Fe65 stimulates proteolytic liberation of the beta-amyloid precursor protein intracellular domain

The beta-amyloid precursor protein (APP)-binding protein Fe65 is involved in APP nuclear signaling and several steps in APP proteolytic processing. In this study, we show that Fe65 stimulates gamma-secretase-mediated liberation of the APP intracellular domain (AICD). The mechanism of Fe65-mediated stimulation of AICD formation appears to be through enhanced production of the carboxyl-terminal fragment substrates of gamma-secretase and direct stimulation of processing by gamma-secretase. The stimulatory capacity of Fe65 is isoform-dependent, as the non-neuronal and a2 isoforms promote APP processing more effectively than the exon 9 inclusive neuronal form of Fe65. Intriguingly, Fe65 stimulation of AICD production appears to be inversely related to pathogenic beta-amyloid production as the Fe65 isoforms profoundly stimulate AICD production and simultaneously decrease Abeta42 production. Despite the capacity of Fe65 to stimulate gamma-secretase-mediated APP proteolysis, it does not rescue the loss of proteolytic function associated with the presenilin-1 familial Alzheimer disease mutations. These data suggest that Fe65 regulation of APP proteolysis may be integrally associated with its nuclear signaling function, as all antecedent proteolytic steps prior to release of Fe65 from the membrane are fostered by the APP-Fe65 interaction.     

Structure of the intracellular domain of the amyloid precursor protein in complex with Fe65-PTB2

Cleavage of the amyloid precursor protein (APP) is a crucial event in Alzheimer disease pathogenesis that creates the amyloid-beta peptide (Abeta) and liberates the carboxy-terminal APP intracellular domain (AICD) into the cytosol. The interaction of the APP C terminus with the adaptor protein Fe65 mediates APP trafficking and signalling, and is thought to regulate APP processing and Abeta generation. We determined the crystal structure of the AICD in complex with the C-terminal phosphotyrosine-binding (PTB) domain of Fe65. The unique interface involves the NPxY PTB-binding motif and two alpha helices. The amino-terminal helix of the AICD is capped by threonine T(668), an Alzheimer disease-relevant phosphorylation site involved in Fe65-binding regulation. The structure together with mutational studies, isothermal titration calorimetry and nuclear magnetic resonance experiments sets the stage for understanding T(668) phosphorylation-dependent complex regulation at a molecular level. A molecular switch model is proposed.     

β-淀粉样前体蛋白胞内端与JKTBP2蛋白的相互作用

β-淀粉样前体蛋白APP(β-amyloid precursor protein)与阿尔茨海默氏症密切相关,它经分泌酶1切割后生成的胞内端AID(APP intracellular domain)能够诱导细胞凋亡。为了研究AID在阿尔茨海默氏症病理过程中的作用,我们以AID为诱饵蛋白用酵母双杂交系统筛选与之有相互作用的蛋白。我们发现人不均一核蛋白D类似蛋白JKTBP2的90-204位肽段可以结合AID。利用293T细胞表达蛋白后进行免疫共沉淀,结果证实二者间存在相互作用。这些结果指出JKTBP2可能在阿尔茨海默氏症形成中有重要作用。     

The beta-amyloid domain is essential for axonal sorting of amyloid precursor protein.

We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild-type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild-type APP and mutations implicated in Alzheimer's disease and another brain beta-amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane-inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11-15 in the carbohydrate domain, the other encoded by exons 16-17 in the juxtamembraneous beta-amyloid domain. Treatment of the cells with the N-glycosylation inhibitor tunicamycin induced missorting of wild-type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta-amyloid-dependent membrane proximal signal was the major contributor to axonal sorting, while N-glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS-insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta-amyloid domain plays a major role in axonal delivery of APP.     

Insulin-degrading enzyme rapidly removes the beta-amyloid precursor protein intracellular domain (AICD).

