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1.
Lai MT Crouthamel MC DiMuzio J Pietrak BL Donoviel DB Bernstein A Gardell SJ Li YM Hazuda D 《Journal of neurochemistry》2006,96(1):118-125
beta-Amyloid peptides (Abeta40 and Abeta42) are the major constituents of amyloid plaques, which are one of the hallmarks of Alzheimer's disease (AD). The Abeta is derived from sequential cleavages of amyloid precursor protein (APP) by beta- and gamma-secretases. gamma-Secretase consists of at least four proteins where presenilins (PS1 and PS2 or PS) are the catalytic subunit involved in the gamma-site cleavage of APP. Secretion of both Abeta40 and Abeta42 is significantly reduced in PS1 knock-out cells and completely abolished in cells deficient for both PS1 and PS2. Consequently, both the PS proteins play essential roles in the production of the secretory of Abeta from cells. Recent studies in primary neurons, however, suggest that PSs are not required for intracellular Abeta42 accumulation; thus the intracellular Abeta42 appears to be generated in a PS-independent manner. Here we present the first biochemical evidence indicating that Abeta, especially Abeta42, can be generated in the absence of PS based on an in vitrogamma-secretase assay employing membranes prepared from PS-deficient Blastocyst-derived (BD) cells. This PS-independent gamma-secretase (PSIG) activity is sensitive to the changes in pH and displays an optimal activity at pH 6.0. Pepstatin A is a potent inhibitor for this proteolytic activity with IC50 of 1.2 nm and 0.4 nm for Abeta40 and Abeta42 generation, respectively. These results indicate that these PS-independent gamma-site cleavages are mediated by an aspartyl protease. More importantly, the PSIG activity displays a distinct preference in mediating the 42-site cleavage over the 40-site cleavage, thereby generating Abeta42 as the predominant product. 相似文献
2.
Gamma-secretase catalyzes the proteolytic processing of a number of integral membrane proteins, including amyloid precursor protein (APP) and Notch. The native gamma-secretase is a heterogeneous population of large membrane protein complexes containing presenilin 1 (PS1) or presenilin 2 (PS2), aph-1a or aph-1b, nicastrin, and pen-2. Here we report the reconstitution of a gamma-secretase complex in Sf9 cells by co-infection with baculoviruses carrying the PS1, nicastrin, pen-2, and aph-1a genes. The reconstituted enzyme processes C99 and the Notch-like substrate N160 and displays the characteristic features of gamma-secretase in terms of sensitivity to a gamma-secretase inhibitor, upregulation of Abeta42 production by a familial Alzheimer's disease (FAD) mutation in the APP gene, and downregulation of Notch processing by PS1 FAD mutations. However, the ratio of Abeta42:Abeta40 production by the reconstituted gamma-secretase is significantly higher than that of the native enzyme from 293 cells. Unlike in mammalian cells where PS1 FAD mutations cause an increase in Abeta42 production, PS1 FAD missense mutations in the reconstitution system alter the cleavage sites in the C99 substrate without changing the Abeta42:Abeta40 ratio. In addition, PS1DeltaE9 is a loss-of-function mutation in both C99 and N160 processing. Reconstitution of gamma-secretase provides a homogeneous system for studying the individual gamma-secretase complexes and their roles in Abeta production, Notch processing and AD pathogenesis. These studies may provide important insight into the development of a new generation of selective gamma-secretase inhibitors with an improved side effect profile. 相似文献
3.
Fraering PC Ye W Strub JM Dolios G LaVoie MJ Ostaszewski BL van Dorsselaer A Wang R Selkoe DJ Wolfe MS 《Biochemistry》2004,43(30):9774-9789
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
4.
Presenilin-dependent transcriptional control of the Abeta-degrading enzyme neprilysin by intracellular domains of betaAPP and APLP 总被引:9,自引:0,他引:9
Pardossi-Piquard R Petit A Kawarai T Sunyach C Alves da Costa C Vincent B Ring S D'Adamio L Shen J Müller U St George Hyslop P Checler F 《Neuron》2005,46(4):541-554
5.
