RESUMO
A major characteristic feature of Alzheimer's disease is the formation of compact, extracellular deposits of beta-amyloid (senile plaques). These deposits are surrounded by reactive astrocytes, microglia and dystrophic neurites. Mutations in three genes have been implicated in early-onset familial Alzheimer's disease. However, inflammatory changes and astrogliosis are also believed to play a role in Alzheimer's pathology. What is unclear is the extent to which these factors initiate or contribute to the disease progression. Previous rat studies demonstrated that heterotopic transplantation of foetal cortical tissue onto the midbrain of neonatal hosts resulted in sustained glial reactivity for many months. Similar changes were not seen in cortex-to-cortex grafts. Using this model of chronic cortical gliosis, we have now measured reactive changes in the levels of the key Alzheimer's disease proteins, namely the amyloid precursor protein, apolipoprotein E and presenilin-1. These changes were visualised immunohistochemically and were quantified by western blot analysis. We report here that chronic cortical gliosis in the rat results in a sustained increase in the levels of apolipoprotein E and total amyloid precursor protein. Reactive astrocytes in heterotopic cortical grafts were immunopositive for both of these proteins. Using a panel of amyloid precursor protein antibodies we demonstrate that chronic reactive gliosis is associated with alternative cleavage of the peptide. No significant changes in apolipoprotein E or amyloid precursor protein expression were seen in non-gliotic cortex-to-cortex transplants. Compared to host cortex, the levels of both N-terminal and C-terminal fragments of presenilin-1 were significantly lower in gliotic heterotopic grafts.The changes described here largely mirror those seen in the cerebral cortex of humans with Alzheimer's disease and are consistent with the proposal that astrogliosis may be an important factor in the pathogenesis of this disease.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Apolipoproteínas E/metabolismo , Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Gliose/metabolismo , Proteínas de Membrana/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Animais , Animais Recém-Nascidos , Astrócitos/patologia , Western Blotting , Transplante de Tecido Encefálico/métodos , Córtex Cerebral/fisiopatologia , Córtex Cerebral/transplante , Doença Crônica , Modelos Animais de Doenças , Feto , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/patologia , Gliose/fisiopatologia , Rejeição de Enxerto/metabolismo , Rejeição de Enxerto/patologia , Rejeição de Enxerto/fisiopatologia , Imuno-Histoquímica , Mesencéfalo/metabolismo , Mesencéfalo/fisiopatologia , Mesencéfalo/cirurgia , Neurônios/metabolismo , Neurônios/patologia , Fragmentos de Peptídeos/metabolismo , Presenilina-1 , RatosRESUMO
The beta-amyloid protein (Abeta) is derived by proteolytic processing of the amyloid protein precursor (APP). Cleavage of APP by beta-secretase generates a C-terminal fragment (APP-CTFbeta), which is subsequently cleaved by gamma-secretase to produce Abeta. The aim of this study was to examine the cleavage of APP-CTFbeta by gamma-secretase in primary cortical neurons from transgenic mice engineered to express the human APP-CTFbeta sequence. Neurons were prepared from transgenic mouse cortex and proteins labelled by incubation with [35S]methionine and [35S]cysteine. Labelled APP-CTFbeta and Abeta were then immunoprecipitated with a monoclonal antibody (WO2) specific for the transgene sequences. Approximately 30% of the human APP-CTFbeta (hAPP-CTFbeta) was converted to human Abeta (hAbeta), which was rapidly secreted. The remaining 70% of the hAPP-CTFbeta was degraded by an alternative pathway. The cleavage of hAPP-CTFbeta to produce hAbeta was inhibited by specific gamma-secretase inhibitors. However, treatment with proteasome inhibitors caused an increase in both hAPP-CTFbeta and hAbeta levels, suggesting that the alternative pathway was proteasome-dependent. A preparation of recombinant 20S proteasome was found to cleave a recombinant cytoplasmic domain fragment of APP (APPcyt) directly. The study suggests that in primary cortical neurons, APP-CTFbeta is degraded by two distinct pathways, one involving gamma-secretase, which produces Abeta, and a second major pathway involving direct cleavage of APP-CTFbeta within the cytoplasmic domain by the proteasome. These results raise the possibility that defective proteasome function could lead to an increase in Abeta production in the AD brain.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Cisteína Endopeptidases/metabolismo , Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Western Blotting , Células Cultivadas , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Complexos Multienzimáticos/antagonistas & inibidores , Neurônios/metabolismo , Inibidores de Proteases/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas Recombinantes/metabolismo , Fatores de TempoRESUMO
