共查询到20条相似文献,搜索用时 495 毫秒
1.
Schöbel S Neumann S Hertweck M Dislich B Kuhn PH Kremmer E Seed B Baumeister R Haass C Lichtenthaler SF 《The Journal of biological chemistry》2008,283(21):14257-14268
Ectodomain shedding of the amyloid precursor protein (APP) by the two proteases alpha- and beta-secretase is a key regulatory event in the generation of the Alzheimer disease amyloid beta peptide (Abeta). beta-Secretase catalyzes the first step in Abeta generation, whereas alpha-secretase cleaves within the Abeta domain, prevents Abeta generation, and generates a secreted form of APP with neuroprotective properties. At present, little is known about the cellular mechanisms that control APP alpha-secretase cleavage and Abeta generation. To explore the contributory pathways, we carried out an expression cloning screen. We identified a novel member of the sorting nexin (SNX) family of endosomal trafficking proteins, called SNX33, as a new activator of APP alpha-secretase cleavage. SNX33 is a homolog of SNX9 and was found to be a ubiquitously expressed phosphoprotein. Exogenous expression of SNX33 in cultured cells increased APP alpha-secretase cleavage 4-fold but surprisingly had little effect on beta-secretase cleavage. This effect was similar to the expression of the dominant negative dynamin-1 mutant K44A. SNX33 bound the endocytic GTPase dynamin and reduced the rate of APP endocytosis in a dynamin-dependent manner. This led to an increase of APP at the plasma membrane, where alpha-secretase cleavage mostly occurs. In summary, our study identifies SNX33 as a new endocytic protein, which modulates APP endocytosis and APP alpha-secretase cleavage, and demonstrates that the rate of APP endocytosis is a major control factor for APP alpha-secretase cleavage. 相似文献
2.
Adlerz L Holback S Multhaup G Iverfeldt K 《The Journal of biological chemistry》2007,282(14):10203-10209
The mammalian amyloid precursor protein (APP) protein family consists of the APP and the amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2). The neurotoxic amyloid beta-peptide (Abeta) originates from APP, which is the only member of this protein family implicated in Alzheimer disease. However, the three homologous proteins have been proposed to be processed in similar ways and to have essential and overlapping functions. Therefore, it is also important to take into account the effects on the processing and function of the APP-like proteins in the development of therapeutic drugs aimed at decreasing the production of Abeta. Insulin and insulin-like growth factor-1 (IGF-1) have been shown to regulate APP processing and the levels of Abeta in the brain. In the present study, we show that IGF-1 increases alpha-secretase processing of endogenous APP and also increases ectodomain shedding of APLP1 and APLP2 in human SH-SY5Y neuroblastoma cells. We also investigated the role of different IGF-1-induced signaling pathways, using specific inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK). Our results indicate that phosphatidylinositol 3-kinase is involved in ectodomain shedding of APP and APLP1, but not APLP2, and that MAPK is involved only in the ectodomain shedding of APLP1. 相似文献
3.
The amyloid precursor protein (APP) belongs to a conserved gene family, also including the amyloid precursor-like proteins, APLP1 and APLP2. We have previously shown that all members of the APP protein family are up-regulated upon retinoic acid (RA)-induced neuronal differentiation of SH-SY5Y neuroblastoma cells. Here, we demonstrate that RA also affects the processing of APLP2 and APP, as shown by increased shedding of both sAPLP2 and sAPPalpha, as well as elevated levels of the APP intracellular domains (AICDs). Brain-derived neurotrophic factor (BDNF) has been reported to induce APP promoter activity and RA induces expression of the tyrosine kinase receptor B (TrkB) in neuroblastoma cells. We show that the increase in shedding of both APLP2 and APP in response to RA is not mediated through the TrkB receptor. However, BDNF concomitant with RA increased the expression of APP even further. In addition, the secretion of sAPLP2 and sAPPalpha as well as the levels of AICDs were increased in response to BDNF. In contrast, the levels of membrane-bound APP C-terminal fragment C99 significantly decreased. Our results suggest that RA and BDNF shifts APP processing towards the alpha-secretase pathway. In addition, we show that RA and BDNF regulate N-linked glycosylation of APLP1. 相似文献
4.
