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1.
Mona Harnasch Sandra Grau Christian Behrends Simon L. Dove Ann Hochschild Maria-karnina Iskandar 《Molecular membrane biology》2013,30(6):373-383
An Escherichia coli system was used to produce the human membrane proteins presenilin 1 and amyloid precursor protein and to analyse their interaction. Our data indicate that the main binding site for amyloid precursor protein is located in the N-terminal three-transmembrane segments of presenilin and not in the proposed active site containing the two conserved aspartate residues. The data also suggest the presence of an additional segment of sufficient hydrophobicity at the C-terminus of PS1 to act potentially as a transmembrane segment. The implications of these findings for the function of γ-secretase are discussed. 相似文献
2.
Sato T Diehl TS Narayanan S Funamoto S Ihara Y De Strooper B Steiner H Haass C Wolfe MS 《The Journal of biological chemistry》2007,282(47):33985-33993
Gamma-secretase is an intramembrane aspartyl protease complex that cleaves type I integral membrane proteins, including the amyloid beta-protein precursor and the Notch receptor, and is composed of presenilin, Pen-2, nicastrin, and Aph-1. Although all four of these membrane proteins are essential for assembly and activity, the stoichiometry of the complex is unknown, with the number of presenilin molecules present being especially controversial. Here we analyze functional gamma-secretase complexes, isolated by immunoprecipitation from solubilized membrane fractions and able to produce amyloid beta-peptides and amyloid beta-protein precursor intracellular domain. We show that the active isolated protease contains only one presenilin per complex, which excludes certain models of the active site that require aspartate dyads formed between two presenilin molecules. We also quantified components in the isolated complexes by Western blot using protein standards and found that the amounts of Pen-2 and nicastrin were the same as that of presenilin. Moreover, we found that one Aph-1 was not co-immunoprecipitated with another in active complexes, evidence that Aph-1 is likewise present as a monomer. Taken together, these results demonstrate that the stoichiometry of gamma-components presenilin:Pen-2:nicastrin:Aph-1 is 1:1:1:1. 相似文献
3.
Proteolytic fragments of Alzheimer's disease-associated presenilin 1 are present in synaptic organelles and growth cone membranes of rat brain 总被引:2,自引:0,他引:2
Beher D Elle C Underwood J Davis JB Ward R Karran E Masters CL Beyreuther K Multhaup G 《Journal of neurochemistry》1999,72(4):1564-1573
Previous studies have demonstrated the molecular linkage of three causative genes for early-onset Alzheimer's disease: the presenilin 1 gene on chromosome 14, the presenilin 2 gene on chromosome 1, and the amyloid precursor protein gene on chromosome 21. In the present study, we have investigated the distributions of the approximately 20-kDa C-terminal and approximately 30-kDa N-terminal fragments of presenilin 1 and the amyloid precursor protein in rat brain and compared them with the distribution of several marker proteins. The fragments of presenilin 1 are present in synaptic plasma membranes, neurite growth cone membranes, and small synaptic vesicles of rat brain. Both proteolytic fragments are coenriched in the corresponding tissue fractions. Based on this observation, it seems likely that N- and C-terminal presenilin 1 fragments form a functional unit while remaining associated. In contrast to a predominant subcellular localization of presenilin 1 to the endoplasmic reticulum and Golgi apparatus in different cell lines, our results indicate that rat brain presenilin 1 fragments exit from these biosynthetic compartments to reach synaptic organelles in neurons. 相似文献
4.
Nina Stemmer Elena Strekalova Nevena Djogo Frank Pl?ger Gabriele Loers David Lutz Friedrich Buck Marek Michalak Melitta Schachner Ralf Kleene 《PloS one》2013,8(4)
Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer''s disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca2+- and N-glycan-independent interaction is mediated by amino acids 330–344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β42 levels in culture supernatants, while transfection with the P-domain increases amyloid-β40 levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330–344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β40 and amyloid-β42 levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the γ-secretase complex regulates the proteolytic processing of amyloid precursor protein by the γ-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer''s disease. 相似文献
5.
