Amino acid sequence RERMS represents the active domain of amyloid beta/A4 protein precursor that promotes fibroblast growth

The growth of A-1 fibroblasts depends on exogenous amyloid beta/A4 protein precursor (APP), providing a simple bioassay to study the function of APP. Our preliminary study, testing the activity of a series of fragments derived from the secreted form of APP-695 (sAPP- 695) on this bioassay, has shown that at least one of the active sites of sAPP-695 was localized within a 40-mer sequence (APP296-335, Kang sequence; Roch, J.-M., I. P. Shapiro, M. P. Sundsmo, D. A. C. Otero, L. M. Refolo, N. K. Robakis, and T. Saitoh. 1992. J. Biol. Chem. 267:2214- 2221). In the present study, to further characterize the growth- promoting activity of sAPP-695 on fibroblasts, we applied a battery of synthetic peptides on this bioassay and found that: (a) the sequence of five amino acids, RERMS (APP328-332), was uniquely required for the growth-promoting activity of sAPP-695; (b) the activity was sequence- specific because the reverse-sequence peptide of the active domain had no activity; and (c) the four-amino-acid peptide RMSQ (APP330-333), which partially overlaps the COOH-terminal side of the active sequence RERMS, could antagonize the activity of sAPP-695. Furthermore, a recombinant protein which lacks this active domain (APP20-591 without 306-335) did not promote fibroblast cell growth, suggesting that this domain is the only site of sAPP-695 involved in the growth stimulation. The availability of these biologically active, short peptides and their antagonists should prove to be an essential step for the elucidation of APP involvement in regulation of cellular homeostasis.     

Glycosylation of the amyloid peptide precursor containing the Kunitz protease inhibitor domain improves the inhibition of trypsin

The amyloid beta peptide (A beta P) is the major constituent of the amyloid deposits that accumulate extracellularly in the brain of patients with Alzheimer's disease. This peptide is obtained from transmembrane amyloid protein precursors (APP) which sometimes contain a Kunitz protease inhibitor (KPI) insert in their extracellular domain and therefore are able to inhibit serine proteases. Expression of the transmembrane and the secreted APP containing the KPI domain was obtained by transient transfection of COS-1 cells. The overexpressed proteins were detected in immunoblotting experiments and inhibition of trypsin was analyzed using reverse enzymography. Our results indicate that post-translational modifications including glycosylation improve the inhibition of trypsin by the APP containing the KPI domain.     

Characterization of a presenilin-mediated amyloid precursor protein carboxyl-terminal fragment gamma. Evidence for distinct mechanisms involved in gamma -secretase processing of the APP and Notch1 transmembrane domains

A variety of investigations have led to the conclusion that presenilins (PS) play a critical role in intramembranous, gamma-secretase proteolysis of selected type I membrane proteins, including Notch1 and amyloid precursor protein (APP). We now show that the generation of the S3/Notch intracellular domain and APP-carboxyl-terminal fragment gamma (CTFgamma) derivatives are dependent on PS expression and inhibited by a highly selective and potent gamma-secretase inhibitor. Unexpectedly, the APP-CTFgamma derivative is generated by processing between Leu-645 and Val-646 (of APP(695)), several amino acids carboxyl-terminal to the scissile bonds for production of amyloid beta protein peptides. Although the relationship of APP-CTFgamma to the production of amyloid beta protein peptides is not known, we conclude that in contrast to the highly selective PS-dependent processing of Notch, the PS-dependent gamma-secretase processing of APP is largely nonselective and occurs at multiple sites within the APP transmembrane domain.     

Polarized sorting of beta-amyloid precursor protein and its proteolytic products in MDCK cells is regulated by two independent signals

Progressive cerebral deposition of the amyloid (A beta) beta-protein is an early and invariant feature of Alzheimer's disease. A beta is derived by proteolysis from the membrane-spanning beta-amyloid precursor protein (beta APP). beta APP is processed into various secreted products, including soluble beta APP (APPs), the 4-kD A beta peptide, and a related 3-kD peptide (p3). We analyzed the mechanisms regulating the polarized basolateral sorting of beta APP and its proteolytic derivatives in MDCK cells. Deletion of the last 32 amino acids (residues 664-695) of the beta APP cytoplasmic tail had no influence on either the constitutive approximately 90% level of basolateral sorting of surface beta APP, or the strong basolateral secretion of APPs, A beta, and p3. However, deleting the last 42 amino acids (residues 654-695) or changing tyrosine 653 to alanine altered the distribution of cell surface beta APP so that approximately 40-50% of the molecules were inserted apically. In parallel, A beta was now secreted from both surfaces. Surprisingly, this change in surface beta APP had no influence on the basolateral secretion of APPs and p3. This result suggests that most beta APP molecules which give rise to APPs in MDCK cells are cleaved intracellularly before reaching the surface. Consistent with this conclusion, we readily detected intracellular APPs in carbonate extracts of isolated membrane vesicles. Moreover, ammonium chloride treatment resulted in the equal secretion of APPs into both compartments, as occurs with other non-membranous, basolaterally secreted proteins, but it did not influence the polarity of cell surface beta APP. These results demonstrate that in epithelial cells two independent mechanisms mediate the polarized trafficking of beta APP holoprotein and its major secreted derivative (APPs) and that A beta peptides are derived in part from beta APP holoprotein targeted to the cell surface by a signal that includes tyrosine 653.     

