RESUMO
Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies. Recently, two point mutations in alpha-synuclein were found to be associated with familial PD, but as of yet no mutations have been described in the homologous genes beta- and gamma-synuclein. alpha-Synuclein forms the major fibrillar component of Lewy bodies, but these do not stain for beta- or gamma-synuclein. This result is very surprising, given the extent of sequence conservation and the high similarity in expression and subcellular localization, in particular between alpha- and beta-synuclein. Here we compare in vitro fibrillogenesis of all three purified synucleins. We show that fresh solutions of alpha-, beta-, and gamma- synuclein show the same natively unfolded structure. While over time alpha-synuclein forms the previously described fibrils, no fibrils could be detected for beta- and gamma-synuclein under the same conditions. Most importantly, beta- and gamma-synuclein could not be cross-seeded with alpha-synuclein fibrils. However, under conditions that drastically accelerate aggregation, gamma-synuclein can form fibrils with a lag phase roughly three times longer than alpha-synuclein. These results indicate that beta- and gamma-synuclein are intrinsically less fibrillogenic than alpha-synuclein and cannot form mixed fibrils with alpha-synuclein, which may explain why they do not appear in the pathological hallmarks of PD, although they are closely related to alpha-synuclein and are also abundant in brain.
Assuntos
Proteínas do Tecido Nervoso/química , Doença de Parkinson/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Dicroísmo Circular , Primers do DNA , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Análise Espectral/métodos , Sinucleínas , alfa-Sinucleína , beta-Sinucleína , gama-SinucleínaRESUMO
Cerebral deposition of amyloid beta peptide (Abeta) is an early and critical feature of Alzheimer's disease. Abeta generation depends on proteolytic cleavage of the amyloid precursor protein (APP) by two unknown proteases: beta-secretase and gamma-secretase. These proteases are prime therapeutic targets. A transmembrane aspartic protease with all the known characteristics of beta-secretase was cloned and characterized. Overexpression of this protease, termed BACE (for beta-site APP-cleaving enzyme) increased the amount of beta-secretase cleavage products, and these were cleaved exactly and only at known beta-secretase positions. Antisense inhibition of endogenous BACE messenger RNA decreased the amount of beta-secretase cleavage products, and purified BACE protein cleaved APP-derived substrates with the same sequence specificity as beta-secretase. Finally, the expression pattern and subcellular localization of BACE were consistent with that expected for beta-secretase. Future development of BACE inhibitors may prove beneficial for the treatment of Alzheimer's disease.
Assuntos
Doença de Alzheimer/enzimologia , Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidases/isolamento & purificação , Ácido Aspártico Endopeptidases/metabolismo , Doença de Alzheimer/tratamento farmacológico , Motivos de Aminoácidos , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Sítios de Ligação , Encéfalo/enzimologia , Encéfalo/metabolismo , Linhagem Celular , Clonagem Molecular , Endopeptidases , Endossomos/enzimologia , Expressão Gênica , Biblioteca Gênica , Complexo de Golgi/enzimologia , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , Peptídeos/metabolismo , Inibidores de Proteases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major components of which are filaments consisting of alpha-synuclein. Two recently identified point mutations in alpha-synuclein are the only known genetic causes of PD. alpha-Synuclein fibrils similar to the Lewy body filaments can be formed in vitro, and we have shown recently that both PD-linked mutations accelerate their formation. This study addresses the mechanism of alpha-synuclein aggregation: we show that (i) it is a nucleation-dependent process that can be seeded by aggregated alpha-synuclein functioning as nuclei, (ii) this fibril growth follows first-order kinetics with respect to alpha-synuclein concentration, and (iii) mutant alpha-synuclein can seed the aggregation of wild type alpha-synuclein, which leads us to predict that the Lewy bodies of familial PD patients with alpha-synuclein mutations will contain both, the mutant and the wild type protein. Finally (iv), we show that wild type and mutant forms of alpha-synuclein do not differ in their critical concentrations. These results suggest that differences in aggregation kinetics of alpha-synucleins cannot be explained by differences in solubility but are due to different nucleation rates. Consequently, alpha-synuclein nucleation may be the rate-limiting step for the formation of Lewy body alpha-synuclein fibrils in Parkinson's disease.