Hiraoka Y Ohno M Yoshida K Okawa K Tomimoto H Kita T Nishi E 《Journal of neurochemistry》2007,102(5):1595-1605
Amyloid-beta (Abeta) peptide, the principal component of senile plaques in the brains of patients with Alzheimer's disease, is derived from proteolytic cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. Alternative cleavage of APP by alpha-secretase occurs within the Abeta domain and precludes generation of Abeta peptide. Three members of the ADAM (a disintegrin and metalloprotease) family of proteases, ADAM9, 10 and 17, are the main candidates for alpha-secretases. However, the mechanism that regulates alpha-secretase activity remains unclear. We have recently demonstrated that nardilysin (EC 3.4.24.61, N-arginine dibasic convertase; NRDc) enhances ectodomain shedding of heparin-binding epidermal growth factor-like growth factor through activation of ADAM17. In this study, we show that NRDc enhances the alpha-secretase activity of ADAMs, which results in a decrease in the amount of Abeta generated. When expressed with ADAMs in cells, NRDc dramatically increased the secretion of alpha-secretase-cleaved soluble APP and reduced the amount of Abeta peptide generated. A peptide cleavage assay in vitro also showed that recombinant NRDc enhances ADAM17-induced cleavage of the peptide substrate corresponding to the alpha-secretase cleavage site of APP. A reduction of endogenous NRDc by RNA interference was accompanied by a decrease in the cleavage by alpha-secretase of APP and increase in the amount of Abeta generated. Notably, NRDc is clearly expressed in cortical neurons in human brain. Our results indicate that NRDc is involved in the metabolism of APP through regulation of the alpha-secretase activity of ADAMs, which may be a novel target for the treatment of Alzheimer's disease. 相似文献
5.
Shedding of the amyloid precursor protein-like protein APLP2 by disintegrin-metalloproteinases 总被引:1,自引:0,他引:1
Cleavage of the amyloid precursor protein (APP) within the amyloid-beta (Abeta) sequence by the alpha-secretase prevents the formation of toxic Abeta peptides. It has been shown that the disintegrin-metalloproteinases ADAM10 and TACE (ADAM17) act as alpha-secretases and stimulate the generation of a soluble neuroprotective fragment of APP, APPsalpha. Here we demonstrate that the related APP-like protein 2 (APLP2), which has been shown to be essential for development and survival of mice, is also a substrate for both proteinases. Overexpression of either ADAM10 or TACE in HEK293 cells increased the release of neurotrophic soluble APLP2 severalfold. The strongest inhibition of APLP2 shedding in neuroblastoma cells was observed with an ADAM10-preferring inhibitor. Transgenic mice with neuron-specific overexpression of ADAM10 showed significantly increased levels of soluble APLP2 and its C-terminal fragments. To elucidate a possible regulatory mechanism of APLP2 shedding in the neuronal context, we examined retinoic acid-induced differentiation of neuroblastoma cells. Retinoic acid treatment of two neuroblastoma cell lines upregulated the expression of both APLP2 and ADAM10, thus leading to an increased release of soluble APLP2. 相似文献
6.
Regulated intramembrane proteolysis of the amyloid precursor protein (APP) by the protease activities α-, β- and γ-secretase controls the generation of the neurotoxic amyloid β peptide. APLP2, the amyloid precursor-like protein 2, is a homolog of APP, which shows functional overlap with APP, but lacks an amyloid β domain. Compared to APP, less is known about the proteolytic processing of APLP2, in particular in neurons, and the cleavage sites have not yet been determined. APLP2 is cleaved by the β-secretase BACE1 and additionally by an α-secretase activity. The two metalloproteases ADAM10 and ADAM17 have been suggested as candidate APLP2 α-secretases in cell lines. Here, we used RNA interference and found that ADAM10, but not ADAM17, is required for the constitutive α-secretase cleavage of APLP2 in HEK293 and SH-SY5Y cells. Likewise, in primary murine neurons knock-down of ADAM10 suppressed APLP2 α-secretase cleavage. Using mass spectrometry we determined the proteolytic cleavage sites in the APLP2 sequence. ADAM10 was found to cleave APLP2 after arginine 670, whereas BACE1 cleaves after leucine 659. Both cleavage sites are located in close proximity to the membrane. γ-secretase cleavage was found to occur at different peptide bonds between alanine 694 and valine 700, which is close to the N-terminus of the predicted APLP2 transmembrane domain. Determination of the APLP2 cleavage sites enables functional studies of the different APLP2 ectodomain fragments and the production of cleavage-site specific antibodies for APLP2, which may be used for biomarker development. 相似文献
7.
Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts 总被引:26,自引:0,他引:26
Formation of senile plaques containing the beta-amyloid peptide (A beta) derived from the amyloid precursor protein (APP) is an invariant feature of Alzheimer's disease (AD). APP is cleaved either by beta-secretase or by alpha-secretase to initiate amyloidogenic (release of A beta) or nonamyloidogenic processing of APP, respectively. A key to understanding AD is to unravel how access of these enzymes to APP is regulated. Here, we demonstrate that lipid rafts are critically involved in regulating A beta generation. Reducing cholesterol levels in N2a cells decreased A beta production. APP and the beta-site APP cleavage enzyme (BACE1) could be induced to copatch at the plasma membrane upon cross-linking with antibodies and to segregate away from nonraft markers. Antibody cross-linking dramatically increased production of A beta in a cholesterol-dependent manner. A beta generation was dependent on endocytosis and was reduced after expression of the dynamin mutant K44A and the Rab5 GTPase-activating protein, RN-tre. This inhibition could be overcome by antibody cross-linking. These observations suggest the existence of two APP pools. Although APP inside raft clusters seems to be cleaved by beta-secretase, APP outside rafts undergoes cleavage by alpha-secretase. Thus, access of alpha- and beta-secretase to APP, and therefore A beta generation, may be determined by dynamic interactions of APP with lipid rafts. 相似文献
8.
Sahlin C Lord A Magnusson K Englund H Almeida CG Greengard P Nyberg F Gouras GK Lannfelt L Nilsson LN 《Journal of neurochemistry》2007,101(3):854-862
Mutations within the amyloid-beta (Abeta) domain of the amyloid precursor protein (APP) typically generate hemorrhagic strokes and vascular amyloid angiopathy. In contrast, the Arctic mutation (APP E693G) results in Alzheimer's disease. Little is known about the pathologic mechanisms that result from the Arctic mutation, although increased formation of Abeta protofibrils in vitro and intraneuronal Abeta aggregates in vivo suggest that early steps in the amyloidogenic pathway are facilitated. Here we show that the Arctic mutation favors proamyloidogenic APP processing by increased beta-secretase cleavage, as demonstrated by altered levels of N- and C-terminal APP fragments. Although the Arctic mutation is located close to the alpha-secretase site, APP harboring the Arctic mutation is not an inferior substrate to a disintegrin and metalloprotease-10, a major alpha-secretase. Instead, the localization of Arctic APP is altered, with reduced levels at the cell surface making Arctic APP less available for alpha-secretase cleavage. As a result, the extent and subcellular location of Abeta formation is changed, as revealed by increased Abeta levels, especially at intracellular locations. Our findings suggest that the unique clinical symptomatology and neuropathology associated with the Arctic mutation, but not with other intra-Abeta mutations, could relate to altered APP processing with increased steady-state levels of Arctic Abeta, particularly at intracellular locations. 相似文献
9.
