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1.
Early pathogenic events in Alzheimer's disease (AD) involve increased production and/or reduced clearance of beta-amyloid (Abeta), especially the 42 amino acid fragment Abeta1-42. The Abeta1-42 peptide is generated through cleavage of the amyloid precursor protein by beta- and gamma-secretase and is catabolised by a variety of proteolytic enzymes such as insulin-degrading enzyme and neprilysin. Here, we describe a method that employs immunoprecipitation combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to determine the pattern of C-terminally truncated Abeta peptides in cerebrospinal fluid (CSF). Using antibodies coupled to magnetic beads, we have detected 18 C-terminally and 2 N-terminally truncated Abeta peptides in CSF. By determining the identity and profile of the truncated Abeta peptides, more insight may be gained about differences in the metabolism and structural properties of Abeta in AD. Finally, the Abeta fragment signatures may prove useful as a diagnostic test for AD.  相似文献   

2.
The presence of senile plaques composed of amyloid-beta (Abeta) polypeptides within brain tissue is normally used as a definitive postmortem diagnosis for Alzheimer's Disease (AD). Therefore, these polypeptides have been investigated as potential biomarkers of the disease state. However, at present, there is a lack of a robust assay for the detection of such polypeptides derived from in vivo sources. Such an assay is essential for analysis of biological samples from model AD systems. To overcome this problem we have developed a new single-step assay utilizing two dimensional-chromatography in conjunction with mass spectrometry. The method consists of on-line size-exclusion chromatography (SEC) to provide initial separation of analytes from the sample (based on their molecular weight) coupled with sample preconcentration prior to analysis by microbore high-performance liquid chromatography-mass spectrometry (HPLC-MS). This provides an extremely versatile and powerful assay which can separate specific analytes from cell lysate in a single step without further sample handling. The use of mass spectrometry as the detection system yields much more structural information than can be obtained from traditional ELISA and sandwich ELISA antibody assays. Furthermore, the on-line sample cleanup protocol minimizes sample handling and facilitates assay automation. Utilizing this new assay we have been able to detect Abeta 1-40 and Abeta 1-42 at cellular concentration levels directly from cell lysates. Moreover, we have detected multiple peptide responses within the same analysis, some of which have been tentatively identified as other ragged C-termini Abeta polypeptides derived from Abeta 1-42, based on their molecular weight, as well as oxidized Abeta polypeptides.  相似文献   

3.
Presenilin 1 (PS1) plays an essential role in intramembranous "gamma-secretase" processing of several type I membrane proteins, including the beta-amyloid precursor proteins (APP) and Notch1. In this report, we examine the activity of two familial Alzheimer's disease-linked PS1 variants on the production of secreted Abeta peptides and the effects of L-685,458, a potent gamma-secretase inhibitor, on inhibition of Abeta peptides from cells expressing these PS1 variants. We now report that PS1 variants enhance the production and secretion of both Abeta1-42 and Abeta1-40 peptides. More surprisingly, whereas the IC(50) for inhibition of Abeta1-40 peptide production from cells expressing wild-type PS1 is approximately 1.5 microm, cells expressing the PS1deltaE9 mutant PS1 exhibit an IC(50) of approximately 4 microm. Immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry reveal that the levels of Abeta1-43 peptides are elevated in medium of PS1deltaE9 cells treated with higher concentrations of inhibitor. The differential effects of wild-type and mutant PS1 on gamma-secretase production of Abeta peptides and the disparity in sensitivity of these peptides to a potent gamma-secretase suggest that PS may be necessary, but not sufficient, to catalyze hydrolysis at the scissile bonds that generate the termini of Abeta1-40 and Abeta1-42 peptides.  相似文献   