The intramembranous gamma-secretase cleavage of the beta-amyloid precursor protein (APP) is dependent on biologically active presenilins (PS). Notch also undergoes a similar PS-dependent gamma-secretase-like cleavage, resulting in the liberation of the Notch intracellular domain (NICD), which is critically required for developmental signal transduction. gamma-Secretase processing of APP results in the production of a similar fragment called AICD (APP intracellular domain), which may function in nuclear signaling as well. AICD, like NICD, is rapidly removed. By using a battery of protease inhibitors we demonstrate that AICD, in contrast to NICD, is degraded by a cytoplasmic metalloprotease. In vitro degradation of AICD can be reconstituted with cytoplasmic fractions obtained from neuronal and non-neuronal cells. Taking into account the inhibition profile and the cytoplasmic localization, we identified three candidate enzymes (neurolysin, thimet oligopeptidase, and insulin-degrading enzyme (IDE), also known as insulysin), which all are involved in the degradation of bioactive peptides in the brain. When insulin, a well characterized substrate of IDE, was added to the in vitro degradation assay, removal of AICD was efficiently blocked. Moreover, overexpression of IDE resulted in enhanced degradation of AICD, whereas overexpression of the inactive IDE E111Q mutant did not affect AICD degradation. Finally, immunodepletion of IDE significantly reduced the AICD degrading activity. Therefore our data demonstrate that IDE, which is one of the proteases implicated in the removal of extracellular Abeta, also removes the cytoplasmic product of gamma-secretase cleaved APP.     

The beta-amyloid precursor protein is not processed by the regulated secretory pathway.

The beta-amyloid peptide is derived from a larger membrane bound protein and accumulates as amyloid in Alzheimer's diseased brains. beta-amyloid precursor protein (beta APP) proteolytically processed during constitutive secretion cannot be a source of deposited amyloid because this processing results in cleavage within the amyloidogenic peptide. To see if other secretory pathways could be responsible for generating potentially amyloidogenic molecules we tested the possibility that beta APP is targeted to the regulated secretory pathway. Stable AtT20 cell lines expressing exogenous human beta APP were genetically engineered. These cells were labeled with [35S]-methionine, and chased in the presence or absence of secretagogue. The beta APP both inside the cells and released from the cells was analyzed by immunoprecipitation and gel analysis. Quantitation of autoradiograms showed that virtually all of the synthesized beta APP was secreted by the constitutive pathway, and that no detectable (less than 1%) beta APP was targeted to the regulated secretory pathway.     

Direct visualization of the gamma secretase-generated carboxyl-terminal domain of the amyloid precursor protein: association with Fe65 and translocation to the nucleus

Amyloid-beta, the peptide that deposits as senile plaques in Alzheimer's disease, is derived from the amyloid precursor protein (APP) by a gamma secretase-mediated intramembranous cleavage. In addition to amyloid-beta, this cleavage produces a carboxyl-terminal intracellular fragment which has an unknown function. The carboxyl-terminal domain of APP interacts in the cytoplasm with an adapter protein, Fe65. We demonstrate by laser scanning confocal microscopy that a gamma secretase generated APP carboxyl-terminal domain, tagged with green fluorescent protein (GFP), translocates to the nucleus in a manner dependent upon stabilization by the adapter protein Fe65; APP which has been mutated to block interactions with Fe65 cannot be detected in the nucleus. The APP-CT domain continues to interact with Fe65 in the nucleus, as determined by both colocalization and fluorescence resonance energy transfer (FRET). Visualization of the APP-CT-Fe65 complex in the nucleus may serve as a readout for processes that modify gamma secretase release of APP-CT.     