Assuntos
Corpos de Lewy/química , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/genética , Escherichia coli/genética , Humanos , Cinética , Mutação , Proteínas do Tecido Nervoso/química , Doença de Parkinson/patologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Solubilidade , Sinucleínas , alfa-SinucleínaRESUMO
Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major component of which are filaments consisting of alpha-synuclein. Two recently identified point mutations in alpha-synuclein are the only known genetic causes of PD, but their pathogenic mechanism is not understood. Here we show that both wild type and mutant alpha-synuclein form insoluble fibrillar aggregates with antiparallel beta-sheet structure upon incubation at physiological temperature in vitro. Importantly, aggregate formation is accelerated by both PD-linked mutations. Under the experimental conditions, the lag time for the formation of precipitable aggregates is about 280 h for the wild type protein, 180 h for the A30P mutant, and only 100 h for the A53T mutant protein. These data suggest that the formation of alpha-synuclein aggregates could be a critical step in PD pathogenesis, which is accelerated by the PD-linked mutations.
Assuntos
Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Doença de Parkinson/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Linhagem Celular , Dicroísmo Circular , Clonagem Molecular , Humanos , Conformação Proteica , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Sinucleínas , alfa-SinucleínaRESUMO
The permeability of normal human, the human Dutch variant, and the rat A beta 1-40 proteins at the blood-brain barrier (BBB) was determined in the normal adult rat by quantifying the permeability coefficient-surface area (PS) product for each protein after correction for the residual plasma volume (Vp) occupied by the protein in the blood vessels of different brain regions. The PS for normal and Dutch A beta ranged from 13 x 10(-6) to 22 x 10(-6) ml/g/s in different brain regions, which is 130 to 220 times greater than albumin. These high PS values compare to that of insulin, whose uptake is decidedly by a receptor-mediated transport process, and suggest a similar mechanism for A beta. Remarkably, the PS for rat A beta was 4 times higher and ranged from 54 x 10(-6) to 82 x 10(-6) ml/g/s for different brain regions, suggesting a distinctive species specificity. While the Vp values of human and rat A beta were comparable, the Dutch variant was 2 to 3 times higher, indicating adherence to the vessel walls in different brain regions, consistent with the heavy A beta deposition that has been described in intracerebral vessel walls with this variant. The high PS values observed for A beta at the BBB suggest that sources outside the nervous system could contribute, at least in part, to the cerebral A beta deposits seen in Alzheimer's disease. SDS-PAGE of 125I-labeled human A beta after 60 min of uptake revealed intact protein in plasma and in different brain regions. In addition, 125I-labeled human A beta binding to a protein of 67,000 in both plasma and brain tissue regions was observed with SDS-PAGE. This protein was tentatively identified as albumin, and it was not detectable in the brain regions of animals that had undergone intracardiac perfusion; hence, a portion of A beta binds tightly to and is likely transported by albumin in plasma. The absence of this A beta-albumin complex in brain regions after perfusion and the low permeability of albumin at the BBB imply that A beta itself is efficiently transported at the BBB to account for the high PS values, although presentation of A beta to the capillary endothelial cell by albumin or other plasma proteins cannot be excluded.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica , Encéfalo/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/síntese química , Peptídeos beta-Amiloides/genética , Animais , Permeabilidade Capilar , Variação Genética , Meia-Vida , Humanos , Insulina/sangue , Insulina/metabolismo , Radioisótopos do Iodo , Dados de Sequência Molecular , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Volume Plasmático , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
The amyloid beta-peptide (Abeta) is the major constituent of neuritic plaques in Alzheimer's disease and occurs as a soluble 40-42-residue peptide in cerebrospinal fluid and blood of both normal and AD subjects. It is unclear whether Abeta, once it is secreted by cells, remains free in biological fluids or is associated with other proteins and thus transported and metabolized with them. Such knowledge of the normal fate of Abeta is a prerequisite for understanding the changes that may lead to the pathological aggregation of soluble Abeta in vivo, the possible influence of certain extracellular proteins, particularly apolipoprotein E, on plaque formation, and the pharmacology of putative Abeta-lowering drugs. To address the question of Abeta distribution in human biological fluids, we incubated fresh human plasma from 38 subjects with physiological concentrations (0.5-0.7 nM) of radioiodinated Abeta1-40 and seven plasma samples with Abeta1-42. Lipoproteins and lipid-free proteins were separated and analyzed for bound iodinated Abeta1-40. We found that up to 5% of Abeta added to plasma is bound to selected lipoproteins: very low density, low density, and high density, but not lipoprotein(a). The large majority ( approximately 89%), however, is bound to albumin, and very little Abeta is free. Abeta distribution in plasma was not significantly influenced by apolipoprotein E genotype. We conclude that Abeta is normally bound to and transported by albumin and specific lipoproteins in human plasma under physiological conditions.
