RESUMEN
Oxidative stress and mitochondrial dysfunction have been implicated in Parkinson's disease (PD) pathology. NADH:ubiquinone oxidoreductase (complex I) (EC 1.6.99.3) enzyme activity is aberrant in both PD and 1-methyl-4-phenylpyridinium (MPP(+)) models of PD. Reverse transcription polymerase chain reaction of RNA isolated from MPP(+)-treated human neuroblastoma SH-SY5Y cells identified changes in steady-state mRNA levels of the mitochondrial transcript for subunit 4 of complex I (ND4). Expression of ND4 decreased to nearly 50% after 72 h of MPP(+) (1 mM) exposure. The expression of other mitochondrial transcripts did not change significantly under the same conditions. Pre-incubation of cells with the free-radical spin-trap, N-tert-butyl-alpha-(2-sulfophenyl)-nitrone prior to MPP(+) exposure, prevented decreases in cell viability and ND4 expression. This suggests that functional defects in complex I enzyme activity in PD and MPP(+) toxicity may result from changes in steady-state mRNA levels and that free radicals may be important in this process.
Asunto(s)
1-Metil-4-fenilpiridinio/toxicidad , Herbicidas/toxicidad , NADH NADPH Oxidorreductasas/genética , Neuronas/fisiología , Complejo I de Transporte de Electrón , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Mitocondrias/enzimología , Neuroblastoma , Neuronas/efectos de los fármacos , Enfermedad de Parkinson/metabolismo , ARN Mensajero/análisis , Células Tumorales CultivadasRESUMEN
Proteolysis of the amyloid beta protein precursor (APP) is a key event in the development of Alzheimer's disease. In our search for proteases that can cleave APP and liberate the amino terminus of the amyloidogenic beta protein, we characterized a calcium-dependent serine protease (CASP) which is present in reactive astrocytes and cross-reacts with anti-cathepsin G antibodies. We wanted to take advantage of this cross-reactivity to clone the cDNA of CASP and eventually evaluate its tissue distribution. Screening of two human fetal brain cDNA libraries with anti-cathepsin G antibodies led to the identification of a cDNA coding for a novel protein whose only homology to known proteins is to the active site of trypsin-type serine proteases. We called this protein the novel serine protease (NSP). NSP exists in at least three differentially spliced forms, one of which is expressed predominantly in brain and testis. Immunohistochemistry and immunoprecipitation with antibodies generated against NSP show that it is expressed and secreted by a variety of cells and that, in brain, it is found primarily in cerebrovascular smooth muscle cells and reactive astrocytes.
Asunto(s)
Encéfalo/enzimología , Péptidos y Proteínas de Señalización Intracelular , Serina Endopeptidasas/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Formación de Anticuerpos , Secuencia de Bases , Mapeo Cromosómico , Escherichia coli/genética , Haplorrinos , Humanos , Inmunohistoquímica , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Péptidos/inmunología , Proteínas Recombinantes/biosíntesis , Serina Endopeptidasas/inmunología , Serina Endopeptidasas/aislamiento & purificaciónRESUMEN
Previous studies have demonstrated that insulin-like growth factor-I (IGF-I) increases elastin gene transcription in aortic smooth muscle cells and that this up-regulation is accompanied by a loss of protein binding to the proximal promoter. Sp1 has been identified as one of the factors whose binding is lost, and in the present study we show that Sp3 binding is also abrogated by IGF-I, but in a selected manner. In functional analyses using Drosophila SL-2 cells, Sp1 expression can drive transcription from the elastin proximal promoter, while co-expression of Sp3 results in a repression of Sp1 activity. Footprint and gel shift analyses position the IGF-I responsive sequences to a putative retinoblastoma control element (RCE). Mutation of the putative RCE sequence as assessed by transient transfection of smooth muscle cells results in an increase in reporter activity equal in magnitude to that conferred by IGF-I on the wild type promoter. Together these results support the hypothesis that IGF-I-mediated increase in elastin transcription occurs via a mechanism of derepression involving the abrogation of a repressor that appears to be Sp3 binding to the RCE.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Elastina/genética , Factor I del Crecimiento Similar a la Insulina/fisiología , Retinoblastoma/genética , Factores de Transcripción/fisiología , Transcripción Genética/fisiología , Animales , Células Cultivadas , Drosophila melanogaster , Humanos , Mutagénesis , Unión Proteica , Ratas , Ratas Sprague-Dawley , Factor de Transcripción Sp3 , TransfecciónRESUMEN
Alzheimer's disease is characterized neuropathologically by the presence of neuritic and amyloid plaques, vascular amyloid, and neurofibrillary tangles in specific brain areas. The main constituent of amyloid deposits is amyloid beta protein, a 40-42 amino acid proteolytic product of the amyloid beta-precursor protein. In our search for proteases that can generate the N-terminus of amyloid beta protein (beta-secretases), we discovered a thiol-dependent metalloprotease that was identified, by peptide sequencing, as metalloendopeptidase EC 3.4.24.15. In vitro, the metalloprotease cleaves the methionine-aspartic acid bond in a 10 amino acid synthetic peptide, indicating that it could generate the N-terminus of amyloid beta protein, and generates amyloidogenic fragments from full-length recombinant amyloid beta-precursor protein. Mouse monoclonal antibodies produced against a unique synthetic peptide from the metalloprotease labeled various monkey tissues as detected by western blots and immunohistochemistry. Unexpectedly, two monoclonal antibodies, IVD6 and IIIF3, immunolabeled strongly intracellular neurofibrillary tangles, neurites of senile plaques, and neuropil threads, but not "ghost" tangles or amyloid in sections taken from Alzheimer's disease brain. This finding provides further evidence for the metalloprotease's relevance to Alzheimer's disease pathology, although the connection between tangle staining and the formation of amyloid beta protein remains to be elucidated.
Asunto(s)
Enfermedad de Alzheimer/patología , Anticuerpos Monoclonales/inmunología , Metaloendopeptidasas/inmunología , Ovillos Neurofibrilares/patología , Animales , Especificidad de Anticuerpos , Western Blotting , Haplorrinos , Humanos , Inmunohistoquímica , RatonesRESUMEN
To determine whether there is a specific receptor for serpin:protease complexes on the astrocyte cell surface, we analyzed the cell-binding characteristics of an 125I-cathepsin G:alpha 1-antichymotrypsin complex. Complex formation is maximal at a 1:1 molar ratio of cathepsin G to alpha 1-antichymotrypsin (alpha 1-ACT) as revealed by sodium dodecyl sulfate-gel electrophoresis and autoradiography. Complex binding to mouse spinal cord astrocytes was inhibited by the presence of excess unlabeled complex, but not by the native protease, cathepsin G, or by the serpin, alpha 1-ACT. Scatchard analysis of the binding curve showed the Kd to be 8 x 10(-8) M. We estimated receptor numbers on astrocytes to be about 2.2 x 10(6) sites per cell. An alpha 1-ACT-derived pentapeptide, Phe-Leu-Met-Ile-Ile (FLMII), homologous to a well-conserved segment in the serpin superfamily, did not inhibit the binding of complexes to cells. These data indicate that a specific receptor for alpha 1-ACT:cathepsin G exists on the CNS glial cell surface. Study of this receptor in astrocytes should facilitate understanding of the serpin:protease balance in the brain.