participate in telomere replication. Mutations in some of these genes are causative of several syndromes previously associated to telomere shortening. CONCLUSION: Novel variants in telomere, DNA repair and replication genes are described that might indicate the contribution of variants in these genes to the development of telomeropathies. Patients carrying variants in telomere-related genes presented worse evolution after diagnosis than the rest of patients analyzed.
Assuntos
Anemia Aplástica/genética , Reparo do DNA/genética , Disceratose Congênita/genética , Fibrose Pulmonar/genética , Encurtamento do Telômero/genética , Telômero/genética , Adolescente , Adulto , Criança , Pré-Escolar , Éxons/genética , Feminino , Humanos , Lactente , Masculino , Linhagem , RNA/genética , Telomerase/genética , Adulto JovemRESUMO
Ataxia telangiectasia (AT) is a genetic disease caused by mutations in the ATM gene but the mechanisms underlying AT are not completely understood. Key functions of the ATM protein are to sense and regulate cellular redox status and to transduce DNA double-strand break signals to downstream effectors. ATM-deficient cells show increased ROS accumulation, activation of p38 protein kinase, and increased levels of DNA damage. GSE24.2 peptide and a short derivative GSE4 peptide corresponding to an internal domain of Dyskerin have proved to induce telomerase activity, decrease oxidative stress, and protect from DNA damage in dyskeratosis congenita (DC) cells. We have found that expression of GSE24.2 and GSE4 in human AT fibroblast is able to decrease DNA damage, detected by γ-H2A.X and 53BP1 foci. However, GSE24.2/GSE4 expression does not improve double-strand break signaling and repair caused by the lack of ATM activity. In contrast, they cause a decrease in 8-oxoguanine and OGG1-derived lesions, particularly at telomeres and mitochondrial DNA, as well as in reactive oxygen species, in parallel with increased expression of SOD1. These cells also showed lower levels of IL6 and decreased p38 phosphorylation, decreased senescence and increased ability to divide for longer times. Additionally, these cells are more resistant to treatment with H202 and the radiomimetic-drug bleomycin. Finally, we found shorter telomere length (TL) in AT cells, lower levels of TERT expression, and telomerase activity that were also partially reverted by GSE4. These observations suggest that GSE4 may be considered as a new therapy for the treatment of AT that counteracts the cellular effects of high ROS levels generated in AT cells and in addition increases telomerase activity contributing to increased cell proliferation.
Assuntos
Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos/metabolismo , Telômero/metabolismo , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/patologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Nanopartículas/química , Proteínas Nucleares/biossíntese , Proteínas Nucleares/química , Proteínas Nucleares/genética , Estresse Oxidativo/fisiologia , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Telomerase/metabolismo , Telômero/genética , Telômero/patologiaRESUMO
Dyskeratosis congenita is an inherited disease caused by mutations in genes coding for telomeric components. It was previously reported that expression of a dyskerin-derived peptide, GSE24.2, increases telomerase activity, regulates gene expression and decreases DNA damage and oxidative stress in dyskeratosis congenita patient cells. The biological activity of short peptides derived from GSE24.2 was tested and one of them, GSE4, that probed to be active, was further characterized in this article. Expression of this eleven amino acids long peptide increased telomerase activity and reduced DNA damage, oxidative stress and cell senescence in dyskerin-mutated cells. GSE4 expression also activated c-myc and TERT promoters and increase of c-myc, TERT and TERC expression. The level of biological activity of GSE4 was similar to that obtained by GSE24.2 expression. Incorporation of a dyskerin nuclear localization signal to GSE24.2 did not change its activity on promoter regulation and DNA damage protection. However, incorporation of a signal that increases the rate of nucleolar localization impaired GSE24.2 activity. Incorporation of the dyskerin nuclear localization signal to GSE4 did not alter its biological activity. Mutation of the Aspartic Acid residue that is conserved in the pseudouridine synthase domain present in GSE4 did not impair its activity, except for the repression of c-myc promoter activity and the decrease of c-myc, TERT and TERC gene expression in dyskerin-mutated cells. These results indicated that GSE4 could be of great therapeutic interest for treatment of dyskeratosis congenita patients.