4.
On the nucleation of amyloid beta-protein monomer folding   总被引:1,自引:0,他引:1  
Neurotoxic assemblies of the amyloid beta-protein (Abeta) have been linked strongly to the pathogenesis of Alzheimer's disease (AD). Here, we sought to monitor the earliest step in Abeta assembly, the creation of a folding nucleus, from which oligomeric and fibrillar assemblies emanate. To do so, limited proteolysis/mass spectrometry was used to identify protease-resistant segments within monomeric Abeta(1-40) and Abeta(1-42). The results revealed a 10-residue, protease-resistant segment, Ala21-Ala30, in both peptides. Remarkably, the homologous decapeptide, Abeta(21-30), displayed identical protease resistance, making it amenable to detailed structural study using solution-state NMR. Structure calculations revealed a turn formed by residues Val24-Lys28. Three factors contribute to the stability of the turn, the intrinsic propensities of the Val-Gly-Ser-Asn and Gly-Ser-Asn-Lys sequences to form a beta-turn, long-range Coulombic interactions between Lys28 and either Glu22 or Asp23, and hydrophobic interaction between the isopropyl and butyl side chains of Val24 and Lys28, respectively. We postulate that turn formation within the Val24-Lys28 region of Abeta nucleates the intramolecular folding of Abeta monomer, and from this step, subsequent assembly proceeds. This model provides a mechanistic basis for the pathologic effects of amino acid substitutions at Glu22 and Asp23 that are linked to familial forms of AD or cerebral amyloid angiopathy. Our studies also revealed that common C-terminal peptide segments within Abeta(1-40) and Abeta(1-42) have distinct structures, an observation of relevance for understanding the strong disease association of increased Abeta(1-42) production. Our results suggest that therapeutic approaches targeting the Val24-Lys28 turn or the Abeta(1-42)-specific C-terminal fold may hold promise.  相似文献   

5.
Deposition of beta-amyloid (Abeta) peptides in the brain is an early and invariant feature of all forms of Alzheimer's disease. As with any secreted protein, the extracellular concentration of Abeta is determined not only by its production but also by its catabolism. A major focus of Alzheimer's research has been the elucidation of the mechanisms responsible for the generation of Abeta. Much less, however, is known about the mechanisms responsible for Abeta removal in the brain. In this report, we describe the identification of endothelin-converting enzyme-1 (ECE-1) as a novel Abeta-degrading enzyme. We show that treatment of endogenous ECE-expressing cell lines with the metalloprotease inhibitor phosphoramidon causes a 2-3-fold elevation in extracellular Abeta concentration that appears to be due to inhibition of intracellular Abeta degradation. Furthermore, we show that overexpression of ECE-1 in Chinese hamster ovary cells, which lack endogenous ECE activity, reduces extracellular Abeta concentration by up to 90% and that this effect is completely reversed by treatment of the cells with phosphoramidon. Finally, we show that recombinant soluble ECE-1 is capable of hydrolyzing synthetic Abeta40 and Abeta42 in vitro at multiple sites.  相似文献   

6.
Gamma-secretase is a member of an unusual class of proteases with intramembrane catalytic sites. This enzyme cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin (PS) composed of its N- and C-terminal fragments (PS-NTF/CTF), a mature glycosylated form of nicastrin (NCT), Aph-1, and Pen-2. Recent data from studies in Drosophila, mammalian, and yeast cells suggest that PS, NCT, Aph-1, and Pen-2 are necessary and sufficient to reconstitute gamma-secretase activity. However, many unresolved issues, in particular the possibility of other structural or regulatory components, would be resolved by actually purifying the enzyme. Here, we report a detailed, multistep purification procedure for active gamma-secretase and an initial characterization of the purified protease. Extensive mass spectrometry of the purified proteins strongly suggests that PS-NTF/CTF, mNCT, Aph-1, and Pen-2 are the components of active gamma-secretase. Using the purified gamma-secretase, we describe factors that modulate the production of specific Abeta species: (1) phosphatidylcholine and sphingomyelin dramatically improve activity without changing cleavage specificity within an APP substrate; (2) increasing CHAPSO concentrations from 0.1 to 0.25% yields a approximately 100% increase in Abeta42 production; (3) exposure of an APP-based recombinant substrate to 0.5% SDS modulates cleavage specificity from a disease-mimicking pattern (high Abeta42/43) to a physiological pattern (high Abeta40); and (4) sulindac sulfide directly and preferentially decreases Abeta42 cleavage within the purified complex. Taken together, our results define a procedure for purifying active gamma-secretase and suggest that the lipid-mediated conformation of both enzyme and substrate regulate the production of the potentially neurotoxic Abeta42 and Abeta43 peptides.  相似文献   