Czirr E Leuchtenberger S Dorner-Ciossek C Schneider A Jucker M Koo EH Pietrzik CU Baumann K Weggen S 《The Journal of biological chemistry》2007,282(34):24504-24513
Abeta42-lowering nonsteroidal anti-inflammatory drugs (NSAIDs) constitute the founding members of a new class of gamma-secretase modulators that avoid side effects of pan-gamma-secretase inhibitors on NOTCH processing and function, holding promise as potential disease-modifying agents for Alzheimer disease (AD). These modulators are active in cell-free gamma-secretase assays indicating that they directly target the gamma-secretase complex. Additional support for this hypothesis was provided by the observation that certain mutations in presenilin-1 (PS1) associated with early-onset familial AD (FAD) change the cellular drug response to Abeta42-lowering NSAIDs. Of particular interest is the PS1-DeltaExon9 mutation, which provokes a pathogenic increase in the Abeta42/Abeta40 ratio and dramatically reduces the cellular response to the Abeta42-lowering NSAID sulindac sulfide. This FAD PS1 mutant is unusual as a splice-site mutation results in deletion of amino acids Thr(291)-Ser(319) including the endoproteolytic cleavage site of PS1, and an additional amino acid exchange (S290C) at the exon 8/10 splice junction. By genetic dissection of the PS1-DeltaExon9 mutation, we now demonstrate that a synergistic effect of the S290C mutation and the lack of endoproteolytic cleavage is sufficient to elevate the Abeta42/Abeta40 ratio and that the attenuated response to sulindac sulfide results partially from the deficiency in endoproteolysis. Importantly, a wider screen revealed that a diminished response to Abeta42-lowering NSAIDs is common among aggressive FAD PS1 mutations. Surprisingly, these mutations were also partially unresponsive to gamma-secretase inhibitors of different structural classes. This was confirmed in a mouse model with transgenic expression of the PS1-L166P mutation, in which the potent gamma-secretase inhibitor LY-411575 failed to reduce brain levels of soluble Abeta42. In summary, these findings highlight the importance of genetic background in drug discovery efforts aimed at gamma-secretase, suggesting that certain AD mouse models harboring aggressive PS mutations may not be informative in assessing in vivo effects of gamma-secretase modulators and inhibitors. 相似文献
6.
Mutations in presenilin 1 (PS1) lead to dominant inheritance of early onset familial Alzheimer disease (FAD). These mutations are known to alter the gamma-secretase cleavage of the amyloid precursor protein, resulting in increased ratio of Abeta42/Abeta40 and accelerated amyloid plaque pathology in transgenic mouse models. To investigate the factors that drive the Abeta42/Abeta40 ratio and amyloid pathogenesis and to investigate the possible interactions between wild-type and FAD mutant PS1, which are co-expressed in transgenic animals, we expressed the PS1 M146V knock-in allele either on wild-type PS1 (PS1M146V/+) or PS1 null (PS1M146V/-) background and crossed these alleles with the Tg2576 APP transgenic mice. Introduction of the PS1 M146V mutation on Tg2576 background resulted in earlier onset of plaque pathology. Surprisingly, removing the wild-type PS1 in the presence of the PS1 M146V mutation (PS1M146V/-) greatly exacerbated the amyloid burden; and this was attributed to a reduction of gamma-secretase activity rather than an increase in Abeta42. Our findings establish a protective role of the wild-type PS1 against the FAD mutation-induced amyloid pathology through a partial loss-of-function mechanism. 相似文献
7.