Assuntos
Proteínas de Ciclo Celular/genética , Senescência Celular , Dano ao DNA , Mutação/genética , Proteínas Nucleares/genética , Estresse Oxidativo , Peptídeos/metabolismo , Telomerase/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Disceratose Congênita/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Sinais de Localização Nuclear , Fragmentos de Peptídeos , Regiões Promotoras Genéticas/genéticaRESUMO
The aim of the present study was to develop a novel strategy to deliver intracellularly the peptide GSE24.2 for the treatment of Dyskeratosis congenita (DC) and other defective telomerase disorders. For this purpose, biodegradable polymeric nanoparticles using poly(lactic-co-glycolic acid) (PLGA NPs) or poly(lactic-co-glycolic acid)-poly ethylene glycol (PLGA-PEG NPs) attached to either polycations or cell-penetrating peptides (CPPs) were prepared in order to increase their cellular uptake. The particles exhibited an adequate size and zeta potential, with good peptide loading and a biphasic pattern obtained in the in vitro release assay, showing an initial burst release and a later sustained release. GSE24.2 structural integrity after encapsulation was assessed using SDS-PAGE, revealing an unaltered peptide after the NPs elaboration. According to the cytotoxicity results, cell viability was not affected by uncoated polymeric NPs, but the incorporation of surface modifiers slightly decreased the viability of cells. The intracellular uptake exhibited a remarkable improvement of the internalization, when the NPs were conjugated to the CPPs. Finally, the bioactivity, addressed by measuring DNA damage rescue and telomerase reactivation, showed that some formulations had the lowest cytotoxicity and highest biological activity. These results proved that GSE24.2-loaded NPs could be delivered to cells, and therefore, become an effective approach for the treatment of DC and other defective telomerase syndromes.
Assuntos
Materiais Biocompatíveis/química , Proteínas de Ciclo Celular/química , Sistemas de Liberação de Medicamentos , Reativadores Enzimáticos/química , Nanopartículas/química , Proteínas Nucleares/química , Fragmentos de Peptídeos/química , Animais , Materiais Biocompatíveis/efeitos adversos , Transporte Biológico , Proteínas de Ciclo Celular/administração & dosagem , Proteínas de Ciclo Celular/efeitos adversos , Proteínas de Ciclo Celular/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/efeitos adversos , Peptídeos Penetradores de Células/química , Células Cultivadas , Fenômenos Químicos , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/efeitos adversos , Preparações de Ação Retardada/química , Preparações de Ação Retardada/uso terapêutico , Composição de Medicamentos , Sistemas de Liberação de Medicamentos/efeitos adversos , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Disceratose Congênita/tratamento farmacológico , Reativadores Enzimáticos/administração & dosagem , Reativadores Enzimáticos/efeitos adversos , Reativadores Enzimáticos/uso terapêutico , Humanos , Ácido Láctico/efeitos adversos , Ácido Láctico/química , Camundongos , Nanopartículas/efeitos adversos , Proteínas Nucleares/administração & dosagem , Proteínas Nucleares/efeitos adversos , Proteínas Nucleares/genética , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/efeitos adversos , Fragmentos de Peptídeos/genética , Poliaminas/efeitos adversos , Poliaminas/química , Polieletrólitos , Polietilenoglicóis/efeitos adversos , Polietilenoglicóis/química , Poliglactina 910/efeitos adversos , Poliglactina 910/química , Ácido Poliglicólico/efeitos adversos , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Estabilidade Proteica , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapêuticoRESUMO
Rhodopsin is the visual photoreceptor responsible for dim light vision. This receptor is located in the rod cell of the retina and is a prototypical member of the G-protein-coupled receptor superfamily. The structural details underlying the molecular recognition event in transducin activation by photoactivated rhodopsin are of key interest to unravel the molecular mechanism of signal transduction in the retina. We constructed and expressed rhodopsin mutants in the second and third cytoplasmic domains of rhodopsin--where the natural amino acids were substituted by the human M3 acetylcholine muscarinic receptor homologous residues--in order to determine their potential involvement in G-protein recognition. These mutants showed normal chromophore formation and a similar photobleaching behavior than WT rhodopsin, but decreased thermal stability in the dark state. The single mutant V138³·5³ and the multiple mutant containing V2275·6² and a combination of mutations at the cytoplasmic end of transmembrane helix 6 caused a reduction in transducin activation upon rhodopsin photoactivation. Furthermore, combination of mutants at the second and third cytoplasmic domains revealed a cooperative role, and partially restored transducin activation. The results indicate that hydrophobic interactions by V138³·5³, V2275·6², V2506·³³, V2546·³7 and I2556·³8 are critical for receptor activation and/or efficient rhodopsin-transducin interaction.