Intramembranous cleavage of the beta-amyloid precursor protein by gamma-secretase is the final processing event generating amyloid-beta peptides, which are thought to be causative agents for Alzheimer's disease. Missense mutations in the presenilin genes co-segregate with early-onset Alzheimer's disease, and, recently, a close biochemical linkage between presenilins and the identity of gamma-secretase has been established. Here we describe for the first time that certain potent gamma-secretase inhibitors are able to interfere with the endoproteolytic processing of presenilin 1 (PS1). In addition, we identified a novel gamma-secretase inhibitor, [1S-benzyl-4R-[1-(5-cyclohexyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-3(R,S)-ylcarbamoyl)-S-ethylcarbamoyl]-2R-hydroxy-5-phenyl-pentyl]-carbamic acid tert-butyl ester (CBAP), which not only physically interacts with PS1, but upon chronic treatment produces a "pharmacological knock-down" of PS1 fragments. This indicates that the observed accumulation of full-length PS1 is caused by a direct inhibition of its endoproteolysis. The subsequent use of CBAP as a biological tool to increase full-length PS1 levels in the absence of exogenous PS1 expression has provided evidence that wild-type PS1 endoproteolysis is not required either for PS1/gamma-secretase complex assembly or trafficking. Furthermore, in cell-based systems CBAP does not completely recapitulate PS1 loss-of-function phenotypes. Even though the beta-amyloid precursor protein cleavage and the S3 cleavage of the Notch receptor are inhibited by CBAP, an impairment of Trk receptor maturation was not observed.
Assuntos
Endopeptidases/química , Inibidores Enzimáticos/farmacologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Membrana Celular/metabolismo , Células Cultivadas , Centrifugação , DNA Complementar/metabolismo , Dimerização , Relação Dose-Resposta a Droga , Endopeptidases/metabolismo , Humanos , Concentração Inibidora 50 , Cinética , Proteínas de Membrana/metabolismo , Mutação de Sentido Incorreto , Fenótipo , Presenilina-1 , Ligação Proteica , Ratos , Relação Estrutura-Atividade , Frações Subcelulares , Fatores de Tempo , Transfecção , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
Mutations in the presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease by altering gamma-secretase cleavage of the amyloid precursor protein, the last step in the generation of Abeta peptide. Ablation of presenilin (PS) genes, or mutation of two critical aspartates, abolishes gamma-secretase cleavage, suggesting that PS may be the gamma-secretases. Independently, inhibition experiments indicate that gamma-secretase is an aspartyl protease. To characterize the putative gamma-secretase activity associated with presenilins, lysates from human neuroblastoma SH-SY5Y and human brain homogenates were incubated with biotin derivatives of pepstatin, followed by immunoprecipitation of PS and associated proteins, and biotin detection by Western blotting. Precipitation with PS1 antibodies, directed to either N-terminal or loop regions, yielded the same 43 kDa band, of apparent molecular mass consistent with that of full-length PS1, although it may represent an aspartyl protease complexed with PS1. Incubation of cell lysates with pepstatin-biotin, followed by streptavidin precipitation and PS1 Western blotting, revealed PS1 fragments and full-length protein, indicating that pepstatin-biotin bound to both cleaved and uncleaved PS1. Binding could be competed by gamma-secretase inhibitor L-685,458 and could not be achieved with a PS1 mutant lacking the two transmembrane aspartates. Pepstatin-biotin was also shown to bind to PS2. PS1 was specifically absorbed to pepstatin-agarose, with an optimal pH of 6. Binding of pepstatin-biotin to PS1 from lymphocytes of a heterozygous carrier of pathologic exon 9 deletion was markedly decreased as compared to control lymphocytes, suggesting that this PS1 mutation altered the pepstatin binding site.