Peptidomimetic probes and molecular modeling suggest that Alzheimer's gamma-secretase is an intramembrane-cleaving aspartyl protease 总被引:9,自引:0,他引:9
Wolfe MS Xia W Moore CL Leatherwood DD Ostaszewski B Rahmati T Donkor IO Selkoe DJ 《Biochemistry》1999,38(15):4720-4727
The amyloid beta-protein (Abeta), implicated in the pathogenesis of Alzheimer's disease (AD), is a proteolytic metabolite generated by the sequential action of beta- and gamma-secretases on the amyloid precursor protein (APP). The two main forms of Abeta are 40- and 42-amino acid C-terminal variants, Abeta40 and Abeta42. We recently described a difluoro ketone peptidomimetic (1) that blocks Abeta production at the gamma-secretase level [Wolfe, M. S., et al. (1998) J. Med. Chem. 41, 6-9]. Although designed to inhibit Abeta42 production, 1 also effectively blocked Abeta40 formation. Various amino acid changes in 1 still resulted in inhibition of Abeta40 and Abeta42 production, suggesting relatively loose sequence specificity by gamma-secretase. The alcohol counterparts of selected difluoro ketones also lowered Abeta levels, indicating that the ketone carbonyl is not essential for activity and suggesting that these compounds inhibit an aspartyl protease. Selected compounds inhibited the aspartyl protease cathepsin D but not the cysteine protease calpain, corroborating previous suggestions that gamma-secretase is an aspartyl protease with some properties similar to those of cathepsin D. Also, since the gamma-secretase cleavage sites on APP are within the transmembrane region, we consider the hypothesis that this region binds to gamma-secretase as an alpha-helix and discuss the implications of this model for the mechanism of certain forms of hereditary AD. 相似文献
8.
Shimojo M Sahara N Mizoroki T Funamoto S Morishima-Kawashima M Kudo T Takeda M Ihara Y Ichinose H Takashima A 《The Journal of biological chemistry》2008,283(24):16488-16496
Presenilin (PS)/gamma-secretase-mediated intramembranous proteolysis of amyloid precursor protein produces amyloid beta (Abeta) peptides in which Abeta species of different lengths are generated through multiple cleavages at the gamma-, zeta-, and epsilon-sites. An increased Abeta42/Abeta40 ratio is a common characteristic of most cases of familial Alzheimer disease (FAD)-linked PS mutations. However, the molecular mechanisms underlying amyloid precursor protein proteolysis leading to increased Abeta42/Abeta40 ratios still remain unclear. Here, we report our findings on the enzymatic analysis of gamma-secretase derived from I213T mutant PS1-expressing PS1/PS2-deficient (PS(-/-)) cells and from the brains of I213T mutant PS1 knock-in mice. Kinetics analyses revealed that the FAD mutation reduced de novo Abeta generation, suggesting that mutation impairs the total catalytic rate of gamma-secretase. Analysis of each Abeta species revealed that the FAD mutation specifically reduced Abeta40 levels more drastically than Abeta42 levels, leading to an increased Abeta42/Abeta40 ratio. By contrast, the FAD mutation increased the generation of longer Abeta species such as Abeta43, Abeta45, and >Abeta46. These results were confirmed by analyses of gamma-secretase derived from I213T knock-in mouse brains, in which the reduction of de novo Abeta generation was mutant allele dose-dependent. Our findings clearly indicate that the mechanism underlying the increased Abeta42/Abeta40 ratio observed in cases of FAD mutations is related to the differential inhibition of gamma-site cleavage reactions, in which the reaction producing Abeta40 is subject to more inhibition than that producing Abeta42. Our results also provide novel insight into how enhancing the generation of longer Abetas may contribute to Alzheimer disease onset. 相似文献
9.
Ikeuchi T Dolios G Kim SH Wang R Sisodia SS 《The Journal of biological chemistry》2003,278(9):7010-7018
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
10.