7.
beta-Amyloid peptides (Abeta) that form the senile plaques of Alzheimer disease consist mainly of 40- and 42-amino acid (Abeta 40 and Abeta 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of Abeta involves beta-secretase and gamma-secretase activities and is regulated by membrane trafficking of the proteins involved in Abeta production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks Abeta 40 and Abeta 42 production but does not impact sAPPalpha levels and does not inhibit beta-secretase. Rather, EHT 1864 modulates APP processing at the level of gamma-secretase to prevent Abeta 40 and Abeta 42 generation. This effect does not result from a direct inhibition of the gamma-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces Abeta 40 and Abeta 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting Abeta formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease.  相似文献   

8.
Strong support for a primary causative role of the Abeta peptides in the development of Alzheimer's disease (AD) neurodegeneration derives from reports that presenilin familial AD (FAD) mutants alter amyloid precursor protein processing, thus increasing production of neurotoxic Abeta 1-42 (Abeta 42). This effect of FAD mutants is also reflected in an increased ratio of peptides Abeta 42 over Abeta 1-40 (Abeta 40). In the present study, we show that several presenilin 1 FAD mutants failed to increase production of Abeta 42 or the Abeta 42/40 ratio. Our data suggest that the mechanism by which FAD mutations promote neurodegeneration and AD may be independent of their effects on Abeta production.  相似文献   

9.
beta-Amyloid efflux mediated by p-glycoprotein   总被引:2,自引:0,他引:2  
A large body of evidence suggests that an increase in the brain beta-amyloid (Abeta) burden contributes to the etiology of Alzheimer's disease (AD). Much is now known about the intracellular processes regulating the production of Abeta, however, less is known regarding its secretion from cells. We now report that p-glycoprotein (p-gp), an ATP-binding cassette (ABC) transporter, is an Abeta efflux pump. Pharmacological blockade of p-gp rapidly decrease extracellular levels of Abeta secretion. In vitro binding studies showed that addition of synthetic human Abeta1-40 and Abeta1-42 peptides to hamster mdr1-enriched vesicles labeled with the fluorophore MIANS resulted in saturable quenching, suggesting that both peptides interact directly with the transporter. Finally, we were able to directly measure transport of Abeta peptides across the plasma membranes of p-gp enriched vesicles, and showed that this phenomenon was both ATP- and p-gp-dependent. Taken together, our study suggests a novel mechanism of Abeta detachment from cellular membranes, and represents an obvious route towards identification of such a mechanism in the brain.  相似文献   

10.
Alzheimer's disease (AD) is marked by the presence of neurofibrillary tangles and amyloid plaques in the brain of patients. To study plaque formation, we report on further quantitative and qualitative analysis of human and mouse amyloid beta peptides (Abeta) from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP). Using enzyme-linked immunosorbant assays (ELISAs) specific for either human or rodent Abeta, we found that the peptides from both species aggregated to form plaques. The ratios of deposited Abeta1-42/1-40 were in the order of 2-3 for human and 8-9 for mouse peptides, indicating preferential deposition of Abeta42. We also determined the identity and relative levels of other Abeta variants present in protein extracts from soluble and insoluble brain fractions. This was done by combined immunoprecipitation and mass spectrometry (IP/MS). The most prominent peptides truncated either at the carboxyl- or the amino-terminus were Abeta1-38 and Abeta11-42, respectively, and the latter was strongly enriched in the extracts of deposited peptides. Taken together, our data indicate that plaques of APP-London transgenic mice consist of aggregates of multiple human and mouse Abeta variants, and the human variants that we identified were previously detected in brain extracts of AD patients.  相似文献   

