RESUMO
The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis.
Assuntos
Proteínas 14-3-3/fisiologia , Antígenos de Fungos/fisiologia , Apoptose , Células Epiteliais/microbiologia , Proteínas Fúngicas/fisiologia , Glicoproteínas/fisiologia , Paracoccidioides/fisiologia , Linhagem Celular/microbiologia , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Reação em Cadeia da Polimerase em Tempo RealRESUMO
At the time of diagnosis, half of lung cancer patients have advanced incurable disease. Different chemotherapy combinations--with or without targeted therapies--yield similar results in spite of the continuous efforts of clinicians. However, molecular biological studies have already shed a great deal of light on the existence of multiple genetic aberrations that can be useful for customizing treatment. mRNA transcripts involved in DNA repair pathways, such as ERCC1 and BRCA1, confer selective resistance to cisplatin or taxanes. Polymorphisms in DNA repair genes and methylation of checkpoint genes in circulating serum DNA could become important predictive markers of survival to certain cisplatin-based regimens. EGFR tyrosine kinase mutations are the crux of targeted therapies.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/terapia , Proteínas 14-3-3/genética , Proteínas 14-3-3/fisiologia , Adenocarcinoma/genética , Antineoplásicos/classificação , Antineoplásicos/farmacologia , Benzamidas , Carcinoma Pulmonar de Células não Pequenas/genética , Dano ao DNA , Reparo do DNA/genética , Epistasia Genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/fisiologia , Cloridrato de Erlotinib , Genes BRCA1 , Genes cdc , Genes erbB-1 , Genes ras , Predisposição Genética para Doença , Humanos , Mesilato de Imatinib , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/fisiologia , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Polimorfismo Genético , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Quinazolinas/farmacologia , Quinazolinas/uso terapêuticoRESUMO
Endopeptidase 24.15 (ep24.15: EC3.4.24.15), a secreted protein involved in peptide metabolism, is unusual in that it does not contain a signal peptide sequence. In this work, we describe the physical interaction between ep24.15 and 14-3-3 epsilon, one isoform of a family of ubiquitous phosphoserine/threonine-scaffold proteins that organizes cell signaling and is involved in exocytosis. The interaction between ep24.15 and 14-3-3 epsilon increased following phosphorylation of ep24.15 at Ser(644) by protein kinase A (PKA). The co-localization of ep24.15 and 14-3-3 epsilon was increased by exposure of HEK293 cells (human embryonic kidney cells) to forskolin (10 microm). Overexpression of 14-3-3 epsilon in HEK293 cells almost doubled the secretion of ep24.15 stimulated by A23187 (7.5 microm) from 10%[1.4 +/- 0.24 AFU/(min 10(6) cells)] to 19%[2.54 +/- 0.24 AFU/(min 10(6) cells)] (p < 0.001) of the total intracellular enzyme activity. Treatment with forskolin had a synergistic effect on the A23187-stimulated secretion of ep24.15 that was totally blocked by the PKA inhibitor KT5720. The ep24.15 point mutation S644A reduced the co-localization of ep24.15 and 14-3-3 in stably transfected HEK293 cells. Indeed, secretion of the ep24.15 S644A mutant from these cells was only slightly stimulated by A23187 and insensitive to forskolin, in contrast to that of the wild type enzyme. Together, these data suggest that prior interaction with 14-3-3 is an important step in the unconventional stimulated secretion of ep24.15.