Phosphorylation-dependent regulation of the interaction of amyloid precursor protein with Fe65 affects the production of beta-amyloid

Neuronal Fe65 is an adapter protein that interacts with the cytoplasmic domain of the beta-amyloid precursor protein (APP). Although the interaction has been reported to occur between the second phosphotyrosine interaction domain of Fe65 and the YENPTY motif in the cytoplasmic domain of APP, the regulatory mechanism and biological function of this interaction remain unknown. We report here that (i) a single amino acid mutation at the Thr-668 residue of APP695, located 14 amino acids toward the amino-terminal end from the (682)YENPTY(687) motif, reduced the interaction between members of the Fe65 family of proteins and APP, whereas interaction of APP with the phosphotyrosine interaction domain of other APP binders such as X11-like and mammalian disabled-1 was not influenced by this mutation; (ii) the phosphorylation of APP at Thr-668 diminished the interaction of APP with Fe65 by causing a conformational change in the cytoplasmic domain that contains the Fe65-binding motif, YENPTY; and (iii) the expression of Fe65 slightly suppressed maturation of APP and decreased production of beta-amyloid (Abeta). Mutation at Thr-668 of APP abolished the effect of Fe65 on APP maturation. This mutation blocked the Fe65-dependent suppression of Abeta production and resulted in the release of increased levels of Abeta in the presence of Fe65. We previously reported that during maturation of APP in neurons, the protein is specifically phosphorylated at Thr-668 and undergoes O-glycosylation. The present results suggest that the phosphorylation of O-glycosylated mature APP at Thr-668 causes a conformational change in its cytoplasmic domain that prevents binding of Fe65 in neurons and may lead to an alteration in the production of Abeta.     

Synthesis and characterization of the Kunitz protease-inhibitor domain of the beta-amyloid precursor protein.

To understand the pathological process by which amyloid is deposited in Alzheimer's disease, it is important to characterize the proteolytic processing events of the beta-amyloid precursor protein (beta-APP) from which the amyloid-forming fragment is excised. A potentially important component in beta-APP processing is the 57-amino acid (aa) Kunitz serine protease inhibitor (KPI) located within the extracellular domain of both the 751- and 770-aa isoforms of beta-APP. We have synthesized DNA encoding the 57-aa KPI domain as a necessary step in identifying the role of the protease inhibitor in beta-APP processing and amyloid formation. A bacterial secretion system directed by the alkaline phosphatase signal peptide of Escherichia coli linked to a synthetic gene encoding KPI was used to produce soluble, extracellular recombinant KPI (reKPI) protein. The reKPI protein was purified to homogeneity from bacterial supernatants and was biochemically and biologically characterized. Complete aa sequence analysis confirmed the fidelity of the reKPI, and fast-atom bombardment mass-spectral analysis was used to document that reKPI was of the predicted Mr. The reKPI is as active on a molar basis as the inhibitor-containing beta-APP when assayed for inhibition of trypsin activity. Together these data suggest that reKPI protein is properly folded and lacking in modified aa. Hence, this reKPI will be an important reagent in gaining a better understanding of the role of the KPI domain in beta-APP function and metabolism, as well as in the proteolytic events involved in beta-amyloid formation.     

Expression of human FE65 in amyloid precursor protein transgenic mice is associated with a reduction in beta-amyloid load

FE65 is an adaptor protein that interacts with the cytoplasmic tail of the amyloid precursor protein (APP). In cultured non-neuronal cells, the formation of the FE65-APP complex is a key element for the modulation of APP processing, signalling and beta-amyloid (Abeta) production. The functions of FE65 in vivo, including its role in the metabolism of neuronal APP, remain to be investigated. In this study, transgenic mice expressing human FE65 were generated and crossbred with APP transgenic mice, known to develop Abeta deposits at 6 months of age. Compared with APP mice, APP/FE65 double transgenic mice exhibited a lower Abeta accumulation in the cerebral cortex as demonstrated by immunohistochemistry and immunoassay, and a lower level of APP-CTFs. The reduced accumulation of Abeta in APP/FE65 double transgenics, compared with APP mice, could be linked to the low Abeta42 level observed at 4 months of age and to the lower APP-CTFs levels. The present work provides evidence that FE65 plays a role in the regulation of APP processing in an in vivo model.     

