共查询到20条相似文献,搜索用时 15 毫秒
1.
S Sudoh G Hua Y Kawamura K Maruyama H Komano K Yanagisawa 《European journal of biochemistry》2000,267(7):2036-2045
Previously, we reported that mutations in presenilin 1 (PS1) increased the intracellular levels of amyloid beta-protein (Abeta)42. However, it is still not known at which cellular site or how PS1 mutations exert their effect of enhancing Abeta42-gamma-secretase cleavage. In this study, to clarify the molecular mechanisms underlying this enhancement of Abeta42-gamma-secretase cleavage, we focused on determining the intracellular site of the cleavage. To address this issue, we used APP-C100 encoding the C-terminal beta-amyloid precursor protein (APP) fragment truncated at the N terminus of Abeta (C100); C100 requires only gamma-secretase cleavage to yield Abeta. Mutated PS1 (M146L)-induced Neuro 2a cells showed enhanced Abeta1-42 generation from transiently expressed C100 as well as from full-length APP, whereas the generation of Abeta1-40 was not increased. The intracellular generation of Abeta1-42 from transiently expressed C100 in both mutated PS1-induced and wild-type Neuro 2a cells was inhibited by brefeldin A. Moreover, the generation of Abeta1-42 and Abeta1-40 from a C100 mutant containing a di-lysine endoplasmic reticulum retention signal was greatly decreased, indicating that the major intracellular site of gamma-secretase cleavage is not the endoplasmic reticulum. The intracellular generation of Abeta1-42/40 from C100 was not influenced by monensin treatment, and the level of Abeta1-42/40 generated from C100 carrying a sorting signal for the trans-Golgi network was higher than that generated from wild-type C100. These results using PS1-mutation-harbouring and wild-type Neuro 2a cells suggest that Abeta42/40-gamma-secretase cleavages occur in the Golgi compartment and the trans-Golgi network, and that the PS1 mutation does not alter the intracelluar site of Abeta42-gamma-secretase cleavage in the normal APP proteolytic processing pathway. 相似文献
2.
Agiostratidou G Muros RM Shioi J Marambaud P Robakis NK 《Journal of neurochemistry》2006,96(4):1182-1188
The presenilin (PS)/gamma-secretase system promotes production of the A beta (A beta) peptides by mediating cleavage of amyloid precursor protein (APP) at the gamma-sites. This system is also involved in the processing of type-I transmembrane proteins, including APP, cadherins and Notch1 receptors, at the epsilon-cleavage site, resulting in the production of peptides containing the intracellular domains (ICDs) of the cleaved proteins. Emerging evidence shows that these peptides have important biological functions, raising the possibility that their inhibition by gamma-secretase inhibitors may be detrimental to the cell. Here, we show that peptide E-Cad/CTF2, produced by the PS1/gamma-secretase processing of E-cadherin, promotes the lysosomal/endosomal degradation of the transmembrane APP derivatives, C99 and C83, and inhibits production of the APP ICD (AICD). In addition, E-Cad/CTF2 decreases accumulation of total secreted A beta. These data suggest a novel method to promote the non-amyloidogenic degradation of A beta precursors and to inhibit A beta production. 相似文献
3.
The gamma-secretase complex, composed by presenilin, nicastrin, APH-1 and PEN-2, is involved in intramembranous proteolysis of membrane proteins, such as amyloid precursor protein or Notch. Cleavage occurs in multiple cellular compartments. Here, nicastrin mutants containing targeting signals to the endoplasmic reticulum, trans-Golgi network, lysosomes, or plasma membrane have been shown to yield active gamma-secretase complexes with different activities and specificities: wild-type and plasma membrane nicastrin complexes yielded the highest amounts of secreted amyloid-beta peptide (Abeta), predominantly Abeta40, whereas intracellular targeted mutants produced intracellular Abeta, with a comparatively higher amount of Abeta42. These results suggest that compartmental microenvironments play a role in gamma-secretase activity and specificity. 相似文献
4.