Cappai R Cheng F Ciccotosto GD Needham BE Masters CL Multhaup G Fransson LA Mani K 《The Journal of biological chemistry》2005,280(14):13913-13920
Processing of the recycling proteoglycan glypican-1 involves the release of its heparan sulfate chains by copper ion- and nitric oxide-catalyzed ascorbate-triggered autodegradation. The Alzheimer disease amyloid precursor protein (APP) and its paralogue, the amyloid precursor-like protein 2 (APLP2), contain copper ion-, zinc ion-, and heparan sulfate-binding domains. We have investigated the possibility that APP and APLP2 regulate glypican-1 processing during endocytosis and recycling. By using cell-free biochemical experiments, confocal laser immunofluorescence microscopy, and flow cytometry of tissues and cells from wild-type and knock-out mice, we find that (a) APP and glypican-1 colocalize in perinuclear compartments of neuroblastoma cells, (b) ascorbate-triggered nitric oxidecatalyzed glypican-1 autodegradation is zinc ion-dependent in the same cells, (c) in cell-free experiments, APP but not APLP2 stimulates glypican-1 autodegradation in the presence of both Cu(II) and Zn(II) ions, whereas the Cu(I) form of APP and the Cu(II) and Cu(I) forms of APLP2 inhibit autodegradation, (d) in primary cortical neurons from APP or APLP2 knock-out mice, there is an increased nitric oxide-catalyzed degradation of heparan sulfate compared with brain tissue and neurons from wild-type mice, and (e) in growth-quiescent fibroblasts from APLP2 knock-out mice, but not from APP knock-out mice, there is also an increased heparan sulfate degradation. We propose that the rate of autoprocessing of glypican-1 is modulated by APP and APLP2 in neurons and by APLP2 in fibroblasts. These observation identify a functional relationship between the heparan sulfate and copper ion binding activities of APP/APLP2 in their modulation of the nitroxyl anion-catalyzed heparan sulfate degradation in glypican-1. 相似文献
10.
Clioquinol mediates copper uptake and counteracts copper efflux activities of the amyloid precursor protein of Alzheimer's disease 总被引:1,自引:0,他引:1
Treiber C Simons A Strauss M Hafner M Cappai R Bayer TA Multhaup G 《The Journal of biological chemistry》2004,279(50):51958-51964
The key protein in Alzheimer's disease, the amyloid precursor protein (APP), is a ubiquitously expressed copper-binding glycoprotein that gives rise to the Abeta amyloid peptide. Whereas overexpression of APP results in significantly reduced brain copper levels in three different lines of transgenic mice, knock-out animals revealed increased copper levels. A provoked rise in peripheral levels of copper reduced concentrations of soluble amyloid peptides and resulted in fewer pathogenic Abeta plaques. Contradictory evidence has been provided by the efficacy of copper chelation treatment with the drug clioquinol. Using a yeast model system, we show that adding clioquinol to the yeast culture medium drastically increased the intracellular copper concentration but there was no significant effect observed on zinc levels. This finding suggests that clioquinol can act therapeutically by changing the distribution of copper or facilitating copper uptake rather than by decreasing copper levels. The overexpression of the human APP or APLP2 extracellular domains but not the extracellular domain of APLP1 decreased intracellular copper levels. The expression of a mutant APP deficient for copper binding increased intracellular copper levels several-fold. These data uncover a novel biological function for APP and APLP2 in copper efflux and provide a new conceptual framework for the formerly diverging theories of copper supplementation and chelation in the treatment of Alzheimer's disease. 相似文献
11.
The amyloid precursor protein (APP) is proteolytically processed predominantly by alpha-secretase to release the ectodomain (sAPPalpha). In this study, we have addressed the cellular location of the constitutive alpha-secretase cleavage of endogenous APP in a neuronal cell line. Incubation of the neuroblastoma cell line IMR32 at 20 degrees C prevented the secretion into the medium of soluble wild-type APP cleaved by alpha-secretase as revealed by both immunoelectrophoretic blot analysis with a site-specific antibody and immunoprecipitation following metabolic labeling of the cells. No sAPPalpha was detected in the cell lysates following incubation of the cells at 20 degrees C, indicating that alpha-secretase does not cleave APP in the secretory pathway prior to or within the trans-Golgi network. Parallel studies using an antibody that recognizes specifically the neoepitope revealed on soluble APP cleaved by beta-secretase indicated that this enzyme was acting intracellularly. alpha-Secretase is a zinc metalloproteinase susceptible to inhibition by hydroxamate-based compounds such as batimastat [Parvathy, S., et al. (1998) Biochemistry 37, 1680-1685]. Incubation of the cells with a cell-impermeant, biotinylated hydroxamate inhibitor inhibited the release of sAPPalpha by >92%, indicating that alpha-secretase is cleaving APP almost exclusively at the cell surface. The observation that alpha-secretase cleaves APP at the cell surface, while beta-secretase can act earlier in the secretory pathway within the neuronal cell line indicates that there must be strict control mechanisms in place to ensure that APP is normally cleaved primarily by alpha-secretase in the nonamyloidogenic pathway to produce the neuroprotective sAPPalpha. 相似文献
12.