Grimm HS Beher D Lichtenthaler SF Shearman MS Beyreuther K Hartmann T 《The Journal of biological chemistry》2003,278(15):13077-13085
The final step in A beta generation is the cleavage of the C-terminal 99 amino acid residues of the amyloid precursor protein by gamma-secretase. gamma-Secretase activity is closely linked to the multi-transmembrane-spanning proteins presenilin 1 and presenilin 2. To elucidate whether the cleavage site specificities of gamma-secretase leading to the formation of secreted and intracellular A beta are identical, we made use of point mutations close to the gamma-cleavage site, known to have a dramatic effect on the 42/40 ratio of secreted A beta. We found that the selected point mutations only marginally influenced the 42/40 ratio of intracellular A beta, suggesting differences in the gamma-secretase cleavage site specificity for the generation of secreted and intracellular A beta. The analysis of the subcellular compartments involved in the generation of intracellular A beta revealed that A beta is not generated in the early secretory pathway in the human SH-SY5Y neuroblastoma cell line. In this study we identified late Golgi compartments to be involved in the generation of intracellular A beta. Moreover, we demonstrate that the presence of processed PS1 is not sufficient to obtain gamma-secretase processing of the truncated amyloid precursor protein construct C99, proposing the existence of an additional factor downstream of the endoplasmic reticulum and early Golgi required for the formation of an active gamma-secretase complex. 相似文献
6.
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
7.
Gu Y Sanjo N Chen F Hasegawa H Petit A Ruan X Li W Shier C Kawarai T Schmitt-Ulms G Westaway D St George-Hyslop P Fraser PE 《The Journal of biological chemistry》2004,279(30):31329-31336
Several lines of evidence have indicated that the presenilin proteins function within macromolecular complexes and are necessary for the regulated intramembranous proteolysis of certain type 1 transmembrane proteins, including the amyloid precursor protein, Notch, and p75. Data from multiple complementary experiments now suggest that there may be several distinct presenilin complexes. We show here that presenilin mutations and certain detergents affect the abundance and componentry of the presenilin complexes, and these structural effects correlate with their effects on gamma-secretase activity. Our data suggest that there are at least three complexes, including a approximately 150-kDa nicastrin-aph-1 complex (which is likely to be a precursor complex). There is a stable and abundant intermediate complex of approximately 440 kDa, which contains aph-1, pen-2, nicastrin, and PS1. However, it is the very low abundance, high mass (>/=670 kDa) heteromeric complexes that are associated with the highest gamma-secretase-specific activity. 相似文献
8.
Dineley KT Xia X Bui D Sweatt JD Zheng H 《The Journal of biological chemistry》2002,277(25):22768-22780
Familial Alzheimer's disease-associated mutations in presenilin 1 or 2 or amyloid precursor protein result in elevated beta-amyloid, beta-amyloid accumulation, and plaque formation in the brains of affected individuals. By crossing presenilin 1 transgenic mice carrying the A246E mutation with plaque-producing amyloid precursor protein K670N/M671L transgenic mice (Tg2576), we show that co-expression of both mutant transgenes results in acceleration of amyloid accumulation and associative learning deficits. At 5 months of age with no detectable plaque pathology, amyloid precursor protein transgenic animals are impaired in contextual fear learning following two pairings of conditioned and unconditioned stimuli but appear normal following a more robust five-pairing training. At 9 months of age when beta-amyloid deposition is evident, these mice are impaired following both two-pairing and five-pairing protocols. Mice carrying both transgenes are impaired in contextual fear conditioning at either age. All transgenic animal groups performed as well as controls in cued fear conditioning, indicating that the contextual fear learning deficits are hippocampus-specific. The associative learning impairments are coincident with elevated alpha 7 nicotinic acetylcholine receptor protein in the dentate gyrus. These findings provide two robust and rapid assays for beta-amyloid-associated effects that can be performed on young animals: impaired contextual fear learning and up-regulation of alpha 7 nicotinic receptors. 相似文献
9.