11.
12.
Prostate apoptosis response-4 (Par-4) is a leucine zipper protein that promotes neuronal cell death in Alzheimer's disease (AD). Neuronal degeneration in AD may result from extracellular accumulation of amyloid beta peptide (Abeta) 1-42. To examine the effect of Par-4 on Abeta secretion and to reconcile amyloid/apoptosis hypotheses of AD, we generated IMR-32 cell lines that overexpress Par-4 and/or its leucine zipper domain. Overexpression of Par-4 did not significantly affect levels of the endogenously expressed beta amyloid precursor protein but drastically increased the Abeta(1-42)/Abeta(total) ratio in the conditioned media about 6-8 h after trophic factor withdrawal. Time course analysis of caspase activation reveals that Par-4 overexpression exacerbated caspase activation, which is detectable within 2 h after trophic factor withdrawal. Furthermore, inhibition of caspase activity by the broad spectrum caspase inhibitor BD-fmk significantly attenuated the Par-4-induced increase in Abeta 1-42 production. In addition, the effects of Par-4 on secretion of Abeta 1-42 were consistently blocked by co-expression of the leucine zipper domain, indicating that the effect of Par-4 on Abeta secretion may require its interaction with other protein(s). These results suggest that Par-4 increases secretion of Abeta 1-42 largely through a caspase-dependent pathway after apoptotic cascades are initiated.  相似文献   

13.
The deposition of amyloid-beta peptides (Abeta) in senile plaques (SPs) is a central pathological feature of Alzheimer's disease (AD). Since SPs are composed predominantly of Abeta1-42, which is more amyloidogenic in vitro, the enzymes involved in generating Abeta1-42 may be particularly important to the pathogenesis of AD. In contrast to Abeta1-40, which is generated in the trans-Golgi network and other cytoplasmic organelles, intracellular Abeta1-42 is produced in the endoplasmic reticulum/intermediate compartment (ER/IC), where it accumulates in a stable insoluble pool. Since this pool of insoluble Abeta1-42 may play a critical role in AD amyloidogenesis, we sought to determine how the production of intracellular Abeta is regulated. Surprisingly, the production of insoluble intracellular Abeta1-42 was increased by a putative gamma-secretase inhibitor as well as by an inhibitor of the proteasome. We further demonstrate that this increased generation of Abeta1-42 in the ER/IC is due to a reduction in the turnover of Abeta-containing APP C-terminal fragments. We conclude that the proteasome is a novel site for degradation of ER/IC-generated APP fragments. Proteasome inhibitors may augment the availability of APP C-terminal fragments for gamma-secretase cleavage and thereby increase production of Abeta1-42 in the ER/IC. Based on the organelle-specific differences in the generation of Abeta by gamma-secretase, we conclude that intracellular ER/IC-generated Abeta1-42 and secreted Abeta1-40 are produced by different gamma-secretases. Further, the fact that a putative gamma-secretase inhibitor had opposite effects on the production of secreted and intracellular Abeta may have important implications for AD drug design.  相似文献   

14.
Immunoprecipitation (IP) combined with matrix-assisted laser desorption ionization (MALDI) time of flight (Tof) mass spectrometry has been used to develop quantitative assays for amyloid-beta (Abeta) peptides in cerebrospinal fluid (CSF). Inclusion of (15)N labelled standard peptides allows for absolute quantification of multiple Abeta isoforms in individual samples. Characterization of variability associated with all steps of the assay indicated that the IP step is the single largest contributor to overall variability. Optimization of the assay resulted in overall coefficient of variation 相似文献   