Assuntos
Doença de Alzheimer/enzimologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Pepstatinas/metabolismo , Inibidores de Proteases/metabolismo , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide , Animais , Ligação Competitiva , Biotina/metabolismo , Células COS , Células Cultivadas , Ácidos Cólicos , Detergentes , Éxons/genética , Humanos , Hidrólise , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Peso Molecular , Testes de Precipitina , Presenilina-1 , Presenilina-2 , Ligação Proteica/genética , Deleção de Sequência , Células Tumorais CultivadasRESUMO
Mutations in the presenilin 1 and 2 (PS1 and PS2) genes cause most cases of early onset Alzheimer's disease. The genes encode two homologous multipass membrane proteins. Since the endogenous expression of PS2 has been poorly analyzed to date, we studied PS2 expression and localization in cultured human neuroblastoma cells and mouse neuronal cells. PS2 was mainly detected as a full-length protein of about 52 kDa in these cells and in brain, in contrast to PS1 that is mainly detected as endoproteolytic N-terminal and C-terminal fragments. Using immunofluorescence we found that like PS1, PS2 colocalized with markers of the endoplasmic reticulum-Golgi intermediate compartment, ERGIC-53 and beta-COP. Double labeling for PS1 and PS2 indicated that both proteins are colocalized in neuroblastoma SH-SY5Y cells. To study PS2 expression during differentiation, mouse embryonic carcinoma P19 cells were treated with retinoic acid. We found minimal PS2 expression in undifferentiated cells, an increase from day 2, and a maximum at day 8 after treatment. PS1 expression remained constant during this period. The differential expression of PS1 and PS2 within the P19 cells following retinoic acid treatment indicates different utilization or temporal requirements for these proteins during neuronal differentiation.
Assuntos
Proteínas de Membrana/biossíntese , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neurônios/metabolismo , Neurônios/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Diferenciação Celular , Imunofluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Camundongos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Presenilina-2 , Células Tumorais CultivadasRESUMO
Presenilin (PS) mutations are associated with early-onset Alzheimer's disease and PS proteins are involved with gamma-secretase cleavage of the amyloid precursor protein, APP. We have shown previously that alpha-, beta- and gamma-secretase cleavages of APP are conserved in Pichia pastoris. Here, we report co-expression of APP and PSI in P. pastoris and show by immunoelectron microscopy colocalization of these two proteins in expanded endoplasmic reticulum. Western blot analysis indicates a drastic reduction of both alpha- and beta-secretase products. A relative increase in beta-secretase product derived from immature APP is also observed, pointing to a beta-secretase activity of P. pastoris associated with the early secretory pathway.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Pichia/enzimologia , Pichia/genética , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/biossíntese , Precursor de Proteína beta-Amiloide/genética , Ácido Aspártico Endopeptidases/metabolismo , Western Blotting , Células Clonais/citologia , Células Clonais/enzimologia , Células Clonais/metabolismo , Endopeptidases , Retículo Endoplasmático/metabolismo , Glicosilação , Imuno-Histoquímica , Microscopia Eletrônica , Modelos Biológicos , Pichia/citologia , Pichia/ultraestrutura , Presenilina-1 , Processamento de Proteína Pós-Traducional , TransfecçãoRESUMO
