RESUMEN
AIMS/HYPOTHESIS: Transplantation of pancreatic islets constitutes a promising alternative treatment for type 1 diabetes. However, it is limited by the shortage of organ donors. Previous results from our laboratory have demonstrated beneficial effects of recombinant human prolactin (rhPRL) treatment on beta cell cultures. We therefore investigated the role of rhPRL action in human beta cell survival, focusing on the molecular mechanisms involved in this process. METHODS: Human pancreatic islets were isolated using an automated method. Islet cultures were pre-treated in the absence or presence of rhPRL and then subjected to serum starvation or cytokine treatment. Beta cells were labelled with Newport green and apoptosis was evaluated using flow cytometry analysis. Levels of BCL2 gene family members were studied by quantitative RT-PCR and western blot. Caspase-8, -9 and -3 activity, as well as nitric oxide production, were evaluated by fluorimetric assays. RESULTS: The proportion of apoptotic beta cells was significantly lowered in the presence of rhPRL under both cell death-induced conditions. We also demonstrated that cytoprotection may involve an increase of BCL2/BAX ratio, as well as inhibition of caspase-8, -9 and -3. CONCLUSIONS/INTERPRETATION: Our study provides relevant evidence for a protective effect of lactogens on human beta cell apoptosis. The results also suggest that the improvement of cell survival may involve, at least in part, inhibition of cell death pathways controlled by the BCL2 gene family members. These findings are highly relevant for improvement of the islet isolation procedure and for clinical islet transplantation.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Prolactina/farmacología , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Adulto , Apoptosis/fisiología , Inhibidores de Caspasas , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/cirugía , Humanos , Células Secretoras de Insulina/metabolismo , Trasplante de Islotes Pancreáticos/métodos , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/fisiología , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Proteolysis-inducing factor/dermcidin (PIF/DCD) is a novel human gene, located on chromosome 12, locus 12q13.1, that encodes a secreted 110-amino acid protein. Two transcripts for the protein have been identified in normal skin, breast, placenta and brain, and in various primary and metastatic tumor cells. The putative native-state structure of PIF/DCD has not been resolved. Here, we describe some biochemical features of the soluble recombinant 11-kDa protein produced in Escherichia coli. The native 11-kDa polypeptide displayed an anomalous mobility on 1% SDS-PAGE under reduced conditions and appeared as a single approximately 16-kDa band. Under nonreduced conditions, we detected by mass spectrometry, the presence of multiple peaks corresponding to m/z values of 21 kDa, which we confirmed as a dimeric form with a disulfide bridge between cysteine 34 of each 11-kDa monomer. The native protein exhibited an unusually high susceptibility to proteolytic attack by trypsin, and up to 13 peptides derived from its C-terminus were produced after 5 min of incubation. The secondary structure analysis of PIF/DCD native protein in aqueous solution, by circular dichroism spectroscopy, revealed regions with non-well-defined secondary structure but that acquired alpha-helix and beta-sheet secondary structures in the presence of TFE/water mixtures and micellar and non-micellar SDS molecules. By using PONDR, DisEMBL, DisProt, and GlobPlot computational predictors, we identified a long disorder region at the N-terminus of PIF/DCD amino acid sequence. This segment (from 19-50 residues) is critical for some of its biological activities, including neuron survival. This result is coherent with successive failure of crystallization of the protein. Taken together, these data suggest that the disorder and order transition may be relevant for some biological functions of PIF/DCD.
Asunto(s)
Péptidos/química , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Programas InformáticosRESUMEN
Mitochondria increase their outer and inner membrane permeability to solutes, protons and metabolites in response to a variety of extrinsic and intrinsic signaling events. The maintenance of cellular and intraorganelle ionic homeostasis, particularly for Ca2+, can determine cell survival or death. Mitochondrial death decision is centered on two processes: inner membrane permeabilization, such as that promoted by the mitochondrial permeability transition pore, formed across inner membranes when Ca2+ reaches a critical threshold, and mitochondrial outer membrane permeabilization, in which the pro-apoptotic proteins BID, BAX, and BAK play active roles. Membrane permeabilization leads to the release of apoptogenic proteins: cytochrome c, apoptosis-inducing factor, Smac/Diablo, HtrA2/Omi, and endonuclease G. Cytochrome c initiates the proteolytic activation of caspases, which in turn cleave hundreds of proteins to produce the morphological and biochemical changes of apoptosis. Voltage-dependent anion channel, cyclophilin D, adenine nucleotide translocase, and the pro-apoptotic proteins BID, BAX, and BAK may be part of the molecular composition of membrane pores leading to mitochondrial permeabilization, but this remains a central question to be resolved. Other transporting pores and channels, including the ceramide channel, the mitochondrial apoptosis-induced channel, as well as a non-specific outer membrane rupture may also be potential release pathways for these apoptogenic factors. In this review, we discuss the mechanistic models by which reactive oxygen species and caspases, via structural and conformational changes of membrane lipids and proteins, promote conditions for inner/outer membrane permeabilization, which may be followed by either opening of pores or a rupture of the outer mitochondrial membrane.