The discrepancy between presenilin subcellular localization and gamma-secretase processing of amyloid precursor protein
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Cupers P Bentahir M Craessaerts K Orlans I Vanderstichele H Saftig P De Strooper B Annaert W 《The Journal of cell biology》2001,154(4):731-740
We investigated the relationship between PS1 and gamma-secretase processing of amyloid precursor protein (APP) in primary cultures of neurons. Increasing the amount of APP at the cell surface or towards endosomes did not significantly affect PS1-dependent gamma-secretase cleavage, although little PS1 is present in those subcellular compartments. In contrast, almost no gamma-secretase processing was observed when holo-APP or APP-C99, a direct substrate for gamma-secretase, were specifically retained in the endoplasmic reticulum (ER) by a double lysine retention motif. Nevertheless, APP-C99-dilysine (KK) colocalized with PS1 in the ER. In contrast, APP-C99 did not colocalize with PS1, but was efficiently processed by PS1-dependent gamma-secretase. APP-C99 resides in a compartment that is negative for ER, intermediate compartment, and Golgi marker proteins. We conclude that gamma-secretase cleavage of APP-C99 occurs in a specialized subcellular compartment where little or no PS1 is detected. This suggests that at least one other factor than PS1, located downstream of the ER, is required for the gamma-cleavage of APP-C99. In agreement, we found that intracellular gamma-secretase processing of APP-C99-KK both at the gamma40 and the gamma42 site could be restored partially after brefeldin A treatment. Our data confirm the "spatial paradox" and raise several questions regarding the PS1 is gamma-secretase hypothesis. 相似文献
5.
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
6.
Alzheimer's disease is characterized by brain deposition of extracellular amyloid beta-peptide (Abeta)-containing plaques. The cellular site of gamma-secretase activity, which releases Abeta and the corresponding amyloid precursor protein intracellular domain (AICD), remains controversial. Proposed cleavage sites range from the endoplasmic reticulum (ER), the Golgi apparatus, and the cell surface to endosomal compartments. We now used C99-green fluorescent protein (GFP), a fluorescent reporter substrate for gamma-secretase activity and monitored AICD production in living cells. C99-GFP is efficiently cleaved by gamma-secretase, and AICD-GFP is released into the cytosol. Inhibiting gamma-secretase results in accumulation of C99-GFP in early endosomes. By blocking selective transport steps along the secretory pathway, we demonstrate that gamma-secretase does not cleave its substrates in the ER, the Golgi/trans-Golgi network, or in secretory vesicles. In contrast, inhibition of endocytosis did not inhibit cleavage of C99-GFP. Similar results were obtained for another gamma-secretase substrate, NotchDeltaE. Our results suggest that intracellular domains are generated by gamma-secretase at the plasma membrane and/or early endosomes. 相似文献
7.
Fluhrer R Multhaup G Schlicksupp A Okochi M Takeda M Lammich S Willem M Westmeyer G Bode W Walter J Haass C 《The Journal of biological chemistry》2003,278(8):5531-5538
The beta-amyloid precursor protein (beta APP) is proteolytically processed by two secretase activities to produce the pathogenic amyloid beta-peptide (A beta). N-terminal cleavage is mediated by beta-secretase (BACE) whereas C-terminal intramembraneous cleavage is exerted by the presenilin (PS) gamma-secretase complex. The A beta-generating gamma-secretase cleavage principally occurs after amino acid 40 or 42 and results in secretion of A beta-(1-40) or A beta-(1-42). Upon overexpression of BACE in cultured cells we unexpectedly noticed a reduction of secreted A beta-(1-40/42). However, mass spectrometry revealed a truncated A beta species, which terminates at amino acid 34 (A beta-(1-34)) suggesting an alternative gamma-secretase cut. Indeed, expression of a loss-of-function variant of PS1 inhibited not only the production of A beta-(1-40) and A beta-(1-42) but also that of A beta-(1-34). However, expression levels of BACE correlate with the amount of A beta-(1-34), and A beta-(1-34) is produced at the expense of A beta-(1-40) and A beta-(1-42). Since this suggested that BACE is involved in a C-terminal truncation of A beta, we incubated purified BACE with A beta-(1-40) in vitro. Under these conditions A beta-(1-34) was generated. Moreover, when conditioned media containing Abeta-(1-40) and A beta-(1-42) were incubated with cells expressing a loss-of-function PS1 variant together with BACE, A beta-(1-34) was efficiently produced in vivo. These data demonstrate that an apparently gamma-secretase-dependent A beta derivative is produced after the generation of the non-truncated A beta via an additional and unexpected activity of BACE. 相似文献
8.