The genes encoding presenilin-1 (PS1) and presenilin-2 (PS2) were identified as the genes that harbour mutations that cause more than 60% of early onset familial Alzheimer's disease cases (FAD) (1-3). So far, more than 40 missense mutations have been described for presenilin-1 and two have been found in the gene coding for presenilin-2 (reviewed in refs. 4 and 5). Carriers of mutated presenilin genes develop in their brain neuropathological changes characteristic of Alzheimer's disease including the deposition of amyloid Aß peptide. The latter is released from its cognate amyloid precursor protein (APP) by a two-step proteolytic conversion: first, proteolysis of APP by ß-secretase, which releases the N-terminus of Aß, and second, conversion of the remaining fragment by γ-secretase, which cleaves within the predicted transmembrane region of APP. This releases the C-terminus of Aß, which may end either at position 40 or, to a lesser extent, at position 42 (reviewed in ref. 6). The latter species, Aß(1-42), is more prone to aggregation and deposition than Aß(1-40) and is produced at higher levels in the brains and primary fibroblasts of FAD patients carrying PS missense mutations (7). The same result was obtained when cultured cells transfected with mutated PS1 orPS2, or transgenic mice harboring missense PS1 were analyzed for the production of Aß(1-42): in every case increased amounts of the longer Aß(1-42) species were observed (8-10). The mechanisms by which mutations in the PS genes affect the proteolytic processing of APP by γ-secretase have not been resolved in detail. There are two possibilities by which the normal processing of APP may be disturbed: either mutations in the presenilins affect APP metabolism in an indirect way by modulation of proteases or interaction with proteins involved in APP intracellular routing, or presenilins may modulate APP processing directly through physical interactions with APP. Such a direct interaction between presenilins and APP was first demonstrated by us for PS2 (11). Later on, formation of stable complexes with APP was reported not only for PS2 but also for PS1 (12,13,13a).
RESUMO
Alzheimer's disease is characterized by neurodegeneration and deposition of betaA4, a peptide that is proteolytically released from the amyloid precursor protein (APP). Missense mutations in the genes coding for APP and for the polytopic membrane proteins presenilin (PS) 1 and PS2 have been linked to familial forms of early-onset Alzheimer's disease. Overexpression of presenilins, especially that of PS2, induces increased susceptibility for apoptosis that is even more pronounced in cells expressing presenilin mutants. Additionally, presenilins themselves are targets for activated caspases in apoptotic cells. When we analyzed APP in COS-7 cells overexpressing PS2, we observed proteolytic processing close to the APP carboxyl terminus. Proteolytic conversion was increased in the presence of PS2-I, which encodes one of the known PS2 pathogenic mutations. The same proteolytic processing occurred in cells treated with chemical inducers of apoptosis, suggesting a participation of activated caspases in the carboxyl-terminal truncation of APP. This was confirmed by showing that specific caspase inhibitors blocked the apoptotic conversion of APP. Sequence analysis of the APP cytosolic domain revealed a consensus motif for group III caspases ((IVL)ExD). Mutation of the corresponding Asp664 residue abolished cleavage, thereby identifying APP as a target molecule for caspase-like proteases in the pathways of programmed cellular death.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Caspases/metabolismo , Citoplasma/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Animais , Apoptose/efeitos dos fármacos , Células COS , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática , Humanos , Hidrólise , Células Jurkat , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismoRESUMO
betaA4 (Abeta) amyloid peptide, a major component of Alzheimer's disease (AD) plaques, is a proteolytic product of the amyloid precursor protein (APP). Endoproteases, termed beta- and gamma-secretase, release respectively the N- and C-termini of the peptide. APP default secretion involves cleavage within the betaA4 domain by alpha-secretase. To study the conservation of APP processing in lower eukaryotes, the yeast Pichia pastoris was transfected with human APP695 cDNA. In addition to the full-length integral transmembrane protein found in the cell lysate, soluble/secreted APP (sAPP) was detected in the culture medium. Most sAPP comprised the N-terminal moiety of betaA4 and corresponds to sAPPalpha, the product of alpha-secretase. The culture medium also contained minor secreted forms detected by a monoclonal antibody specific for sAPPbeta (the ectodomain released by beta-secretase cleavage). Analysis of the cell lysates with specific antibodies also detected membrane-associated C-terminal fragments corresponding to the products of alpha and beta cleavages. Moreover, immunoprecipitation of the culture medium with three antibodies directed at distinct epitopes of the betaA4 domain yielded a 4 kDa product with the same electrophoretic mobility as betaA4 synthetic peptide. These results suggest that the alpha-, beta-, and gamma-secretase cleavages are conserved in yeast and that P. pastoris may offer an alternative to mammalian cells to identify the proteases involved in the generation of AD betaA4 amyloid.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidases/metabolismo , Pichia/genética , Pichia/metabolismo , Processamento de Proteína Pós-Traducional , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/imunologia , Ácido Aspártico Endopeptidases , Western Blotting , Clonagem Molecular , Endopeptidases/imunologia , Humanos , Hidrólise , Neuroblastoma , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Testes de Precipitina , Processamento de Proteína Pós-Traducional/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Células Tumorais CultivadasRESUMO
Different isoforms and derived polypeptides of the Alzheimer's disease amyloid protein precursor (A beta PP) have been expressed in the yeast Pichia pastoris. The expression characteristics of the different A beta PP polypeptides were studied by post-embedding immunogold electron microscopy with various A beta PP antibodies. The site of intracellular expression could be readily identified with specific antibodies. Full length A beta PP was expressed in association with the nuclear membrane and the endoplasmic reticulum. Secretory derivatives of A beta PP were localized in membrane-bound secretory vesicles. A construct encoding two copies of beta A4[1-42] linked head-to-tail (beta A4duplex) accumulated as irregular dense cytoplasmic and intranuclear inclusions which reacted with all beta A4 antibodies tested. A beta A4-C-terminal construct accumulated into membranous structures in the cytoplasm and nucleus and reacted with most antibodies to beta A4 and the cytoplasmic domain of A beta PP. The two shorter constructs containing the beta A4 sequence formed similar intranuclear aggregates to those reported for intranuclear inclusions of polyglutamine peptides from huntingtin (in Huntington's disease) and ataxin protein fragments (in spinocerebellar ataxia). This is of interest because intracellular aggregation of the polyglutamine and beta A4 peptides may affect cells by similar toxic mechanisms. These studies demonstrate clear differences in the expression properties of different A beta PP polypeptides.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Peptídeos/genética , Frações Subcelulares/química , Precursor de Proteína beta-Amiloide/biossíntese , Humanos , Imuno-Histoquímica , Microscopia Imunoeletrônica , Peptídeos/metabolismo , Pichia , Proteínas Recombinantes/biossínteseRESUMO