15.
Although the pathogenesis of Alzheimer's disease (AD) is not fully understood, growing evidence indicates that the deposition of beta-amyloid (Abeta) and the local reactions of various cell types to this protein play major roles in the development of the disease. Immunization with the Abeta 1-42 peptide has been reported to decrease Abeta deposits in the brains of mutant amyloid precursor protein (APP/V717F) transgenic (tg) mice (Schenk et al. Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature 1999;400:173-177). We have replicated this finding in APPswe/PS1DeltaE9 tg mice, which also develop Abeta deposits in the brain. The immunized animals developed high titers of antibodies against Abeta 1-42 in serum, and Abeta deposits in the brains were significantly reduced. Using surface-enhanced laser desorption/ionization (SELDI) mass spectrometry and ProteinChip((R)) technology, we detected trends toward increased soluble Abeta peptide in the brain and a decrease in assayable Abeta peptide in the serum of immunized compared with control animals. This last finding raises the possibility that anti-Abeta antibodies in the periphery sequester Abeta peptides or target them for degradation and in this way contribute to the enhanced Abeta clearance from the brain in immunized animals.  相似文献   

16.
We studied the processing of amyloid beta-peptides (Abetas) including Abeta(1-40), Abeta(1-42) and pAbeta(3-42) by rat neutral cysteine protease bleomycin hydrolase (BH) according to the methods of SDS-PAGE, HPLC and matrix-assisted laser desorption/inonization time-of-flight mass spectrometry (MALDI-TOF MS). BH significantly processed them by novel features of its diverse activities. It initially cleaved at two sites, His(14)-Gln(15) and Phe(19)-Phe(20) degraded to short intermediates then to amino acids by aminopeptidase and/or carboxypeptidase activities. Also, full-length Abetas were clipped at the carboxyl(C)-terminal region. On the other hand, BH cleaved at only the His(14)-Gln(15) bond in pbetaA(3-42) within a short period of the reaction by endopeptidase activity, and processed the intermediates in order by carboxypeptidase activity. On processing by BH, it found that both fibrillar Abeta(1-40) and Abeta(1-42) were more resistant than non-fibrillar peptides. These results indicate that the processing specificity of BH depends upon the structure and sequence of Abetas.  相似文献   

17.
Insoluble pools of the amyloid-beta peptide (Abeta) in brains of Alzheimer's disease patients exhibit considerable N- and C-terminal heterogeneity. Mounting evidence suggests that both C-terminal extensions and N-terminal truncations help precipitate amyloid plaque formation. Although mechanisms underlying the increased generation of C-terminally extended peptides have been extensively studied, relatively little is known about the cellular mechanisms underlying production of N-terminally truncated Abeta. Thus, we used human NT2N neurons to investigate the production of Abeta11-40/42 from amyloid-beta precursor protein (APP) by beta-site APP-cleaving enzyme (BACE). When comparing undifferentiated human embryonal carcinoma NT2- cells and differentiated NT2N neurons, the secretion of sAPP and Abeta correlated with BACE expression. To study the effects of BACE expression on endogenous APP metabolism in human cells, we overexpressed BACE in undifferentiated NT2- cells and NT2N neurons. Whereas NT2N neurons produced both full-length and truncated Abeta as a result of normal processing of endogenous APP, BACE overexpression increased the secretion of Abeta1-40/42 and Abeta11-40/42 in both NT2- cells and NT2N neurons. Furthermore, BACE overexpression resulted in increased intracellular Abeta1-40/42 and Abeta11-40/42. Therefore, we conclude that Abeta11-40/42 is generated prior to deposition in senile plaques and that N-terminally truncated Abeta peptides may contribute to the downstream effects of amyloid accumulation in Alzheimer's disease.  相似文献   