Huse JT Liu K Pijak DS Carlin D Lee VM Doms RW 《The Journal of biological chemistry》2002,277(18):16278-16284
The amyloid beta (A beta) peptide that accumulates in Alzheimer's disease brain is derived from the proteolytic processing of the amyloid precursor protein by beta- and gamma-secretase activities. The beta-secretase enzyme beta-site amyloid precursor protein-cleaving enzyme (BACE) generates the N terminus of A beta by cleavage at either Asp(1) (beta-site) or Glu(11) (beta'-site), ultimately leading to the production of full-length A beta 1-40/42 or truncated A beta 11-40/42. The functional significance of this variable cleavage site specificity as well as the relative pathological impact of full-length versus N-terminally truncated A beta remains largely unknown. In our analysis of BACE reactivity in cell culture, we found that the preference of the protease for either beta- or beta'-cleavage was strongly dependent on intracellular localization. Within the endoplasmic reticulum, beta-site proteolysis predominated, whereas in the trans-Golgi network, beta'-cleavage was favored. Furthermore, the contrasting cleavage site specificities of BACE were not simply due to differences in organelle pH or the oligosaccharide composition of the glycoproteins involved. Examination of post-mortem brain specimens revealed significant levels of A beta 11-40/42 within insoluble amyloid pools. Taken together, these data support an important role for beta'-cleavage in the process of cerebral amyloid deposition and localize the processing event to the trans-Golgi network. 相似文献
9.
Glu11 site cleavage and N-terminally truncated A beta production upon BACE overexpression 总被引:6,自引:0,他引:6
Amyloid beta peptides (A beta) are generated by the proteolytic processing of the amyloid beta precursor protein (APP). The newly identified beta-site APP-cleaving enzyme (BACE) cleaves APP at Asp1 as well as between Tyr10 and Glu11 of A beta, producing C-terminal fragments (CTFs) C99 and C89, respectively. Subsequent cleavage by gamma-secretase gives rise to A beta 1-40/42 and A beta 11-40/42. Although both full-length and A beta peptides truncated at residue 11 have been identified in amyloid plaques in the AD brain, the relative proportion of these two cleavage products produced by BACE and secreted into the medium by cultured cells is unknown. Using cell lines stably overexpressing BACE, we found that A beta 11-40 and A beta 11-42 are major A beta cleavage products generated by BACE. We further showed that BACE utilizes both full-length APP as well as C99 as substrates for the production of C89, and that A beta 11-40/42 can be generated by sequential cleavage of single APP molecules by BACE and gamma-secretase. Taken together, the abundance of A beta 11-40/42 produced by BACE suggests that their roles in AD pathogenesis may be underestimated. 相似文献
10.
Alzheimer's disease (AD) neuropathology is characterized by accumulation of "senile" plaques (SPs) and neurofibrillary tangles (NFTs) in vulnerable brain regions. SPs are principally composed of aggregates of up to 42/43 amino acid beta-amyloid (A beta) peptides. The discovery of familial AD (FAD) mutations in the genes for the amyloid precursor protein (APP) and presenilins (PSs), all of which increase A beta42 production, support the view that A beta is centrally involved in the pathogenesis of AD. A beta42 aggregates readily, and is thought to seed the formation of fibrils, which then act as templates for plaque formation. A beta is generated by the sequential intracellular cleavage of APP by beta-secretase to generate the N-terminal end of A beta, and intramembranous cleavage by gamma-secretase to generate the C-terminal end. Cell biological studies have demonstrated that A beta is generated in the ER, Golgi, and endosomal/lysosomal system. A central question involving the role of A beta in AD concerns how A beta causes disease and whether it is extracellular A beta deposition and/or intracellular A beta accumulation that initiates the disease process. The most prevalent view is that SPs are composed of extracellular deposits of secreted A beta and that A beta causes toxicity to surrounding neurons as extracellular SP. The recent emphasis on the intracellular biology of APP and A beta has led some investigators to consider the possibility that intraneuronal A beta may directly cause toxicity. In this review we will outline current knowledge of the localization of both intracellular and extracellular A beta. 相似文献
11.
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
12.
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
13.