Alzheimer's disease (AD) is characterized by the deposition of Abeta (betaA4) peptides of 39 to 43 amino acid residues, which are normal cellular metabolic products derived by proteolysis of the amyloid precursor protein (APP). The physiologic function of Abeta/APP in vivo is poorly understood. We analyzed human platelets for Abeta production by immunoprecipitation coupled to immunoblotting. A 4-kd Abeta fragment that comigrates with an Abeta40 synthetic peptide and reacts with several antibodies specific for the N- and C-termini of Abeta is detected. The majority of platelet Abeta appears to end at residue 40, as determined by immunoreactivity with an Abeta40-specific antibody. Furthermore, Abeta is secreted upon platelet stimulation with the physiologic agonists thrombin and collagen, together with secretion of soluble APP (sAPP). A comparison between serum and plasma shows a 1.6-fold increase in Abeta levels and a 2.4-fold increase in sAPP levels in serum. This is consistent with the view that platelets are the primary source of circulating Abeta and APP. The release of platelet Abeta by physiologic stimuli suggests that it may play a role in platelet aggregation and coagulation or in the repair mechanisms associated with injury.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Plaquetas/metabolismo , Ativação Plaquetária , Peptídeos beta-Amiloides/sangue , HumanosRESUMO
The recently identified Alzheimer's disease-associated presenilin 1 and 2 (PS1 and PS2) genes encode two homologous multi membrane-spanning proteins. Rabbit antibodies to the N-terminal domain of PS1 detected PS1 in human neuroblastoma SH-SY5Y wild type and PS1 transfectants (SY5Y-PS1) as well as in mouse P19, in CHO-K1 and CHO-APP770 transfected cells, in rat cerebellar granule and hippocampal neurons, and astrocytes. Immunoblotting detected full-length protein of 50 kDa, and a major presumptive cleavage product of 30 kDa. The immunofluorescence pattern resembled labeling of the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) marker protein ERGIC-53. PS1 distribution showed slight condensation after brefeldin A and more marked condensation after incubation of cells at 16 degrees C, characteristic of the ERGIC compartment. Double labeling showed colocalization of ERGIC-53 with PS1 in the SY5Y-PS1 cells. PS1 labeling of SY5Y-PS1 and P19 cells showed overlap of the cis-Golgi marker p210 and colocalization with p210 after brefeldin A which causes redistribution of p210 to the ERGIC. Expression of PS1 did not change in level or cellular distribution during development of neurons in culture. Double labeling for the amyloid precursor protein (APP) and PS1 on SY5Y-PS1 cells and CHO-APP770 cells showed some overlap under control conditions. These results indicate that PS1 is a resident protein of the ERGIC and could be involved in trafficking of proteins, including APP, between the ER and Golgi compartments.
Assuntos
Retículo Endoplasmático/química , Complexo de Golgi/química , Lectinas de Ligação a Manose , Proteínas de Membrana/análise , Neurônios/química , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/análise , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Transporte Biológico/fisiologia , Biomarcadores , Brefeldina A , Células CHO , Compartimento Celular/fisiologia , Cerebelo/citologia , Cricetinae , Ciclopentanos/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Imunofluorescência , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Hipocampo/citologia , Humanos , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Neuroblastoma , Neurônios/metabolismo , Neurônios/ultraestrutura , Presenilina-1 , Inibidores da Síntese de Proteínas/farmacologia , Coelhos , Ratos , Ratos Sprague-Dawley , Células Tumorais CultivadasRESUMO
Mutations in the presenilin genes are associated with early onset familial Alzheimer's disease and lead to increased accumulation of beta A4 peptide, the proteolytic product of the amyloid precursor protein (APP). To test whether presenilins interfere with APP metabolism, presenilin-2 (PS2) was coexpressed with APP in mammalian cells. Analysis of PS2 immunoprecipitates revealed that a fraction of APP was associated with the PS2 immunocomplexes. This non-covalent association was specific for the APP family of proteins and restricted to immature forms, occurring probably during transit through the endoplasmic reticulum. Additionally, coexpression with PS2 resulted in a decrease of APP secretion, suggesting a direct participation of presenilins in the intracellular sorting, trafficking and processing of APP molecules.