18.
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the presence in the brain of senile plaques which contain an amyloid core made of beta-amyloid peptide (Abeta). Abeta is produced by the cleavage of the amyloid precursor protein (APP). Since impairment of neuronal calcium signalling has been causally implicated in ageing and AD, we have investigated the influence of an influx of extracellular calcium on the metabolism of human APP in rat cortical neurones. We report that a high cytosolic calcium concentration, induced by neuronal depolarization, inhibits the alpha-secretase cleavage of APP and triggers the accumulation of intraneuronal C-terminal fragments produced by the beta-cleavage of the protein (CTFbeta). Increase in cytosolic calcium concentration specifically induces the production of large amounts of intraneuronal Abeta1-42, which is inhibited by nimodipine, a specific antagonist of l-type calcium channels. Moreover, calcium release from endoplasmic reticulum is not sufficient to induce the production of intraneuronal Abeta, which requires influx of extracellular calcium mediated by the capacitative calcium entry mechanism. Therefore, a sustained high concentration of cytosolic calcium is needed to induce the production of intraneuronal Abeta1-42 from human APP. Our results show that this accumulation of intraneuronal Abeta1-42 induces neuronal death, which is prevented by a functional gamma-secretase inhibitor.  相似文献   

19.
Rangan SK  Liu R  Brune D  Planque S  Paul S  Sierks MR 《Biochemistry》2003,42(48):14328-14334
Deposition of beta-amyloid (Abeta) is considered an important early event in the pathogenesis of Alzheimer's disease (AD). Clearance of Abeta thus represents a potential therapeutic approach. Antibody-mediated clearance of Abeta by vaccination inhibited and cleared Abeta deposition in animal models; however, inflammatory side effects were observed in humans. An alternative potentially noninflammatory approach to facilitate clearance is to proteolytically cleave Abeta. We screened 12 proteolytic recombinant antibody fragments for potential alpha-secretase activity, a naturally occurring enzyme that cleaves between the Lys16 and Leu17 residues of the amyloid precursor protein (APP). We utilized the synthetic alpha-secretase substrate, benzyloxycarbonyl-l-lysine o-nitrophenyl ester (Z-lys-o-Np) as a preliminary screen for alpha-secretase activity. Two antibody light chain fragments that hydrolyzed Z-lys-o-Np were identified. Abeta hydrolysis was studied using mass spectrometry to identify the cleavage patterns of the antibodies. The recombinant antibody light chain antibody fragment, c23.5, showed alpha-secretase-like activity, producing the 1-16 and 17-40 amino acid fragments of Abeta. The second light chain antibody fragment, hk14, demonstrated carboxypeptidase-like activity, cleaving sequentially from the carboxyl terminal of Abeta. These antibody light chains provide a novel route toward engineering efficient therapeutic antibodies capable of cleaving Abeta in vivo.  相似文献   

20.
Amyloid beta-peptide (Abeta)(1-42) oligomers have recently been discussed as intermediate toxic species in Alzheimer's disease (AD) pathology. Here we describe a new and highly stable Abeta(1-42) oligomer species which can easily be prepared in vitro and is present in the brains of patients with AD and Abeta(1-42)-overproducing transgenic mice. Physicochemical characterization reveals a pure, highly water-soluble globular 60-kDa oligomer which we named 'Abeta(1-42) globulomer'. Our data indicate that Abeta(1-42) globulomer is a persistent structural entity formed independently of the fibrillar aggregation pathway. It is a potent antigen in mice and rabbits eliciting generation of Abeta(1-42) globulomer-specific antibodies that do not cross-react with amyloid precursor protein, Abeta(1-40) and Abeta(1-42) monomers and Abeta fibrils. Abeta(1-42) globulomer binds specifically to dendritic processes of neurons but not glia in hippocampal cell cultures and completely blocks long-term potentiation in rat hippocampal slices. Our data suggest that Abeta(1-42) globulomer represents a basic pathogenic structural principle also present to a minor extent in previously described oligomer preparations and that its formation is an early pathological event in AD. Selective neutralization of the Abeta globulomer structure epitope is expected to have a high potential for treatment of AD.  相似文献   

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