Laudon H Karlström H Mathews PM Farmery MR Gandy SE Lundkvist J Lendahl U Näslund J 《The Journal of biological chemistry》2004,279(23):23925-23932
Processing of the Alzheimer amyloid precursor protein (APP) into the amyloid beta-protein and the APP intracellular domain is a proteolysis event mediated by the gamma-secretase complex where presenilin (PS) proteins are key constituents. PS is subjected to an endoproteolytic cleavage, generating a stable heterodimer composed of an N-terminal and a C-terminal fragment. Here we aimed at further understanding the role of PS in endoproteolysis, in proteolytic processing of APP and Notch, and in assembly of the gamma-secretase complex. By using a truncation protocol and alanine scanning, we identified Tyr-288 in the PS1 N-terminal fragment as critical for PS-dependent intramembrane proteolysis. Further mutagenesis of the 288 site identified mutants differentially affecting endoproteolysis and gamma-secretase activity. The Y288F mutant was endoproteolyzed to the same extent as wild type PS but increased the amyloid beta-protein 42/40 ratio by approximately 75%. In contrast, the Y288N mutant was also endoproteolytically processed but was inactive in reconstituting gamma-secretase in PS null cells. The Y288D mutant was deficient in both endoproteolysis and gamma-secretase activity. All three mutant PS1 molecules were incorporated into gamma-secretase complexes and stabilized Pen-2 in PS null cells. Thus, mutations at Tyr-288 do not affect gamma-secretase complex assembly but can differentially control endoproteolysis and gamma-secretase activity. 相似文献
14.
The Alzheimer's disease-linked protein, presenilin, forms the active site of the gamma-secretase enzyme complex. However, three other proteins, nicastrin (NCT), PEN-2 and APH-1, are required for enzyme activity. This complex is responsible for cleaving the beta-amyloid precursor protein to produce amyloid beta and the intracellular domain (AICD). Although much research has focused on the regions of presenilin that are important for gamma-secretase function, less is known about NCT. To further our understanding of the role of NCT in gamma-secretase activity and complex formation, we have undertaken a systematic evaluation of conserved residues in the juxtamembrane region of the extracellular domain of NCT. Two mutants, S632A and W648A, greatly reduce gamma-secretase activity, as seen by a reduction in amyloid beta and AICD levels. Several lines of evidence suggest that these mutations result in reduced gamma-secretase activity because they affect the ability of NCT to stably associate with the other gamma-secretase components. Since NCT and APH-1 must first bind in order for presenilin and PEN-2 to stably join the complex, we propose that S632 and W648 are essential for a stable interaction with APH-1. 相似文献
15.
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
16.
Petit A Bihel F Alvès da Costa C Pourquié O Checler F Kraus JL 《Nature cell biology》2001,3(5):507-511
We have designed new non-peptidic potential inhibitors of gamma-secretase and examined their ability to prevent production of amyloid-beta 40 (Abeta40) and Abeta42 by human cells expressing wild-type and Swedish-mutant beta-amyloid precursor protein (betaAPP). Here we identify three such agents that markedly reduce recovery of both Abeta40 and Abeta42 produced by both cell lines, and increase that of C99 and C83, the carboxy-terminal fragments of betaAPP that are derived from beta-and alpha-secretase, respectively. Furthermore, we show that these inhibitors do not affect endoproteolysis of endogenous or overexpressed presenilins. These inhibitors are totally unable to affect the mDeltaEnotch-1 cleavage that leads to generation of the Notch intracellular domain (NICD). These represent the first non-peptidic inhibitors that are able to prevent gamma-secretase cleavage of betaAPP without affecting processing of mDeltaEnotch-1 or endoproteolysis of presenilins. The distinction between these two proteolytic events, which are both prevented by disruption of presenilin genes, indicates that although they are intimately linked with betaAPP and Notch maturation, presenilins are probably involved in the control of maturation processes upstream of enzymes that cleave gamma-secretase and Notch. 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.