Li J Pauley AM Myers RL Shuang R Brashler JR Yan R Buhl AE Ruble C Gurney ME 《Journal of neurochemistry》2002,82(6):1540-1548
Mutations in the human presenilin genes (PS1 or PS2) have been linked to autosomal dominant, early onset Alzheimer's disease (AD). Presenilins, probably as an essential part of gamma-secretase, modulate gamma-cleavage of the amyloid protein precursor (APP) to the amyloid beta-peptide (Abeta). Mutations in sel-12, a Caenorhabditis elegans presenilin homologue, cause a defect in egg laying that can be suppressed by loss of function mutations in a second gene, SEL-10. SEL-10 protein is a homologue of yeast Cdc4, a member of the SCF (Skp1-Cdc53/CUL1-F-box protein) E2-E3 ubiquitin ligase family. In this study, we show that human SEL-10 interacts with PS1 and enhances PS1 ubiquitination, thus altering cellular levels of unprocessed PS1 and its N- and C-terminal fragments. Co-transfection of sel-10 and APP cDNAs in HEK293 cells leads to an alteration in the metabolism of APP and to an increase in the production of amyloid beta-peptide, the principal component of amyloid plaque in Alzheimer's disease. 相似文献
10.
Molecular biology and genetics of Alzheimer's disease 总被引:4,自引:0,他引:4
Like several other adult onset neurodegenerative diseases, Alzheimer's disease is a multifactorial illness with both genetic and non-genetic causes. Recent genetic studies have identified four genes associated with inherited risk for AD (presenilin 1, presenilin 2, amyloid precursor protein, and apolipoprotein E). These genes account for about half of the total genetic risk for Alzheimer's disease. It is suspected that several other Alzheimer's disease-susceptibility genes exist, and their identification is the subject of ongoing research. Nevertheless, biological studies on the effects of mutations in the four known genes has led to the conclusion that all of these genes cause dysregulation of amyloid precursor protein processing and in particular dysregulation of the handling of a proteolytic derivative termed Abeta. The accumulation of Abeta appears to be an early and initiating event that triggers a series of downstream processes including misprocessing of the tau protein. This cascade ultimately causes neuronal dysfunction and death, and leads to the clinical and pathological features of Alzheimer's disease. Knowledge of this biochemical cascade now provides several potential targets for the development of diagnostics and therapeutics. 相似文献
11.
Yang DS Tandon A Chen F Yu G Yu H Arawaka S Hasegawa H Duthie M Schmidt SD Ramabhadran TV Nixon RA Mathews PM Gandy SE Mount HT St George-Hyslop P Fraser PE 《The Journal of biological chemistry》2002,277(31):28135-28142
Nicastrin is an integral component of the high molecular weight presenilin complexes that control proteolytic processing of the amyloid precursor protein and Notch. We report here that nicastrin is most probably a type 1 transmembrane glycoprotein that is expressed at moderate levels in the brain and in cultured neurons. Immunofluorescence studies demonstrate that nicastrin is localized in the endoplasmic reticulum, Golgi, and a discrete population of vesicles. Glycosidase analyses reveal that endogenous nicastrin undergoes a conventional, trafficking-dependent maturation process. However, when highly expressed in transfected cells, there is a disproportionate accumulation of the endo-beta-N-acetylglucosaminidase H-sensitive, immature form, with no significant increase in the levels of the fully mature species. Immunoprecipitation revealed that presenilin-1 interacts preferentially with mature nicastrin, suggesting that correct trafficking and co-localization of the presenilin complex components are essential for activity. These findings demonstrate that trafficking and post-translational modifications of nicastrin are tightly regulated processes that accompany the assembly of the active presenilin complexes that execute gamma-secretase cleavage. These results also underscore the caveat that simple overexpression of nicastrin in transfected cells may result in the accumulation of large amounts of the immature protein, which is apparently unable to assemble into the active complexes capable of processing amyloid precursor protein and Notch. 相似文献
12.
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
13.