The Alzheimer amyloid precursor protein. Identification of a stable intermediate in the biosynthetic/degradative pathway

The amyloid forming beta-peptide of Alzheimer's disease is synthesized as part of a larger integral membrane precursor protein (beta APP) of which three alternatively spliced versions of 695, 751, and 770 amino acids have been described. A fourth beta APP form of 563 amino acids does not contain the beta-peptide region. Recent experiments using transient expression in HeLa cells (Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum, J.M., Masters, C.L., and Beyreuther, K. (1989) Cell 57, 115-126) indicate that the beta APP undergoes several posttranslational modifications including the cleavage and secretion of a large portion of its extracellular domain. The nature and fate of the fragment that remains cell-associated following this cleavage has not heretofore been described. The metabolism of this fragment may have particular significance in Alzheimer's disease since it must contain at least part of the beta-peptide. To study the metabolic fate of this fragment, we have established cell lines overexpressing the 695- and 751-amino acid versions of beta APP. Pulse-chase studies show that this system is similar to the HeLa cell system in that both proteins are synthesized first as membrane-bound proteins of approximately 98 and 108 kDa carrying asparagine-linked sugar side chains and are subsequently processed into higher molecular mass forms by the attachment of sulfate, phosphate, and further sugar groups including sialic acid, adding approximately 20 kDa in apparent molecular mass. The mature form of beta APP is cleaved and rapidly secreted, leaving an 11.5-kDa fragment with the transmembrane region and the cytoplasmic domain behind in the cell. This fragment is stable with a half-life of at least 4 h.     

Secretion of the beta/A4 amyloid precursor protein. Identification of a cleavage site in cultured mammalian cells

Alzheimer's disease, a progressive neurodegenerative disorder, affects greater than 10% of the population of individuals greater than 65 years of age. A principal neuropathological feature of this disease is the senile plaque, a fibrillar extracellular deposit primarily composed of a approximately 4-kDa peptide, beta/A4, derived from the amyloid precursor protein (APP). Studies in cultured cells have documented that APP matures through a constitutive secretory pathway and is cleaved at or near the cell surface to release a large ectodomain into the extracellular space. To define the APP cleavage site, we constructed a Chinese hamster ovary cell line, which constitutively overexpresses human APP-770, and analyzed the COOH termini of secreted APP-770-related molecules. Using plasma desorption mass spectrometry and chemical microsequencing, we document that an APP cleavage site in Chinese hamster ovary cells leading to secretion occurs immediately COOH-terminal to lysine residue 687, which lies adjacent to the hydrophobic membrane-spanning domain.     

Antisera to an amino-terminal peptide detect the amyloid protein precursor of Alzheimer's disease and recognize senile plaques

The cerebral amyloid deposited in Alzheimer's disease (AD) contains a 4.2 kDa beta amyloid polypeptide (beta AP) that is derived from a larger beta amyloid protein precursor (beta APP). Three beta APP mRNAs encoding proteins of 695, 751, and 770 amino acids have previously been identified. In each of these, there is a single membrane-spanning domain close to the carboxyl-terminus of the beta APP, and the 42 amino acid beta AP sequence extends from within the membrane-spanning domain into the large extracellular region of the beta APP. We raised rabbit antisera to a peptide corresponding to amino acids 45-62 near the amino-terminus of the beta APP. We show that these antisera detect the beta APP by demonstrating that they (i) label a set of approximately 120 kDa membrane-associated proteins in human brain previously detected by antisera to the carboxyl-terminus of beta APP and (ii) label a set of approximately 120 kDa membrane-associated proteins that are selectively overexpressed in cells transfected with a full length beta APP expression construct. The beta APP45-62 antisera specifically stain senile plaques in AD brains. This finding, along with the previous demonstration that antisera to the carboxyl-terminus of the beta APP label senile plaques, indicates that both near amino-terminal and carboxyl-terminal domains of the beta APP are present in senile plaques and suggests that proteolytic processing of the full length beta APP molecule into insoluble amyloid fibrils occurs in a highly localized fashion at the sites of amyloid deposition in AD brains.     