Assuntos
Peptídeos beta-Amiloides/sangue , Lipoproteínas/sangue , Albumina Sérica/metabolismo , Apolipoproteínas E/metabolismo , Transporte Biológico , Humanos , Fragmentos de Peptídeos/sangue , Ligação ProteicaRESUMO
Cerebral deposition of the amyloid beta protein (A beta) is an early and invariant feature of Alzheimer disease (AD). Whereas the 40-amino acid form of A beta (A beta 40) accounts for approximately 90% of all A beta normally released from cells, it appears to contribute only to later phases of the pathology. In contrast, the longer more amyloidogenic 42-residue form (A beta 42), accounting for only approximately 10% of secreted A beta, is deposited in the earliest phase of AD and remains the major constituent of most amyloid plaques throughout the disease. Moreover, its levels have been shown to be increased in all known forms of early-onset familial AD. Thus, inhibition of A beta 42 production is a prime therapeutic goal. The same protease, gamma-secretase, is assumed to generate the C termini of both A beta 40 and A beta 42. Herein, we analyze the effect of the compound MDL 28170, previously suggested to inhibit gamma-secretase, on beta-amyloid precursor protein processing. By immunoprecipitating conditioned medium of different cell lines with various A beta 40- and A beta 42-specific antibodies, we demonstrate a much stronger inhibition of the gamma-secretase cleavage at residue 40 than of that at residue 42. These data suggest that different proteases generate the A beta 40 and A beta 42 C termini. Further, they raise the possibility of identifying compounds that do not interfere with general beta-amyloid precursor protein metabolism, including A beta 40 production, but specifically block the generation of the pathogenic A beta 42 peptide.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidases/metabolismo , Fragmentos de Peptídeos/biossíntese , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Animais , Anticorpos Monoclonais , Ácido Aspártico Endopeptidases , Células CHO , Linhagem Celular , Cricetinae , Feminino , Humanos , Camundongos , Camundongos Endogâmicos A , Neuroblastoma , Oligopeptídeos/química , Oligopeptídeos/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Apolipoprotein E (ApoE) is the major genetic risk factor for Alzheimer's disease (AD). The ApoE4 allele is associated with earlier disease onset and greater cerebral deposition of the amyloid beta peptide (Abeta), the major constituent of senile (amyloid) plaques. The molecular mechanism underlying these effects of ApoE4 remains unclear; ApoE alleles could have different influences on Abeta production, extracellular aggregation, or clearance. Because the missense mutations on chromosomes 14 and 21 that cause familial forms of AD appear to lead to increased secretion of Abeta, it is important to determine whether ApoE4 has a similar effect. Here, we have examined the effects of all three ApoE alleles on the processing of betaAPP and the secretion of Abeta in intact cells. We established neural (HS683 human glioma) and non-neural (Chinese hamster ovary) cell culture systems that constitutively secrete both ApoE and Abeta at concentrations like those in human cerebrospinal fluid. betaAPP metabolites, generated in the presence of each ApoE allele, were analysed and quantified by two methods: immunoprecipitation and phosphorimaging, and ELISA. We detected no consistent allele-specific effects of ApoE on betaAPP processing in either cell type. Our data suggest that the higher amyloid burden found in AD subjects expressing ApoE4 is not due to increased amyloidogenic processing of betaAPP, in contrast to findings in AD linked to chromosome 14 or 21. These co-expressing cell lines will be useful in the further search for the effects of ApoE on Abeta aggregation or clearance under physiologically relevant conditions.
Assuntos
Precursor de Proteína beta-Amiloide/análise , Apolipoproteínas E/análise , Química Encefálica , Técnicas de Cultura de Células , Doença de Alzheimer/parasitologia , Animais , Sequência de Bases , Western Blotting , Encéfalo/patologia , Células Cultivadas , Cromossomos Humanos Par 14 , Clonagem Molecular , Cricetinae , Ensaio de Imunoadsorção Enzimática , Humanos , Dados de Sequência Molecular , Mutação Puntual , Testes de PrecipitinaRESUMO
The c4 repressors of bacteriophages P1 and P7 are antisense RNAs that inhibit antirepressor synthesis. This antisense inhibition is unusual in that the c4 repressor and the repressed genes orfx and ant are cotranscribed in that order from the same promoter, and c4 RNA is processed from a precursor RNA. Here, we show that c4 RNA directly represses translation of orfx, a small open reading frame, to which ant is translationally coupled. This translational repression blocks ant transcription via a rho-dependent terminator. Thus, c4 RNA controls expression of the ant gene by a novel indirect mechanism combining translational repression, translational coupling, and rho-dependent termination.