Page RM Baumann K Tomioka M Pérez-Revuelta BI Fukumori A Jacobsen H Flohr A Luebbers T Ozmen L Steiner H Haass C 《The Journal of biological chemistry》2008,283(2):677-683
Alzheimer disease amyloid beta-peptide (Abeta) is generated via proteolytic processing of the beta-amyloid precursor protein by beta- and gamma-secretase. Gamma-secretase can be blocked by selective inhibitors but can also be modulated by a subset of non-steroidal anti-inflammatory drugs, including sulindac sulfide. These drugs selectively reduce the generation of the aggregation-prone 42-amino acid Abeta(42) and concomitantly increase the levels of the rather benign Abeta(38). Here we show that Abeta(42) and Abeta(38) generation occur independently from each other. The amount of Abeta(42) produced by cells expressing 10 different familial Alzheimer disease (FAD)-associated mutations in presenilin (PS) 1, the catalytic subunit of gamma-secretase, appeared to correlate with the respective age of onset in patients. However, Abeta(38) levels did not show a negative correlation with the age of onset. Modulation of gamma-secretase activity by sulindac sulfide reduced Abeta(42) in the case of wild type PS1 and two FAD-associated PS1 mutations (M146L and A285V). The remaining eight PS1 FAD mutants showed either no reduction of Abeta(42) or only rather subtle effects. Strikingly, even the mutations that showed no effect on Abeta(42) levels allowed a robust increase of Abeta(38) upon treatment with sulindac sulfide. Similar observations were made for fenofibrate, a compound known to increase Abeta(42) and to decrease Abeta(38). For mutants that predominantly produce Abeta(42), the ability of fenofibrate to further increase Abeta(42) levels became diminished, whereas Abeta(38) levels were altered to varying extents for all mutants analyzed. Thus, we conclude that Abeta(38) and Abeta(42) production do not depend on each other. Using an independent non-steroidal anti-inflammatory drug derivative, we obtained similar results for PS1 as well as for PS2. These in vitro results were confirmed by in vivo experiments in transgenic mice expressing the PS2 N141I FAD mutant. Our findings therefore have strong implications on the selection of transgenic mouse models used for screening of the Abeta(42)-lowering capacity of gamma-secretase modulators. Furthermore, human patients with certain PS mutations may not respond to gamma-secretase modulators. 相似文献
11.
Czirr E Cottrell BA Leuchtenberger S Kukar T Ladd TB Esselmann H Paul S Schubenel R Torpey JW Pietrzik CU Golde TE Wiltfang J Baumann K Koo EH Weggen S 《The Journal of biological chemistry》2008,283(25):17049-17054
Proteolytic processing of the amyloid precursor protein by beta- and gamma-secretase generates the amyloid-beta (Abeta) peptides, which are principal drug targets in Alzheimer disease therapeutics. gamma-Secretase has imprecise cleavage specificity and generates the most abundant Abeta40 and Abeta42 species together with longer and shorter peptides such as Abeta38. Several mechanisms could explain the production of multiple Abeta peptides by gamma-secretase, including sequential processing of longer into shorter Abeta peptides. A novel class of gamma-secretase modulators (GSMs) that includes some non-steroidal anti-inflammatory drugs has been shown to selectively lower Abeta42 levels without a change in Abeta40 levels. A signature of GSMs is the concomitant increase in shorter Abeta peptides, such as Abeta38, leading to the suggestion that generation of Abeta42 and Abeta38 peptide species by gamma-secretase is coordinately regulated. However, no evidence for or against such a precursor-product relationship has been provided. We have previously shown that stable overexpression of aggressive presenilin-1 (PS1) mutations associated with early-onset familial Alzheimer disease attenuated the cellular response to GSMs, resulting in greatly diminished Abeta42 reductions as compared with wild type PS1. We have now used this model system to investigate whether Abeta38 production would be similarly affected indicating coupled generation of Abeta42 and Abeta38 peptides. Surprisingly, treatment with the GSM sulindac sulfide increased Abeta38 production to similar levels in four different PS1 mutant cell lines as compared with wild type PS1 cells. This was confirmed with the structurally divergent GSMs ibuprofen and indomethacin. Mass spectrometry analysis and high resolution urea gel electrophoresis further demonstrated that sulindac sulfide did not induce detectable compensatory changes in levels of other Abeta peptide species. These data provide evidence that Abeta42 and Abeta38 species can be independently generated by gamma-secretase and argue against a precursor-product relationship between these peptides. 相似文献
12.