Parvathy S Ehrlich M Pedrini S Diaz N Refolo L Buxbaum JD Bogush A Petanceska S Gandy S 《Journal of neurochemistry》2004,90(4):1005-1010
Studies of metabolism of the Alzheimer amyloid precursor protein (APP) have focused much recent attention on the biology of juxta- and intra-membranous proteases. Release or 'shedding' of the large APP ectodomain can occur via one of two competing pathways, the alpha- and beta-secretase pathways, that are distinguished both by subcellular site of proteolysis and by site of cleavage within APP. The alpha-secretase pathway cleaves within the amyloidogenic Abeta domain of APP, precluding the formation of toxic amyloid aggregates. The relative utilization of the alpha- and beta-secretase pathways is controlled by the activation of certain protein phosphorylation signal transduction pathways including protein kinase C (PKC) and extracellular signal regulated protein kinase [ERK/mitogen-activated protein kinase (MAP kinase)], although the relevant substrates for phosphorylation remain obscure. Because of their apparent ability to decrease the risk for Alzheimer disease, the effects of statins (HMG CoA reductase inhibitors) on APP metabolism were studied. Statin treatment induced an APP processing phenocopy of PKC or ERK activation, raising the possibility that statin effects on APP processing might involve protein phosphorylation. In cultured neuroblastoma cells transfected with human Swedish mutant APP, atorvastatin stimulated the release of alpha-secretase-released, soluble APP (sAPPalpha). However, statin-induced stimulation of sAPPalpha release was not antagonized by inhibitors of either PKC or ERK, or by the co-expression of a dominant negative isoform of ERK (dnERK), indicating that PKC and ERK do not play key roles in mediating the effect of atorvastatin on sAPPalpha secretion. These results suggest that statins may regulate alpha-secretase activity either by altering the biophysical properties of plasma membranes or by modulating the function of as-yet unidentified protein kinases that respond to either cholesterol or to some intermediate in the cholesterol metabolic pathway. A 'phospho-proteomic' analysis of N2a cells with and without statin treatment was performed, revealing changes in the phosphorylation state of several protein kinases plausibly related to APP processing. A systematic evaluation of the possible role of these protein kinases in statin-regulated APP ectodomain shedding is underway. 相似文献
13.
14.
The role of presenilin-1 in the gamma-secretase cleavage of the amyloid precursor protein of Alzheimer's disease 总被引:3,自引:0,他引:3
Octave JN Essalmani R Tasiaux B Menager J Czech C Mercken L 《The Journal of biological chemistry》2000,275(3):1525-1528
Presenilin-1 (PS1) is required for the release of the intracellular domain of Notch from the plasma membrane as well as for the cleavage of the amyloid precursor protein (APP) at the gamma-secretase cleavage site. It remains to be demonstrated whether PS1 acts by facilitating the activity of the protease concerned or is the protease itself. PS1 could have a gamma-secretase activity by itself or could traffic APP and Notch to the appropriate cellular compartment for processing. Human APP 695 and PS1 were coexpressed in Sf9 insect cells, in which endogenous gamma-secretase activity is not detected. In baculovirus-infected Sf9 cells, PS1 undergoes endoproteolysis and interacts with APP. However, PS1 does not cleave APP in Sf9 cells. In CHO cells, endocytosis of APP is required for Abeta secretion. Deletion of the cytoplasmic sequence of APP (APPDeltaC) inhibits both APP endocytosis and Abeta production. When APPDeltaC and PS1 are coexpressed in CHO cells, Abeta is secreted without endocytosis of APP. Taken together, these results conclusively show that, although PS1 does not cleave APP in Sf9 cells, PS1 allows the secretion of Abeta without endocytosis of APP by CHO cells. 相似文献
15.