Loss-of-function presenilin mutations in Alzheimer disease. Talking Point on the role of presenilin mutations in Alzheimer disease
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De Strooper B 《EMBO reports》2007,8(2):141-146
Presenilin mutations are the main cause of familial Alzheimer disease. From a genetic point of view, these mutations seem to result in a gain of toxic function; however, biochemically, they result in a partial loss of function in the gamma-secretase complex, which affects several downstream signalling pathways. Consequently, the current genetic terminology is misleading. In fact, the available data indicate that several clinical presenilin mutations also lead to a decrease in amyloid precursor protein-derived amyloid beta-peptide generation, further implying that presenilin mutations are indeed loss-of-function mutations. The loss of function of presenilin causes incomplete digestion of the amyloid beta-peptide and might contribute to an increased vulnerability of the brain, thereby explaining the early onset of the inherited form of Alzheimer disease. In this review, I evaluate the implications of this model for the amyloid-cascade hypothesis and for the efficacy of presenilin/gamma-secretase as a drug target. 相似文献
14.
Shioi J Georgakopoulos A Mehta P Kouchi Z Litterst CM Baki L Robakis NK 《Journal of neurochemistry》2007,101(3):674-681
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
15.
Nonsteroidal anti-inflammatory drugs lower Abeta42 and change presenilin 1 conformation 总被引:8,自引:0,他引:8
Lleó A Berezovska O Herl L Raju S Deng A Bacskai BJ Frosch MP Irizarry M Hyman BT 《Nature medicine》2004,10(10):1065-1066
Recent reports suggest that some commonly used nonsteroidal anti-inflammatory drugs (NSAIDs) unexpectedly shift the cleavage products of amyloid precursor protein (APP) to shorter, less fibrillogenic forms, although the underlying mechanism remains unknown. We now demonstrate, using a fluorescence resonance energy transfer method, that Abeta(42)-lowering NSAIDs specifically affect the proximity between APP and presenilin 1 and alter presenilin 1 conformation both in vitro and in vivo, suggesting a novel allosteric mechanism of action. 相似文献
16.
Guozhang Mao Mei‐Zhen Cui Tong Li Yipeng Jin Xuemin Xu 《Journal of neurochemistry》2012,123(5):837-844
γ‐secretase is a protease complex with at least four components: presenilin, nicastrin (NCT), anterior pharynx‐defective 1 (Aph‐1), and presenilin enhancer 2 (Pen‐2). In this study, using knockout cell lines and small interfering RNA technology, our data demonstrated that the disappeared presenilin 1 C‐terminal fragment (PS1C) caused by knockdown of pen‐2 or knockout of NCT or Aph‐1 was recovered by the addition of proteasome inhibitors, indicating that Pen‐2, as well as NCT and Aph‐1α, is dispensable for presenilin endoproteolysis. Our data also demonstrate that the formation of the nicastrin‐Aph‐1 subcomplex plays not only an important role in γ‐secretase complex assembly but also in recruiting substrate C‐terminal fragment of amyloid precursor protein generated by β‐cleavage. Ablating any one component resulted in the instability of other components of the γ‐secretase complex, and the presence of all three of the other components is required for full maturation of NCT. 相似文献
17.
Ishrut Hussain Julien Fabrègue Laurence Anderes Solenne Ousson Frédéric Borlat Valérie Eligert Sébastien Berger Mitko Dimitrov Jean-René Alattia Patrick C. Fraering Dirk Beher 《The Journal of biological chemistry》2013,288(4):2521-2531
γ-Secretase is a large enzyme complex comprising presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1 that mediates the intramembrane proteolysis of a large number of proteins including amyloid precursor protein and Notch. Recently, a novel γ-secretase activating protein (GSAP) was identified that interacts with γ-secretase and the C-terminal fragment of amyloid precursor protein to selectively increase amyloid-β production. In this study we have further characterized the role of endogenous and exogenous GSAP in the regulation of γ-secretase activity and amyloid-β production in vitro. Knockdown of GSAP expression in N2a cells decreased amyloid-β levels. In contrast, overexpression of GSAP in HEK cells expressing amyloid precursor protein or in N2a cells had no overt effect on amyloid-β generation. Likewise, purified recombinant GSAP had no effect on amyloid-β generation in two distinct in vitro γ-secretase assays. In subsequent cellular studies with imatinib, a kinase inhibitor that reportedly prevents the interaction of GSAP with the C-terminal fragment of amyloid precursor protein, a concentration-dependent decrease in amyloid-β levels was observed. However, no interaction between GSAP and the C-terminal fragment of amyloid precursor protein was evident in co-immunoprecipitation studies. In addition, subchronic administration of imatinib to rats had no effect on brain amyloid-β levels. In summary, these findings suggest the roles of GSAP and imatinib in the regulation of γ-secretase activity and amyloid-β generation are uncertain. 相似文献
18.