Presenilins mediate a dual intramembranous gamma-secretase cleavage of Notch-1 总被引:8,自引:0,他引:8
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Okochi M Steiner H Fukumori A Tanii H Tomita T Tanaka T Iwatsubo T Kudo T Takeda M Haass C 《The EMBO journal》2002,21(20):5408-5416
Following ectodomain shedding, Notch-1 undergoes presenilin (PS)-dependent constitutive intramembranous endoproteolysis at site-3. This cleavage is similar to the PS-dependent gamma-secretase cleavage of the beta-amyloid precursor protein (betaAPP). However, topological differences in cleavage resulting in amyloid beta-peptide (Abeta) or the Notch-1 intracellular domain (NICD) indicated independent mechanisms of proteolytic cleavage. We now demonstrate the secretion of an N-terminal Notch-1 Abeta-like fragment (Nbeta). Analysis of Nbeta by MALDI-TOF MS revealed that Nbeta is cleaved at a novel site (site-4, S4) near the middle of the transmembrane domain. Like the corresponding cleavage of betaAPP at position 40 and 42 of the Abeta domain, S4 cleavage is PS dependent. The precision of this cleavage is affected by familial Alzheimer's disease-associated PS1 mutations similar to the pathological endoproteolysis of betaAPP. Considering these similarities between intramembranous processing of Notch and betaAPP, we conclude that these proteins are cleaved by a common mechanism utilizing the same protease, i.e. PS/gamma-secretase. 相似文献
19.
Abeta46 is processed to Abeta40 and Abeta43, but not to Abeta42, in the low density membrane domains
Yagishita S Morishima-Kawashima M Ishiura S Ihara Y 《The Journal of biological chemistry》2008,283(2):733-738
Gamma-secretase cleaves the transmembrane domain of beta-amyloid precursor protein at multiple sites referred to as gamma-, epsilon-, and zeta-cleavage sites. We previously showed that N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a potent dipeptide gamma-secretase inhibitor, causes differential accumulation of longer amyloid beta-proteins (Abetas) within Chinese hamster ovary cells co-expressing beta C-terminal fragment and wild-type presenilin 1 (C99/wtPS1 cells). In this study, we used sucrose density gradient centrifugation to fractionate the membranes from C99/wtPS1 cells that had been pretreated with DAPT. We found that accumulating Abeta46 localized exclusively to low density membrane (LDM) domains. Incubating the Abeta46-accumulating LDM domains at 37 degrees C produced Abeta40, Abeta42, Abeta43, and beta-amyloid precursor protein intracellular domain. The addition of L685,458 completely prevented beta-amyloid precursor protein intracellular domain generation and resulted in a large decrease in the level of Abeta46 and the concomitant appearance of Abeta40 and Abeta43 but not Abeta42. Further addition of DAPT suppressed the production of Abeta40/43 and abolished the decrease in the amount of Abeta46. These data indicate that preaccumulated Abeta46 is processed by gamma-secretase to Abeta40/43 but not to Abeta42 in the LDM domains. The amount of newly produced Abeta40 and Abeta43 was roughly equivalent to the decrease in the amount of Abeta46. Temporal profiles did not show a maximal concentration for Abeta43, suggesting that Abeta46 is processed to Abeta40 and Abeta43 through a nonsuccessive process. 相似文献
20.
Hoke DE Tan JL Ilaya NT Culvenor JG Smith SJ White AR Masters CL Evin GM 《The FEBS journal》2005,272(21):5544-5557
The gamma-secretase complex mediates the final proteolytic event in Alzheimer's disease amyloid-beta biogenesis. This membrane complex of presenilin, anterior pharynx defective, nicastrin, and presenilin enhancer-2 cleaves the C-terminal 99-amino acid fragment of the amyloid precursor protein intramembranously at gamma-sites to form C-terminally heterogeneous amyloid-beta and cleaves at an epsilon-site to release the intracellular domain or epsilon-C-terminal fragment. In this work, two novel in vitro gamma-secretase assays are developed to further explore the biochemical characteristics of gamma-secretase activity. During development of a bacterial expression system for a substrate based on the amyloid precursor protein C-terminal 99-amino acid sequence, fragments similar to amyloid-beta and an epsilon-C-terminal fragment were observed. Upon purification this substrate was used in parallel with a transfected source of substrate to measure gamma-secretase activity from detergent extracted membranes. With these systems, it was determined that recovery of size-fractionated cellular and tissue-derived gamma-secretase activity is dependent upon detergent concentration and that activity correlates to a subset of high molecular mass presenilin complexes. We also show that by changing the solvent environment with dimethyl sulfoxide, detection of epsilon-C-terminal fragments can be elevated. Lastly, we show that zinc causes an increase in the apparent molecular mass of an amyloid precursor protein gamma-secretase substrate and inhibits its cleavage. These studies further refine our knowledge of the complexes and biochemical factors needed for gamma-secretase activity and suggest a mechanism by which zinc dysregulation may contribute to Alzheimer's disease pathogenesis. 相似文献