The secretory beta-amyloid precursor protein is a motogen for human epidermal keratinocytes

Cell migration is known to be triggered by constituents of the extracellular matrix such as fibronectin and by soluble mediators commonly summarized as motogens. Many growth factors such as the epidermal growth factor (EGF) have been shown to act as motogens. Recently, the secretory N-terminal portion of the beta-amyloid precursor protein (sAPP) has been identified as a keratinocyte growth factor. Hence, in this study we analysed whether sAPP stimulates also keratinocyte migration employing the stroboscopic cell motility assay. The migration velocity as well as the frequency of lamellipodia protrusion and ruffle formation were increased about two-fold thus corresponding to the effect of EGF. Using a newly developed beta1-integrin migration track assay we observed that sAPP increased the proportion of migrating keratinocytes and their directional persistence. sAPP appeared to operate synergistically with fibronectin with respect to its motogenic effect. Using a modified Boyden chamber assay we showed that sAPP besides its chemokinetic effect functions as a chemoattractant. Like EGF, sAPP exerted its motogenic effect through the activation of Rac kinase but the receptor for sAPP appears to be distinct. The results suggest that sAPP operates as a motogen in the human epidermis, where it may participate in the regulation of reepithelialization during wound healing.     

The metabolism of beta-amyloid converting enzyme and beta-amyloid precursor protein processing

Herein we investigated the processing of beta-secretase (BACE), implicated in Alzheimer's disease through processing of beta-amyloid precursor protein (betaAPP), into smaller metabolites. Four products of approximately 34, approximately 12, approximately 8, and approximately 5kDa were identified, none of which were generated autocatalytically. The approximately 34 and approximately 12kDa forms are held together by disulfide bridges. The approximately 34kDa form results from two cleavages: an N-terminal processing at RLPR(45) downward arrow by furin/PC5, and a C-terminal cleavage at SQDD(379) downward arrow by an unknown enzyme that also releases the C-terminal approximately 12kDa product. Microsequencing of the approximately 8 and approximately 5kDa fragments showed that they are the result of processing at VVFD(407) downward arrow and DMED(442) downward arrow, respectively. Mutagenesis of the identified cleavage sites revealed that the mutants D379A, D379L or D379E block the degradation of BACE into the approximately 12kDa product, confirming the importance of Asp(379). Notably, the D379E mutant results in higher betaAPP derived C99 levels. In contrast, D442A or D442E did not affect the production of the approximately 8 or approximately 5kDa products. The levels of the approximately 8 and approximately 5kDa products are significantly lower in the mutant D407A but less so D407E, likely due to the low efficacy of ER exit of the D407A mutant. Indeed, while co-expression of betaAPP with BACE results in enhanced production of Abeta(11-40), the D407A mutant produces mostly Abeta(40).     

Presenilin-1 regulates intracellular trafficking and cell surface delivery of beta-amyloid precursor protein

Presenilins (PS1/PS2) play a critical role in proteolysis of beta-amyloid precursor protein (beta APP) to generate beta-amyloid, a peptide important in the pathogenesis of Alzheimer's disease. Nevertheless, several regulatory functions of PS1 have also been reported. Here we demonstrate, in neuroblastoma cells, that PS1 regulates the biogenesis of beta APP-containing vesicles from the trans-Golgi network and the endoplasmic reticulum. PS1 deficiency or the expression of loss-of-function variants leads to robust vesicle formation, concomitant with increased maturation and/or cell surface accumulation of beta APP. In contrast, release of vesicles containing beta APP is impaired in familial Alzheimer's disease (FAD)-linked PS1 mutant cells, resulting in reduced beta APP delivery to the cell surface. Moreover, diminution of surface beta APP is profound at axonal terminals in neurons expressing a PS1 FAD variant. These results suggest that PS1 regulation of beta APP trafficking may represent an alternative mechanism by which FAD-linked PS1 variants modulate beta APP processing.