Secretory processing of the Alzheimer amyloid beta/A4 protein precursor is increased by protein phosphorylation.

The 39-43 residue polypeptide (amyloid beta protein, beta A4) deposited as amyloid in Alzheimer's disease (AD) is derived from a set of 695-770 residue precursors referred to as the amyloid beta A4 protein precursor (beta APP). In each of the 695, 751, and 770 residue precursors, the 43 residue beta A4 is an internal peptide that begins 99 residues from the COOH-terminus of the beta APP. Each holoform is normally cleaved within the beta A4 to produce a large secreted derivative as well as a small membrane associated fragment. Neither of these derivatives can produce amyloid because neither contains the entire beta A4 peptide. In this study, we employ cells stably transfected with full length beta APP695, beta APP751, or beta APP770 expression constructs to show that phorbol ester activation of protein kinase C substantially increases the production of secreted forms from each isoform. By increasing processing of beta APP in the secretory pathway, PKC phosphorylation may help to prevent amyloid deposition.     

High level expression, purification, and characterization of the Kunitz-type protease inhibitor domain of protease nexin-2/amyloid beta-protein precursor.

The protease inhibitor, protease nexin-2 (PN-2), is the secreted isoform of the Alzheimer's amyloid beta-protein precursor (A beta PP) that contains the Kunitz-type protease inhibitor (KPI) domain. Here we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the KPI domain of PN-2/A beta PP. In addition to the 57 amino acid KPI domain, the expression product contained an additional four amino acid residues at its amino terminus that correspond to amino acids 285-288 of A beta PP (Ponte et al. 1988 Nature 311:525-527). This expression system generated yields of greater than 1.0 gram of KPI domain per liter of fermentation media. The secreted 61 amino acid product was purified to homogeneity and biochemically characterized. Amino acid analysis and sequencing of the entire expressed KPI domain verified its integrity. Similar to native PN-2/A beta PP, the purified KPI domain potently inhibited trypsin, chymotrypsin, and coagulation factor XIa. Although heparin augments the inhibition of factor XIa by native PN-2/A beta PP it had no effect on the inhibition of factor XIa by expressed KPI domain suggesting that heparin binds to regions on native PN-2/A beta PP outside of the protease inhibitory domain. This KPI domain expression product should be useful in studying the physiologic and pathophysiologic functions of PN-2/A beta PP.     

Structural features of the KPI domain control APP dimerization, trafficking, and processing

The two major isoforms of human APP, APP695 and APP751, differ by the presence of a Kunitz-type protease inhibitor (KPI) domain in the extracellular region. APP processing and function is thought to be regulated by homodimerization. We used bimolecular fluorescence complementation (BiFC) to study dimerization of different APP isoforms and mutants. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain did not affect fluorescence complementation, but native folding of KPI is critical for APP751 homodimerization. APP751 and APP695 dimers were mostly localized at steady state in the Golgi region, suggesting that most of the APP751 and 695 dimers are in the secretory pathway. Mutation of the KPI led to the retention of the APP homodimers in the endoplasmic reticulum. We finally showed that APP751 is more efficiently processed through the nonamyloidogenic pathway than APP695. These findings provide new insight on the particular role of KPI domain in APP dimerization. The correlation observed between dimerization, subcellular localization, and processing suggests that dimerization acts as an efficient regulator of APP trafficking in the secretory compartments that has major consequences on its processing.     

Folding and stability of the extracellular domain of the human amyloid precursor protein

The beta-amyloid peptide (A beta), the major component of the senile plaques found in the brains of Alzheimer's disease patients, is derived from proteolytic processing of a transmembrane glycoprotein known as the amyloid precursor protein (APP). Human APP exists in various isoforms, of which the major ones contain 695, 751, and 770 amino acids. Proteolytic cleavage of APP by alpha- or beta-secretases releases the extracellular soluble fragments sAPP alpha or sAPP beta, respectively. Despite the fact that sAPP alpha plays important roles in both physiological and pathological processes in the brain, very little is known about its structure and stability. We have recently presented a structural model of sAPP alpha 695 obtained from small-angle x-ray scattering measurements (Gralle, M., Botelho, M. M., Oliveira, C. L. P., Torriani, I., and Ferreira, S. T. (2002) Biophys. J. 83, 3513-3524). We now report studies on the folding and stabilities of sAPP alpha 695 and sAPP alpha 770. The combined use of intrinsic fluorescence, 4-4'-Dianilino-1,1'binaphthyl-5,5'-disulfonic acid (bis-ANS) fluorescence, circular dichroism, differential ultraviolet absorption, and small-angle x-ray scattering measurements of the equilibrium unfolding of sAPP alpha 695 and sAPP alpha 770 by GdnHCl and urea revealed multistep folding pathways for both sAPP alpha isoforms. Such stepwise folding processes may be related to the identification of distinct structural domains in the three-dimensional model of sAPP alpha. Furthermore, the relatively low stability of the native state of sAPP alpha suggests that conformational plasticity may play a role in allowing APP to interact with a number of distinct physiological ligands.     