The influence of cholesterol and the lovastatin (cholesterol-lowering drug) on secretion of alpha-secretase cleavage product of amyloid precursor protein (APP) and expression of nicotinic acetylcholine receptors (nAChRs) was investigated in human HTB-15 astrocytes. The results showed that exposure of cholesterol to astrocytes inhibited the secretion of alpha-form of secreted APP (alphaAPPs) and reduced cell viability, while lovastatin enhanced the alpha-secretase processing on astrocytes; cholesterol treatment decreased expression of alpha7 nAChR, whereas lovastatin induced an up-regulation of the receptor; the increase in alphaAPPs resulted from lovastatin was partially inhibited by the alpha7 nAChR antagonists, alpha-bungarotoxin or methyllycaconitine; cholesterol or lovastatin did not influence either whole APP level or expression of alpha4 nAChR. We suggest that high dose of cholesterol may inhibit both the activity of alpha-secretase in APP metabolic processing and the expression of alpha7 nAChR, while lovastatin may stimulate alpha-secretase cleavage processing that might be regulated by alpha7 nAChR. 相似文献
16.
Amyloid precursor protein (APP) and amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2) are members of a large gene family. Although APP is known to be the source of the beta-amyloid peptides involved in the development of Alzheimer's disease, the normal functions of APP, APLP1 and APLP2 in cells are poorly understood. In this study, we carried out gene silencing analysis by means of RNA interference with synthetic small interfering RNA duplexes targeting the App, Aplp1 and Aplp2 genes in Neuro2a (N2a) cells, a mouse neuroblastoma cell line. The results demonstrated that cell viability and neurite outgrowth of N2a cells undergoing knockdown of Aplp1 were significantly reduced, compared with N2a cells undergoing knockdown of either App or Aplp2. 相似文献
17.
Bai Y Markham K Chen F Weerasekera R Watts J Horne P Wakutani Y Bagshaw R Mathews PM Fraser PE Westaway D St George-Hyslop P Schmitt-Ulms G 《Molecular & cellular proteomics : MCP》2008,7(1):15-34
Despite intense research efforts, the physiological function and molecular environment of the amyloid precursor protein has remained enigmatic. Here we describe the application of time-controlled transcardiac perfusion cross-linking, a method for the in vivo mapping of protein interactions in intact tissue, to study the interactome of the amyloid precursor protein (APP). To gain insights into the specificity of reported protein interactions the study was extended to the mammalian amyloid precursor-like proteins (APLP1 and APLP2). To rule out sampling bias as an explanation for differences in the individual datasets, a small scale quantitative iTRAQ (isobaric tags for relative and absolute quantitation)-based comparison of APP, APLP1, and APLP2 interactomes was carried out. An interactome map was derived that confirmed eight previously reported interactions of APP and revealed the identity of more than 30 additional proteins that reside in spatial proximity to APP in the brain. Subsequent validation studies confirmed a physiological interaction between APP and leucine-rich repeat and Ig domain-containing protein 1, demonstrated a strong influence of Ig domain-containing protein 1 on the proteolytic processing of APP, and consolidated similarities in the biology of APP and p75. 相似文献
18.