Studies demonstrating that accumulation and aggregation of the amyloid beta protein (Abeta) within the brain is likely to cause Alzheimer's disease (AD) have provided the rationale for therapeutic strategies aimed at influencing Abeta production, aggregation and clearance. gamma-secretase catalyzes the final cleavage that releases the Abeta from its precursor; therefore, it is a potential therapeutic target for the treatment of AD. Recent data show that the polytopic membrane proteins presenilin 1 and presenilin 2 are either catalytic components or essential co-factors of a membrane-bound proteolytic complex that possesses gamma-secretase activity. Although recent findings demonstrating that gamma-secretase inhibitors bind directly to presenilins (PSs) further support a catalytic role for PSs in gamma-secretase cleavage, additional studies are still needed to clarify the role of PSs in gamma-secretase cleavage and the use of targeting PSs to reduce Abeta production. 相似文献
19.
Chen F Gu Y Hasegawa H Ruan X Arawaka S Fraser P Westaway D Mount H St George-Hyslop P 《The Journal of biological chemistry》2002,277(39):36521-36526
The presenilin 1 (PS1) and presenilin 2 (PS2) proteins are necessary for proteolytic cleavage of the amyloid precursor protein (APP) within its transmembrane domain. One of these cleavage events (termed gamma-secretase) generates the C-terminal end of the Abeta-peptide by proteolysis near residue 710 or 712 of APP(770). Another event (termed gamma-like or epsilon-secretase cleavage) cleaves near residue 721 at approximately 2-5 residues inside the cytoplasmic membrane boundary to generate a series of stable, C-terminal APP fragments. This latter cleavage is analogous to S3-cleavage of Notch. We report here that specific mutations in the N terminus, loop, or C terminus of PS1 all increase the production of Abeta(42) but cause inhibition of both epsilon-secretase cleavage of APP and S3-cleavage of Notch. These data support the hypothesis that epsilon-cleavage of APP and S3-cleavage of Notch are similar events. They also argue that, although both the gamma-site and the epsilon-site cleavage of APP are presenilin-dependent, they are likely to be independent catalytic events. 相似文献
20.
Wolfe MS 《Biochemistry》2006,45(26):7931-7939
Gamma-secretase is responsible for the proteolytic processing of a variety of membrane-associated fragments derived from type I integral membrane proteins, including the amyloid beta-protein precursor and the Notch receptor. This enzyme is composed of four different integral membrane proteins: presenilin, nicastrin, Aph-1, and Pen-2. During assembly and maturation of the protease complex, presenilin is endoproteolyzed into two subunits, each of which contributes one aspartate to the active site of an aspartyl protease. Substrate apparently interacts with an initial docking site before passing in whole or in part between the two presenilin subunits to the internal water-containing active site. The ectodomain of nicastrin also interacts with the N-terminus of the substrate as an essential step in substrate recognition and processing. Sites for allosteric regulation on the protease complex allow selective inhibition or modulation of APP processing without interfering with Notch signaling, and such selective agents may represent promising leads for the development of Alzheimer's disease therapeutics. Elucidation of detailed structural features of gamma-secretase and other membrane-embedded proteases is the next frontier in understanding how these enzymes carry out hydrolysis within the lipid bilayer. 相似文献