Assuntos
Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Proteínas de Membrana/genética , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Presenilina-2 , Ligação Proteica , TransfecçãoRESUMO
The amyloid protein (A beta or beta A4) is the major constituent of amyloid plaques in the Alzheimer's disease brain. A beta is cleaved from the amyloid precursor protein (APP) by a mechanism which is poorly understood. Cell culture studies suggest that APP may be cleaved by secretases within the late Golgi compartment. Studies performed so far have mainly used exogenous APP and synthetic peptides as substrates. For this study, a Golgi and plasma membrane-enriched fraction was isolated from rat brain and incubated at 37 degrees C at pH 7.2 to study the degradation of endogenous APP. The breakdown of APP was accompanied by the concomitant generation of A beta-containing C-terminal fragments, in a time-dependent fashion. The metal ion chelators EDTA and 1,10-phenanthroline inhibited this degradation. The inhibition by EDTA was reversed by 50 microM Zn2+ but not by other metal ions. The protease activity was not inhibited by cysteine, serine or aspartic protease inhibitors nor was it inhibited by compounds which are inhibitors of known metalloendopeptidases and matrix metalloproteinases (cFP, phosphoramidon and TIMP-2). Our data suggest that a novel Zn(2+)-dependent metalloprotease activity associated with a Golgi and plasma membrane-enriched fraction can degrade endogenous APP to generate A beta containing C-terminal fragments. This protease may generate amyloidogenic fragments of APP which may serve as precursors for A beta.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Metaloendopeptidases/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Animais , Western Blotting , Fracionamento Celular , Membrana Celular/química , Membrana Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Concentração de Íons de Hidrogênio , Metais/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ratos , Zinco/metabolismoRESUMO
The ability of the 72 kDa gelatinase A to cleave the amyloid protein precursor (APP) was investigated. HeLa cells were transfected with an APP695 plasmid. The cells were incubated with gelatinase A, which cleaved the 110 kDa cell-surface APP, releasing a 100 kDa form of the protein. A peptide homologous to the beta-secretase site was cleaved by gelatinase A adjacent to a glutamate residue at position -3 (beta A4 numbering system). A peptide homologous to the alpha-secretase site was not cleaved. The results demonstrate that 72 kDa gelatinase A is not an alpha-secretase, but that it may have a beta-secretase activity.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidases/metabolismo , Proteínas da Matriz Extracelular , Gelatinases/metabolismo , Metaloendopeptidases/metabolismo , Agrecanas , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/genética , Ácido Aspártico Endopeptidases , Sítios de Ligação , Células HeLa , Humanos , Lectinas Tipo C , Metaloproteinase 2 da Matriz , Dados de Sequência Molecular , Proteoglicanas/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
beta A4 amyloid peptide, the main constituent of amyloid plaques and cerebrovascular amyloid deposits associated with Alzheimer's disease, derives from a large precursor protein (APP) by the action of beta- and gamma-secretases, the unidentified endoproteases which release the amino and carboxyl termini of beta A4, respectively. Several gamma-secretase cleavage sites exist which yield the more soluble (1-39/40) forms of beta A4 and the longer forms (1-42/43) which have a greater tendency to aggregate into amyloid plaques. gamma-Secretase activity may therefore be critical in amyloid formation. In this study, a synthetic peptide which encompasses the various gamma-secretase cleavage sites was used as a substrate to probe proteases of various classes and specificities. Elastase, collagenase, and cathepsin D cleaved at the amyloidogenic sites (after Ala42 or after Thr43) to release the carboxyl termini of the aggregating forms. In addition, collagenase and pepsin released the carboxyl terminus of the more soluble forms. Human brain fractions enriched in lysosomes contained a proteolytic activity that cleaved the substrate at the amyloidogenic site(s). This activity was more active at acidic pH and was inhibited by pepstatin, two characteristics of the lysosomal aspartyl proteinase cathepsin D. The same lysosomal fractions were found to contain APP carboxyl-terminal fragments which are potentially amyloidogenic. These were degraded, only in acidic conditions, by an endogenous protease activity inhibited by pepstatin. Thus, a cathepsin D-like activity from human brain is a candidate for APP gamma-secretase(s).