Murphy MP Uljon SN Fraser PE Fauq A Lookingbill HA Findlay KA Smith TE Lewis PA McLendon DC Wang R Golde TE 《The Journal of biological chemistry》2000,275(34):26277-26284
Presenilins (PSs) are polytopic membrane proteins that have been implicated as potential therapeutic targets in Alzheimer's disease because of their role in regulating the gamma-secretase cleavage that generates the amyloid beta protein (Abeta). It is not clear how PSs regulate gamma-secretase cleavage, but there is evidence that PSs could be either essential cofactors in the gamma-secretase cleavage, gamma-secretase themselves, or regulators of intracellular trafficking that indirectly influence gamma-secretase cleavage. Using presenilin 1 (PS1) mutants that inhibit Abeta production in conjunction with transmembrane domain mutants of the amyloid protein precursor that are cleaved by pharmacologically distinct gamma-secretases, we show that PS1 regulates multiple pharmacologically distinct gamma-secretase activities as well as inducible alpha-secretase activity. It is likely that PS1 acts indirectly to regulate these activities (as in a trafficking or chaperone role), because these data indicate that for PS1 to be gamma-secretase it must either have multiple active sites or exist in a variety of catalytically active forms that are altered to an equivalent extent by the mutations we have studied. 相似文献
13.
{gamma}-Secretase Substrate Concentration Modulates the Abeta42/Abeta40 Ratio: IMPLICATIONS FOR ALZHEIMER DISEASE 总被引:1,自引:0,他引:1
Yin YI Bassit B Zhu L Yang X Wang C Li YM 《The Journal of biological chemistry》2007,282(32):23639-23644
Mutation of the amyloid precursor protein (APP), presenilin-1, or presenilin-2 results in the development of early onset autosomal dominant forms of Alzheimer disease (AD). These mutations lead to an increased Abeta42/Abeta40 ratio that correlates with the onset of disease. However, it remains unknown how these mutations affect gamma-secretase, a protease that generates the termini of Abeta40 and Abeta42. Here we have determined the reaction mechanism of gamma-secretase with wild type and three mutated APP substrates. Our findings indicate that despite the overall outcome of an increased Abeta42/Abeta40 ratio, these mutations each display rather distinct reactivity to gamma-secretase. Intriguingly, we found that the ratio of Abeta42/Abeta40 is variable with substrate concentration; increased substrate concentrations result in higher ratios of Abeta42/Abeta40. Moreover, we demonstrated that reduction of gamma-secretase substrate concentration by BACE1 inhibition in cells decreased the Abeta42/Abeta40 ratio. This study indicates that biological factors affecting targets such as BACE1 and APP, which ultimately cause an increased concentration of gamma-secretase substrate, can augment the Abeta42/Abeta40 ratio and may play a causative role in sporadic AD. Therefore, strategies lowering the Abeta42/Abeta40 ratio through partial reduction of gamma-secretase substrate production may introduce a practical therapeutic modality for treatment of AD. 相似文献
14.
Désiré L Bourdin J Loiseau N Peillon H Picard V De Oliveira C Bachelot F Leblond B Taverne T Beausoleil E Lacombe S Drouin D Schweighoffer F 《The Journal of biological chemistry》2005,280(45):37516-37525
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
15.