Production of intracellular amyloid-containing fragments in hippocampal neurons expressing human amyloid precursor protein and protection against amyloidogenesis by subtle amino acid substitutions in the rodent sequence.

A distinguishing feature of Alzheimer's disease (AD) is the deposition of amyloid plaques in brain parenchyma. These plaques arise by the abnormal accumulation of beta A4, a proteolytic fragment of amyloid precursor protein (APP). Despite the fact that neurons are dramatically affected in the course of the disease, little is known about the neuronal processing of APP. To address this question we have expressed in fully mature, synaptically active rat hippocampal neurons, the neuronal form of human APP (APP695), two mutant forms of human APP associated with AD, and the mouse form of APP (a species known not to develop amyloid plaques). Protein expression was achieved via the Semliki Forest Virus system. Expression of wild type human APP695 resulted in the secretion of beta A4-amyloid peptide and the intracellular accumulation of potential amyloidogenic and non-amyloidogenic fragments. The relative amount of amyloid-containing fragments increased dramatically during expression of the clinical mutants, while it decreased strongly when the mouse form of APP was expressed. 'Humanizing' the rodent APP sequence by introducing three mutations in the beta A4-region also led to increased production of amyloid peptide to levels similar to those obtained with human APP. The single Gly601 to Arg substitution alone was sufficient to triple the ratio of beta A4-peptide to non-amyloidogenic p3-peptide. Due to the capacity of these cells to secrete and accumulate intracellular amyloid fragments, we hypothesize that in the pathogenesis of AD there is a positive feed-back loop where neurons are both producers and victims of amyloid, leading to neuronal degeneration and dementia.(ABSTRACT TRUNCATED AT 250 WORDS)     

APP N端片段的神经营养作用

APP是β－淀粉样肽的前体蛋白,由695－770个氨基酸经,春N端水解产物可分沁至细胞外环境。AP具有促进神经细胞生长作用,其中319－335肽段即APP17肽能提高动物的学习记忆能力。其他APP片段是否具有神经营养功能未见报道。本研究通过观察APP N端片段对人神经母细胞瘤株SY5Y生长的影响以及对实验性糖尿病动物行为的影响,希望APP促进神经细胞生长的其他肽段。用化学合肥APP N端多肽片段,以SY5Y细胞MTT代谢率、细胞计数、LDH漏出率和实验性糖尿病小鼠水迷宫试验结果为观察指标。结果APP64肽、29肽、11肽均有促进SY5Y细胞生长的作用,APP11肽可提高糖尿病动物水迷宫测试成绩,说明可溶性APP的N端可能具有神经营养作用,我们认为保持此作用的最短片段为APP11肽,此肽段的发现为进一步研究APP的构效关系奠定了基础。     

Isolation of baculovirus-derived secreted and full-length beta-amyloid precursor protein

We have expressed two forms of the Alzheimer's beta-amyloid precursor protein (beta APP), the 695-amino acid form (695 beta APP), and the 751-amino acid form (751 beta APP) in a baculovirus system. Both forms were expressed as full-length precursor, and were subsequently processed in vivo to release extracellular secreted proteins. The secreted forms were cleaved from the full-length beta APP in a manner analogous to the cleavage of beta APP during constitutive secretion in mammalian cells (Weidemann, A., K?nig, G., Bunke, D., Fischer, P., Salbaum, J. M., Masters, C. L., Beyreuther, K. (1989) Cell 57, 115-126; Oltersdorf, T., Ward, P. J., Henriksson, T., Beattie, E. C., Neve, R., Lieberburg, I., and Fritz, L. J. (1990) J. Biol. Chem. 265, 4492-4497). High levels of expression of 20-50 mg/liter were achieved. Both full-length and secreted forms of the beta-amyloid precursor proteins were purified using a combination of ion-exchange and immunoaffinity chromatography using a monoclonal antibody directed against beta APP. The 751 beta APP-derived full-length and secreted forms, which contain the Kunitz protease inhibitor domain, were shown to be as active in the inhibition of trypsin as is mammalian-derived secreted beta APP. The availability of purified full-length beta APP from the baculovirus system will be valuable for biochemical and cell biological analyses that may elucidate the mechanism of the inappropriate processing that leads to beta-amyloid formation in Alzheimer's disease.     