ADAM10 activation is required for green tea (-)-epigallocatechin-3-gallate-induced alpha-secretase cleavage of amyloid precursor protein 总被引:4,自引:0,他引:4
Obregon DF Rezai-Zadeh K Bai Y Sun N Hou H Ehrhart J Zeng J Mori T Arendash GW Shytle D Town T Tan J 《The Journal of biological chemistry》2006,281(24):16419-16427
Recently, we have shown that green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) exerts a beneficial role on reducing brain Abeta levels, resulting in mitigation of cerebral amyloidosis in a mouse model of Alzheimer disease. EGCG seems to accomplish this by modulating amyloid precursor protein (APP) processing, resulting in enhanced cleavage of the alpha-COOH-terminal fragment (alpha-CTF) of APP and corresponding elevation of the NH(2)-terminal APP product, soluble APP-alpha (sAPP-alpha). These beneficial effects were associated with increased alpha-secretase cleavage activity, but no significant alteration in beta-or gamma-secretase activities. To gain insight into the molecular mechanism whereby EGCG modulates APP processing, we evaluated the involvement of three candidate alpha-secretase enzymes, a-disintegrin and metalloprotease (ADAM) 9, 10, or 17, in EGCG-induced non-amyloidogenic APP metabolism. Results show that EGCG treatment of N2a cells stably transfected with "Swedish" mutant human APP (SweAPP N2a cells) leads to markedly elevated active ( approximately 60 kDa mature form) ADAM10 protein. Elevation of active ADAM10 correlates with increased alpha-CTF cleavage, and elevated sAPP-alpha. To specifically test the contribution of ADAM10 to non-amyloidogenic APP metabolism, small interfering RNA knockdown of ADAM9, -10, or -17 mRNA was employed. Results show that ADAM10 (but not ADAM9 or -17) is critical for EGCG-mediated alpha-secretase cleavage activity. In summary, ADAM10 activation is necessary for EGCG promotion of non-amyloidogenic (alpha-secretase cleavage) APP processing. Thus, ADAM10 represents an important pharmacotherapeutic target for the treatment of cerebral amyloidosis in Alzheimer disease. 相似文献
19.
Cleavage of amyloid precursor protein (APP) by beta-secretase generates beta-amyloid (Abeta), the major component of senile plaques in Alzheimer's disease. Cleavage of APP by alpha-secretase prevents Abeta formation, producing nonamyloidogenic secreted APPs products. PKC-regulated APP alpha-secretase cleavage has been shown to involve tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE). To determine the location of APP cleavage, we examined PKC-regulated APPs secretion by examining cell surface versus intracellular APP in CHO cells stably expressing APP(695) (CHO695). We demonstrate that PKC regulates cell surface and intracellular APP cleavage. The majority of secreted APPs originates from the intracellular compartment, and PKC does not cause an increase in APP trafficking to the cell surface for cleavage. Therefore, intracellular APP regulated by PKC must be cleaved at an intracellular site. Experiments utilizing Brefeldin A suggest APP cleavage occurs at the Golgi or late in the secretory pathway. Experiments using TAPI, an inhibitor of TACE, demonstrate PKC-regulated APPs secretion from the cell surface is inhibited after pretreatment with TAPI, and APPs secretion from the intracellular pool is partially inhibited after pretreatment with TAPI. These findings suggest PKC-regulated APP cleavage occurs at multiple locations within the cell and both events appear to involve TACE. 相似文献
20.
Tobias M J Allinson Edward T Parkin Thomas P Condon Sylva L U Schwager Edward D Sturrock Anthony J Turner Nigel M Hooper 《European journal of biochemistry》2004,271(12):2539-2547
Numerous transmembrane proteins, including the blood pressure regulating angiotensin converting enzyme (ACE) and the Alzheimer's disease amyloid precursor protein (APP), are proteolytically shed from the plasma membrane by metalloproteases. We have used an antisense oligonucleotide (ASO) approach to delineate the role of ADAM10 and tumour necrosis factor-alpha converting enzyme (TACE; ADAM17) in the ectodomain shedding of ACE and APP from human SH-SY5Y cells. Although the ADAM10 ASO and TACE ASO significantly reduced (> 81%) their respective mRNA levels and reduced the alpha-secretase shedding of APP by 60% and 30%, respectively, neither ASO reduced the shedding of ACE. The mercurial compound 4-aminophenylmercuric acetate (APMA) stimulated the shedding of ACE but not of APP. The APMA-stimulated secretase cleaved ACE at the same Arg-Ser bond in the juxtamembrane stalk as the constitutive secretase but was more sensitive to inhibition by a hydroxamate-based compound. The APMA-activated shedding of ACE was not reduced by the ADAM10 or TACE ASOs. These results indicate that neither ADAM10 nor TACE are involved in the shedding of ACE and that APMA, which activates a distinct ACE secretase, is the first pharmacological agent to distinguish between the shedding of ACE and APP. 相似文献