Asunto(s)
Apoptosis/fisiología , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Animales , Caspasas/metabolismo , Permeabilidad de la Membrana Celular , Citocromos c/metabolismo , Mitocondrias/fisiología , Proteínas de Transporte de Membrana Mitocondrial/fisiología , Poro de Transición de la Permeabilidad Mitocondrial , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
Mitochondria increase their outer and inner membrane permeability to solutes, protons and metabolites in response to a variety of extrinsic and intrinsic signaling events. The maintenance of cellular and intraorganelle ionic homeostasis, particularly for Ca2+, can determine cell survival or death. Mitochondrial death decision is centered on two processes: inner membrane permeabilization, such as that promoted by the mitochondrial permeability transition pore, formed across inner membranes when Ca2+ reaches a critical threshold, and mitochondrial outer membrane permeabilization, in which the pro-apoptotic proteins BID, BAX, and BAK play active roles. Membrane permeabilization leads to the release of apoptogenic proteins: cytochrome c, apoptosis-inducing factor, Smac/Diablo, HtrA2/Omi, and endonuclease G. Cytochrome c initiates the proteolytic activation of caspases, which in turn cleave hundreds of proteins to produce the morphological and biochemical changes of apoptosis. Voltage-dependent anion channel, cyclophilin D, adenine nucleotide translocase, and the pro-apoptotic proteins BID, BAX, and BAK may be part of the molecular composition of membrane pores leading to mitochondrial permeabilization, but this remains a central question to be resolved. Other transporting pores and channels, including the ceramide channel, the mitochondrial apoptosis-induced channel, as well as a non-specific outer membrane rupture may also be potential release pathways for these apoptogenic factors. In this review, we discuss the mechanistic models by which reactive oxygen species and caspases, via structural and conformational changes of membrane lipids and proteins, promote conditions for inner/outer membrane permeabilization, which may be followed by either opening of pores or a rupture of the outer mitochondrial membrane.
Asunto(s)
Animales , Apoptosis/fisiología , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Permeabilidad de la Membrana Celular , Caspasas/metabolismo , Citocromos c/metabolismo , Mitocondrias/fisiología , Proteínas de Transporte de Membrana Mitocondrial/fisiología , /metabolismo , Especies Reactivas de Oxígeno/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
Proteolysis-inducing factor/dermcidin (PIF/DCD) is a novel human gene, located on chromosome 12, locus 12q13.1, that encodes a secreted 110-amino acid protein. Two transcripts for the protein have been identified in normal skin, breast, placenta and brain, and in various primary and metastatic tumor cells. The putative native-state structure of PIF/DCD has not been resolved. Here, we describe some biochemical features of the soluble recombinant 11-kDa protein produced in Escherichia coli. The native 11-kDa polypeptide displayed an anomalous mobility on 1% SDS-PAGE under reduced conditions and appeared as a single ~16-kDa band. Under nonreduced conditions, we detected by mass spectrometry, the presence of multiple peaks corresponding to m/z values of 21 kDa, which we confirmed as a dimeric form with a disulfide bridge between cysteine 34 of each 11-kDa monomer. The native protein exhibited an unusually high susceptibility to proteolytic attack by trypsin, and up to 13 peptides derived from its C-terminus were produced after 5 min of incubation. The secondary structure analysis of PIF/DCD native protein in aqueous solution, by circular dichroism spectroscopy, revealed regions with non-well-defined secondary structure but that acquired á-helix and â-sheet secondary structures in the presence of TFE/water mixtures and micellar and non-micellar SDS molecules. By using PONDR®, DisEMBL, DisProt, and GlobPlot computational predictors, we identified a long disorder region at the N-terminus of PIF/DCD amino acid sequence. This segment (from 19-50 residues) is critical for some of its biological activities, including neuron survival. This result is coherent with successive failure of crystallization of the protein. Taken together, these data suggest that the disorder and order transition may be relevant for some biological functions of PIF/DCD.