Regulation of cholesterol and sphingomyelin metabolism by amyloid-beta and presenilin 总被引:6,自引:0,他引:6
Grimm MO Grimm HS Pätzold AJ Zinser EG Halonen R Duering M Tschäpe JA De Strooper B Müller U Shen J Hartmann T 《Nature cell biology》2005,7(11):1118-1123
Amyloid beta peptide (Abeta) has a key role in the pathological process of Alzheimer's disease (AD), but the physiological function of Abeta and of the amyloid precursor protein (APP) is unknown. Recently, it was shown that APP processing is sensitive to cholesterol and other lipids. Hydroxymethylglutaryl-CoA reductase (HMGR) and sphingomyelinases (SMases) are the main enzymes that regulate cholesterol biosynthesis and sphingomyelin (SM) levels, respectively. We show that control of cholesterol and SM metabolism involves APP processing. Abeta42 directly activates neutral SMase and downregulates SM levels, whereas Abeta40 reduces cholesterol de novo synthesis by inhibition of HMGR activity. This process strictly depends on gamma-secretase activity. In line with altered Abeta40/42 generation, pathological presenilin mutations result in increased cholesterol and decreased SM levels. Our results demonstrate a biological function for APP processing and also a functional basis for the link that has been observed between lipids and Alzheimer's disease (AD). 相似文献
16.
The final proteolytic step to generate the amyloid beta-protein (Abeta) of Alzheimer's disease (AD) from beta-amyloid precursor protein (APP) is achieved by presenilin (PS)-dependent gamma-secretase cleavage. AD-causing mutations in PS1 and PS2 result in a selective and significant increase in production of the more amyloidogenic Abeta42 peptide. PS1 and PS2 undergo endoproteolysis by an unknown enzyme termed presenilinase to generate the functional complex of N- and C-terminal fragments (NTF/CTF). To investigate the endoproteolytic activity that generates active PS, we used a mammalian cell-free system that allows de novo human PS NTF and CTF generation. PS NTF and CTF generation in vitro was observed in endoplasmic reticulum (ER)-enriched fractions of membrane vesicles and to a lesser extent in Golgi/trans-Golgi-network (TGN)-enriched fractions. AD-causing mutations in PS1 and PS2 did not alter de novo generation of PS fragments. Removal of peripheral membrane-associated and cytosolic proteins did not prevent de novo generation of fragments, indicating that presenilinase activity corresponds to an integral membrane protein. Among several general inhibitors of different protease classes that blocked the presenilinase activity, pepstatin A was the most potent inhibitor. Screening available transition state analogue gamma-secretase inhibitors led to the identification of two compounds that were able to prevent the de novo generation of PS fragments, with an expected inhibition of Abeta generation. Our studies provide a biochemical approach to characterize and identify this elusive presenilinase. 相似文献
17.
Maltese WA Wilson S Tan Y Suomensaari S Sinha S Barbour R McConlogue L 《The Journal of biological chemistry》2001,276(23):20267-20279
gamma-Secretase is a membrane-associated endoprotease that catalyzes the final step in the processing of Alzheimer's beta-amyloid precursor protein (APP), resulting in the release of amyloid beta-peptide (Abeta). The molecular identity of gamma-secretase remains in question, although recent studies have implicated the presenilins, which are membrane-spanning proteins localized predominantly in the endoplasmic reticulum (ER). Based on these observations, we have tested the hypothesis that gamma-secretase cleavage of the membrane-anchored C-terminal stump of APP (i.e. C99) occurs in the ER compartment. When recombinant C99 was expressed in 293 cells, it was localized mainly in the Golgi apparatus and gave rise to abundant amounts of Abeta. Co-expression of C99 with mutant forms of presenilin-1 (PS1) found in familial Alzheimer's disease resulted in a characteristic elevation of the Abeta(42)/Abeta(40) ratio, indicating that the N-terminal exodomain of APP is not required for mutant PS1 to influence the site of gamma-secretase cleavage. Biogenesis of both Abeta(40) and Abeta(42) was almost completely eliminated when C99 was prevented from leaving the ER by addition of a di-lysine retention motif (KKQN) or by co-expression with a dominant-negative mutant of the Rab1B GTPase. These findings indicate that the ER is not a major intracellular site for gamma-secretase cleavage of C99. Thus, by inference, PS1 localized in this compartment does not appear to be active as gamma-secretase. The results suggest that presenilins may acquire the characteristics of gamma-secretase after leaving the ER, possibly by assembling with other proteins in peripheral membranes. 相似文献
18.
