RESUMEN
A more detailed insight into disease mechanisms of multiple sclerosis (MS) is crucial for the development of new and more effective therapies. MS is a chronic inflammatory autoimmune disease of the central nervous system. The aim of this study is to identify novel disease associated proteins involved in the development of inflammatory brain lesions, to help unravel underlying disease processes. Brainstem proteins were obtained from rats with MBP induced acute experimental autoimmune encephalomyelitis (EAE), a well characterized disease model of MS. Samples were collected at different time points: just before onset of symptoms, at the top of the disease and following recovery. To analyze changes in the brainstem proteome during the disease course, a quantitative proteomics study was performed using two-dimensional difference in-gel electrophoresis (2D-DIGE) followed by mass spectrometry. We identified 75 unique proteins in 92 spots with a significant abundance difference between the experimental groups. To find disease-related networks, these regulated proteins were mapped to existing biological networks by Ingenuity Pathway Analysis (IPA). The analysis revealed that 70% of these proteins have been described to take part in neurological disease. Furthermore, some focus networks were created by IPA. These networks suggest an integrated regulation of the identified proteins with the addition of some putative regulators. Post-synaptic density protein 95 (DLG4), a key player in neuronal signalling and calcium-activated potassium channel alpha 1 (KCNMA1), involved in neurotransmitter release, are 2 putative regulators connecting 64% of the identified proteins. Functional blocking of the KCNMA1 in macrophages was able to alter myelin phagocytosis, a disease mechanism highly involved in EAE and MS pathology. Quantitative analysis of differentially expressed brainstem proteins in an animal model of MS is a first step to identify disease-associated proteins and networks that warrant further research to study their actual contribution to disease pathology.
Asunto(s)
Encefalomielitis Autoinmune Experimental/metabolismo , Esclerosis Múltiple/metabolismo , Mapeo de Interacción de Proteínas , Proteoma , Animales , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Homólogo 4 de la Proteína Discs Large , Femenino , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Análisis de Componente Principal , Mapas de Interacción de Proteínas , Proteómica/métodos , Ratas , Ratas Endogámicas Lew , Reproducibilidad de los ResultadosRESUMEN
Leukemia inhibitory factor (LIF) promotes the survival of oligodendrocytes (OLG) both in vitro and in an animal model of multiple sclerosis. Here, we show that LIF protects mature rat OLG cultures selectively against the combined insult of the proinflammatory cytokines interferon-gamma and tumor necrosis factor-alpha, but it does not protect against oxidative stress nor against staurosporine induced apoptosis. We further demonstrate that LIF activates the janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) and the phosphatidylinositol 3 kinase/Akt pathway in mature OLG. We show that LIF protection is independent of suppressors of cytokine signaling and Bcl-2 mRNA expression levels. To gain further insight into the protective mechanism, a quantitative proteomic approach (DIGE) was applied to identify differentially expressed proteins in LIF-treated OLG. Our results indicate that LIF induces a shift in the cellular machinery toward a prosurvival execution program, illustrated by an enhanced expression of isoforms of the antiapoptotic molecule 14-3-3. These data provide further insight into the mechanisms of LIF-mediated protection of mature OLGs.
Asunto(s)
Proteínas 14-3-3/metabolismo , Apoptosis/efectos de los fármacos , Factor Inhibidor de Leucemia/farmacología , Oligodendroglía/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Western Blotting , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Oligodendroglía/citología , Oligodendroglía/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Espectrometría de Masa por Ionización de Electrospray , Proteínas Supresoras de la Señalización de Citocinas/genética , Espectrometría de Masas en Tándem , Transcripción Genética/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Oligodendrocytes are glial cells responsible for the synthesis and maintenance of myelin in the central nervous system (CNS). Oligodendrocytes are vulnerable to damage occurring in a variety of neurological diseases. Understanding oligodendrocyte biology is crucial for the dissemination of de- and remyelination mechanisms. The goal of the present study is the construction of a protein database of mature rat oligodendrocytes. Post-mitotic oligodendrocytes were isolated from mature Wistar rats and subjected to immunocytochemistry. Proteins were extracted and analyzed by means of two-dimensional gel electrophoresis and two-dimensional liquid chromatography, both coupled to mass spectrometry. The combination of the gel-based and gel-free approach resulted in confident identification of a total of 200 proteins. A minority of proteins were identified in both proteomic strategies. The identified proteins represent a variety of functional groups, including novel oligodendrocyte proteins. The results of this study emphasize the power of the applied proteomic strategy to study known or to reveal new proteins and to investigate their regulation in oligodendrocytes in different disease models.