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Catepsina B/metabolismo , Endopeptidases/metabolismo , Doença de Alzheimer/enzimologia , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases , Western Blotting , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Humanos , Hidrólise , Microscopia Eletrônica , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The processing of amyloid precursor protein (APP) and production of beta A4 amyloid are events likely to influence the development and progression of Alzheimer's disease, since beta A4 is the major constituent of amyloid deposited in this disorder. Our previous studies showed that human platelets contain full-length APP (APPFL) and are a suitable substrate to study normal APP processing. In the present study, we show that a 22-kDa beta A4-containing carboxyl-terminal fragment (22-CTF) of APP is present in unstimulated platelets. Both APPFL and 22-CTF are proteolytically degraded when platelets are activated with thrombin, collagen, or calcium ionophore A23187. Complete cleavage of APPFL and 22-CTF require the presence of extracellular calcium. Following stimulation in the presence of calcium, a new CTF of 17 kDa is generated, and the NH2-terminal epitope of beta A4 amyloid is lost. Preincubation of platelets with the cell-permeable cysteine protease inhibitors calpeptin, (2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane ethyl ester (E64d), Na alpha-p-tosyl-L-lysine chloromethyl ketone, or calcium chelator EGTA before platelet stimulation inhibits the degradation of both APPFL and 22-CTF. Divalent metal ions including zinc, copper, and cobalt inhibit the degradation of APPFL and 22-CTF. This study suggests that a calcium-dependent neutral cysteine protease is involved in the proteolytic processing of an amyloidogenic species of APP in human platelets.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Plaquetas/metabolismo , Cisteína Endopeptidases/fisiologia , Calcimicina/farmacologia , Cálcio/farmacologia , Calpaína/farmacologia , Ácido Egtázico/farmacologia , Humanos , Fragmentos de Peptídeos/análise , Inibidores de Proteases/farmacologia , Zinco/farmacologiaRESUMO
gamma-secretase, the endoprotease which releases the C-terminus of beta A4 amyloid peptide, cleaves within the hydrophobic transmembrane domain of the amyloid precursor protein. In order to obtain a substrate for gamma-secretase, a dodecapeptide which spans the cleavage site was synthesized, labelled with 125-iodine and conjugated to an agarose gel. A radiometric solid-phase assay was developed using this immobilized substrate. Peptide products were separated by reverse-phase HPLC and TLC to allow characterization of the cleavage site(s).
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Endopeptidases/análise , Endopeptidases/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Ácido Aspártico Endopeptidases , Sítios de Ligação/genética , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Humanos , Radioisótopos do Iodo , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Especificidade por SubstratoRESUMO
Synthetic peptides meeting certain guidelines have been used as immunogens to generate antibodies with predefined specificity. We have raised and characterized using established methods a monoclonal antibody against a synthetic peptide corresponding to the 18-amino acid carboxyterminal sequence (A194-211) of the platelet-derived growth factor (PDGF) A chain expressed by the U343 human glioma cell line. This antibody was generated in order to carry out structure-function studies on this region of PDGF whose biological significance is not yet clear. Anti-PDGF-A194-211 was found to be a low titre, IgM kappa molecule, with a Kd of 2.8 x 10(-7) M. When antibody reactivity was tested with parent PDGF-AAL (A chain homodimer containing a carboxyterminal extension) significant binding was observed. Surprisingly, 125I-PDGF-AAS, consisting of truncated A chains but lacking the extension was also bound. Moreover, poly-L-lysine, beta-thromboglobulin, PDGF-A194-211, and myoglobin competed dose-dependently with 125I-PDGF-AAL for antibody. 125I-bovine serum albumin was also bound. Examination of the primary sequence of proteins and peptides bound by the antibody revealed only one shared structural motif: a lysyl-lysine moiety. Selected small synthetic peptides containing this and other sequences were used as potential competitors of 125I-PDGF-A194-211 in antibody binding. Lysyl-lysyl-glycyl-glutamic acid [corrected] and lysyl-lysine competed, whereas lysyl-leucine did not. These results suggest that as few as two amino acid residues constitute a functional antigenic determinant and contrast with most previous estimates of the minimum number of residues required. Furthermore, we show that guidelines governing the design of synthetic peptides for their use as antigens to produce monoclonal antibodies of predetermined specificity may be unreliable.