Proteolytic processing of amyloid beta protein precursor (APP) by thrombin.

Search for proteases responsible for an altered processing of APP which generates intermediates containing beta/A4 peptide is preceding to understand the formation of beta amyloid deposits characteristic of Alzheimer's disease, since many studies reveal that APP is ordinarily processed so as not to generate beta amyloid. Here, we have examined the action of thrombin, a serine protease in the blood clotting, in APP processing. Thrombin cleaved the mouse recombinant APP695 in vitro, resulting in the accumulation of 28 kDa fragment. The immunoblot analysis showed that the fragment is derived from the carboxy-terminal side of the recombinant APP695. Further, amino acid sequencing exhibited that the fragment is generated by the cleavage at Arg 510-Ile 511 and therefore includes entire beta/A4 peptide. We consider that the 28 kDa fragment is a possible intermediate for beta/A4 peptide. Thus thrombin may be involved in the altered processing of APP.     

Frame-shifted amyloid precursor protein found in Alzheimer's disease and Down's syndrome increases levels of secreted amyloid beta40

Frame-shifted amyloid precursor protein (APP(+1)), which has a truncated out-of-frame C-terminus, accumulates in the neuropathological hallmarks of patients with Alzheimer's disease pathology. To study a possible involvement of APP(+1) in the pathogenesis of Alzheimer's disease, we expressed APP695 and APP(+1) in the HEK293 cell-line and studied whether the processing of APP695 was affected. APP(+1) is a secretory protein, but high expression of APP695 and APP(+1) results in the formation of intracellular aggregate-like structures containing both proteins and Fe65, an adaptor protein that interacts with APP695. APP(+1) is shown to interact with APP695, suggesting that these structures consist of functional protein complexes. Such an interaction can also be anticipated in post-mortem brains of young Down's syndrome patients without any sign of neuropathology. Here we observed APP(+1) immunoreactivity in beaded fibres. Additional support for functional consequences on the processing of APP695 comes from a 1.4-fold increase in levels of secreted amyloid beta40 in cells co-expressing APP695 and APP(+1), although APP(+1) itself does not contain the amyloid beta sequence. Taken together, these data show that co-expression of APP695 and APP(+1) affects the processing of APP695 in a pro-amyloidogenic way and this could gradually contribute to Alzheimer's disease pathology, as has been implicated in Down's syndrome patients.     

Membrane-microdomain localization of amyloid β-precursor protein (APP) C-terminal fragments is regulated by phosphorylation of the cytoplasmic Thr668 residue

Amyloid β-precursor protein (APP) is primarily cleaved by α- or β-secretase to generate membrane-bound, C-terminal fragments (CTFs). In turn, CTFs are potentially subject to a second, intramembrane cleavage by γ-secretase, which is active in a lipid raft-like membrane microdomain. Mature APP (N- and O-glycosylated APP), the actual substrate of these secretases, is phosphorylated at the cytoplasmic residue Thr(668) and this phosphorylation changes the overall conformation of the cytoplasmic domain of APP. We found that phosphorylated and nonphosphorylated CTFs exist equally in mouse brain and are kinetically equivalent as substrates for γ-secretase, in vitro. However, in vivo, the level of the phosphorylated APP intracellular domain peptide (pAICD) generated by γ-cleavage of CTFs was very low when compared with the level of nonphosphorylated AICD (nAICD). Phosphorylated CTFs (pCTFs), rather than nonphosphorylated CTFs (nCTFs), were preferentially located outside of detergent-resistant, lipid raft-like membrane microdomains. The APP cytoplasmic domain peptide (APP(648-695)) with Thr(P)(668) did not associate with liposomes composed of membrane lipids from mouse brain to which the nonphosphorylated peptide preferentially bound. In addition, APP lacking the C-terminal 8 amino acids (APP-ΔC8), which are essential for membrane association, decreased Aβ generation in N2a cells. These observations suggest that the pCTFs and CTFΔC8 are relatively movable within the membrane, whereas the nCTFs are susceptible to being anchored into the membrane, an interaction made available as a consequence of not being phosphorylated. By this mechanism, nCTFs can be preferentially captured and cleaved by γ-secretase. Preservation of the phosphorylated state of APP-CTFs may be a potential treatment to lower the generation of Aβ in Alzheimer disease.