Aggregates of beta-amyloid peptide (Abeta) are the major component of the amyloid core of the senile plaques observed in Alzheimer's disease (AD). Abeta results from the amyloidogenic processing of its precursor, the amyloid precursor protein (APP), by beta- and gamma-secretase activities. If beta-secretase has recently been identified and termed BACE, the identity of gamma-secretase is still obscure. Studies with knock-out mice showed that presenilin 1 (PS1), of which mutations are known to be the first cause of inherited AD, is mandatory for the gamma-secretase activity. However, the proteolytic activity of PS1 remains a matter of debate. Here we used transfected Sf9 insect cells, a cellular model lacking endogenous beta- and/or gamma-secretase activities, to characterize the role of BACE and PS1 in the amyloidogenic processing of human APP. We show that, in Sf9 cells, BACE performs the expected beta-secretase cleavage of APP, generating C99. We also show that C99, which is a substrate of gamma-secretase, tightly binds to the human PS1. Despite this interaction, Sf9 cells still do not produce Abeta. This strongly argues against a direct proteolytic activity of PS1 in APP processing, and points toward an implication of PS1 in trafficking/presenting its substrate to the gamma-secretase. 相似文献
19.
The cleavage of the transmembrane amyloid precursor protein (APP) by beta-secretase leaves the C-terminal fragment of APP, C99, anchored in the plasma membrane. C99 is subsequently processed by gamma-secretase, an unusual aspartyl protease activity largely dependent on presenilin (PS), generating the amyloid beta-peptide (Abeta) that accumulates in the brain of patients with Alzheimer's disease. It has been suggested that PS proteins are the catalytic core of this proteolytic activity, but a number of other proteins mandatory for gamma-secretase cleavage have also been discovered. The exact role of PS in the gamma-secretase activity remains a matter of debate, because cells devoid of PS still produce some forms of Abeta. Here, we used insect cells expressing C99 to demonstrate that the expression of presenilin 1 (PS1), which binds C99, not only increases the production of Abeta by these cells but also increases the intracellular levels of C99 to the same extent. Using pulse-chase experiments, we established that this results from an increased half-life of C99 in cells expressing PS1. In Chinese hamster ovary cells producing C99 from full-length human APP, similar results were observed. Finally, we show that a functional inhibitor of gamma-secretase does not alter the ability of PS1 to increase the intracellular levels of C99. This finding suggests that the binding of PS1 to C99 does not necessarily lead to its immediate cleavage by gamma-secretase, which could be a spatio-temporally regulated or an induced event, and provides biochemical evidence for the existence of a substrate-docking site on PS1. 相似文献
20.
Presenilin 2 familial Alzheimer's disease mutations result in partial loss of function and dramatic changes in Abeta 42/40 ratios 总被引:1,自引:0,他引:1
Gene knockout studies in mice suggest that presenilin 1 (PS1) is the major gamma-secretase and that it contributes disproportionately to amyloid beta (Abeta) peptide generation from beta-amyloid precursor protein (APP), whereas PS2 plays a more minor role. Based on this and other observations we hypothesized that familial Alzheimer's disease (FAD) mutations in PS2 would have a dramatic effect on function in order to have an observable effect on Abeta levels in the presence of normal PS1 alleles. Only four of the eight reported FAD mutations in PS2 have altered function in vitro suggesting that the other variants represent rare polymorphisms rather than disease-causing mutations. In support of our hypothesis, the four verified PS2 FAD mutations cause substantial changes in the Abeta 42/40 ratio, comparable with PS1 mutations that cause very-early-onset FAD. Most of the PS2 mutations also cause a significant decrease in Abeta 40, APP C-terminal fragment (CTF)gamma and Notch intracellular domain (NICD) production suggesting that they are partial loss of function mutations. PS2 M239V, its PS1 homolog M233V, and other FAD mutations within transmembrane (TM) 5 of PS1 differentially affect CTFgamma and NICD production suggesting that TM5 of PS are important for gamma-secretase cleavage of APP but not Notch. 相似文献