Asunto(s)
Encéfalo/metabolismo , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/metabolismo , Proteómica/métodos , Animales , Encéfalo/citología , Células Cultivadas , Cromatografía Liquida/métodos , Electroforesis en Gel Bidimensional/métodos , Inmunohistoquímica/métodos , Espectrometría de Masas/métodos , Proteínas del Tejido Nervioso/clasificación , Oligodendroglía/citología , Ratas , Ratas WistarRESUMEN
This paper reports on the findings of the Biomedical Research Institute, as one of the participants in the pilot study of the HUPO Brain Proteome Project. A biopsy and autopsy study sample derived from human brain was distributed among the participants for proteomic analysis. In our laboratory, attention was focused on protein identification using the bottom-up shotgun approach. Protein extracts derived from both samples were trypsinized and analyzed separately by 2-D LC and MS. In a complementary approach, the tryptic digests were analyzed directly by LC-ESI-MS/MS and gas-phase fractionation in the mass spectrometer. Taken together, both proteomic approaches in combination with a stringent evaluation process, resulted in the confident identification of 209 proteins in the human brain samples under investigation.
Asunto(s)
Encéfalo/metabolismo , Proteoma/metabolismo , Adulto , Autopsia , Biopsia , Encéfalo/patología , Cromatografía Liquida , Epilepsia del Lóbulo Temporal/metabolismo , Epilepsia del Lóbulo Temporal/patología , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Persona de Mediana Edad , Proyectos Piloto , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Neurological diseases, including multiple sclerosis (M.S.), often provoke changes in the functioning of the endothelial and epithelial brain barriers and give rise to disease-associated alterations of the cerebrospinal fluid (CSF) proteome. In the present study, pooled and ultrafiltered CSF of M.S. and non-M.S. patients was digested with trypsin and analyzed by off-line strong cation-exchange chromatography (SCX) coupled to on-line reversed-phase LC-ESI-MS/MS. In an alternative approach, the trypsin-treated subproteomes were analyzed directly by LC-ESI-MS/MS and gas-phase fractionation in the mass spectrometer. Taken together, both proteomic approaches in combination with a three-step evaluation process including the search engines Sequest and Mascot, and the validation software Scaffold, resulted in the identification of 148 proteins. Sixty proteins were identified in CSF for the first time by mass spectrometry. For validation purposes, the concentration of cystatin A was determined in individual CSF and serum samples of M.S. and non-M.S. patients using ELISA.
Asunto(s)
Líquido Cefalorraquídeo/química , Esclerosis Múltiple/líquido cefalorraquídeo , Proteoma/análisis , Proteómica/métodos , Adulto , Anciano , Estudios de Casos y Controles , Cromatografía Liquida , Femenino , Humanos , Vértebras Lumbares/anatomía & histología , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Reproducibilidad de los Resultados , UltrafiltraciónRESUMEN
The structural proteome of phiKMV, a lytic bacteriophage infecting Pseudomonas aeruginosa, was analysed using two approaches. In one approach, structural proteins of the phage were fractionated by SDS-PAGE for identification by liquid chromatography-mass spectrometry (LC-MS). In a second approach, a whole-phage shotgun analysis (WSA) was applied. WSA uses trypsin digestion of whole phage particles, followed by reversed-phase HPLC and gas-phase fractionation of the complex peptide mixture prior to MS. The results yield a comprehensive view of structure-related proteins in phiKMV and suggest subtle structural differences from phage T7.
Asunto(s)
Bacteriófagos/metabolismo , Genoma Viral , Proteoma , Pseudomonas aeruginosa/virología , Proteínas Virales/metabolismo , Bacteriófago T7/genética , Bacteriófagos/enzimología , Bacteriófagos/genética , Cromatografía Líquida de Alta Presión , Espectrometría de Masas , Proteínas Virales/genéticaRESUMEN
Multiple sclerosis is an autoimmune inflammatory demyelinating disease of the central nervous system. Disease mechanisms in multiple sclerosis at the molecular level remain poorly understood and no reliable proteinaceous disease markers are available yet. The goal of the present study is the construction of a protein database of two-dimensional gel electrophoresis (2-DE) separated cerebrospinal fluid (CSF) proteins from multiple sclerosis patients. By means of liquid chromatography tandem mass spectrometry 65 different proteins were identified from 300 spots. Eighteen of these proteins have not been reported previously on 2-DE gels of CSF. Here we report on the identification of these proteins and discuss their potential relation to multiple sclerosis.