A second cytotoxic proteolytic peptide derived from amyloid beta-protein precursor

The amyloid beta-protein precursor gives rise to the amyloid beta-protein, the principal constituent of senile plaques and a cytotoxic fragment involved in the pathogenesis of Alzheimer disease. Here we show that amyloid beta-protein precursor was proteolytically cleaved by caspases in the C terminus to generate a second unrelated peptide, called C31. The resultant C31 peptide was a potent inducer of apoptosis. Both caspase-cleaved amyloid beta-protein precursor and activated caspase-9 were present in brains of Alzheimer disease patients but not in control brains. These findings indicate the possibility that caspase cleavage of amyloid beta-protein precursor with the generation of C31 may be involved in the neuronal death associated with Alzheimer disease.     

The epithelial Na+ channel is inhibited by a peptide derived from proteolytic processing of its alpha subunit

Epithelial sodium channels (ENaCs) mediate Na(+) entry across the apical membrane of high resistance epithelia that line the distal nephron, airway and alveoli, and distal colon. These channels are composed of three homologous subunits, termed alpha, beta, and gamma, which have intracellular amino and carboxyl termini and two membrane-spanning domains connected by large extracellular loops. Maturation of ENaC subunits involves furin-dependent cleavage of the extracellular loops at two sites within the alpha subunit and at a single site within the gamma subunit. The alpha subunits must be cleaved twice, immediately following Arg-205 and Arg-231, in order for channels to be fully active. Channels lacking alpha subunit cleavage are inactive with a very low open probability. In contrast, channels lacking both alpha subunit cleavage and the tract alphaAsp-206-Arg-231 are active when expressed in oocytes, suggesting that alphaAsp-206-Arg-231 functions as an inhibitor that stabilizes the channel in the closed conformation. A synthetic 26-mer peptide (alpha-26), corresponding to alphaAsp-206-Arg-231, reversibly inhibits wild-type mouse ENaCs expressed in Xenopus oocytes, as well as endogenous Na(+) channels expressed in either a mouse collecting duct cell line or primary cultures of human airway epithelial cells. The IC(50) for amiloride block of ENaC was not affected by the presence of alpha-26, indicating that alpha-26 does not bind to or interact with the amiloride binding site. Substitution of Arg residues within alpha-26 with Glu, or substitution of Pro residues with Ala, significantly reduced the efficacy of alpha-26. The peptide inhibits ENaC by reducing channel open probability. Our results suggest that proteolysis of the alpha subunit activates ENaC by disassociating an inhibitory domain (alphaAsp-206-Arg-231) from its effector site within the channel complex.     

Atypical processing of amyloid precursor fusion protein by proteolytic activity in Pichia pastoris.

Secretases catalyze the production of important proteolytic products of the amyloid precursor protein. We expressed a fusion protein that contained horseradish peroxidase, fragment 590-695 of amyloid precursor protein, and c-myc and polyhistidine tags in Pichia pastoris. It secreted a 50-kDa N-terminal fragment; a 15-kDa C-terminal fragment accumulated in cells. The N-terminal fragment exhibited peroxidase activity and reacted with antibodies specific for peptides within the sequences -2 to 15 and 21-37 of beta-amyloid peptide. The C-terminal fragment reacted with antibodies that recognize the sequences 649-664 and 676-695 of amyloid precursor protein and the C-terminal c-myc tag. To locate the cut site, the C-terminal fragment was metabolically labeled with either [(35)S]Met or [(3)H]Lys and radiosequenced. A major component, derived from a cleavage at Gly(25)-Ser(26) of beta-amyloid, was detected. Results suggest a predominant atypical cleavage, like that observed in Down Syndrome fibroblasts, occurs between the alpha- and gamma-sites.     

Interaction between amyloid beta peptide and an aggregation blocker peptide mimicking islet amyloid polypeptide

Assembly of amyloid-beta peptide (Aβ) into cytotoxic oligomeric and fibrillar aggregates is believed to be a major pathologic event in Alzheimer's disease (AD) and interfering with Aβ aggregation is an important strategy in the development of novel therapeutic approaches. Prior studies have shown that the double N-methylated analogue of islet amyloid polypeptide (IAPP) IAPP-GI, which is a conformationally constrained IAPP analogue mimicking a non-amyloidogenic IAPP conformation, is capable of blocking cytotoxic self-assembly of Aβ. Here we investigate the interaction of IAPP-GI with Aβ40 and Aβ42 using NMR spectroscopy. The most pronounced NMR chemical shift changes were observed for residues 13-20, while residues 7-9, 15-16 as well as the C-terminal half of Aβ--that is both regions of the Aβ sequence that are converted into β-strands in amyloid fibrils--were less accessible to solvent in the presence of IAPP-GI. At the same time, interaction of IAPP-GI with Aβ resulted in a concentration-dependent co-aggregation of Aβ and IAPP-GI that was enhanced for the more aggregation prone Aβ42 peptide. On the basis of the reduced toxicity of the Aβ peptide in the presence of IAPP-GI, our data are consistent with the suggestion that IAPP-GI redirects Aβ into nontoxic "off-pathway" aggregates.     

gamma-Cleavage is dependent on zeta-cleavage during the proteolytic processing of amyloid precursor protein within its transmembrane domain

beta-Amyloid precursor protein apparently undergoes at least three major cleavages, gamma-, epsilon-, and the newly identified zeta-cleavage, within its transmembrane domain to produce secreted beta-amyloid protein (Abeta). However, the roles of epsilon- and zeta-cleavages in the formation of secreted Abeta and the relationship among these three cleavages, namely epsilon-, zeta-, and gamma-cleavages, remain elusive. We investigated these issues by attempting to determine the formation and turnover of the intermediate products generated by these cleavages, in the presence or absence of known gamma-secretase inhibitors. By using a differential inhibition strategy, our data demonstrate that Abeta(46) is an intermediate precursor of secreted Abeta. Our co-immunoprecipitation data also reveal that, as an intermediate, Abeta(46) is tightly associated with presenilin in intact cells. Furthermore, we identified a long Abeta species that is most likely the long sought after intermediate product, Abeta(49), generated by epsilon-cleavage, and this Abeta(49) is further processed by zeta- and gamma-cleavages to generate Abeta(46) and ultimately the secreted Abeta(40/42). More interestingly, our data demonstrate that gamma-cleavage not only occurs last but also depends on zeta-cleavage occurring prior to it, indicating that zeta-cleavage is crucial for the formation of secreted Abeta. Thus, we conclude that the C terminus of secreted Abeta is most likely generated by a series of sequential cleavages, namely first epsilon-cleavage which is then followed by zeta- and gamma-cleavages, and that Abeta(46) produced by zeta-cleavage is the precursor of secreted Abeta(40/42).     

N-Terminal amino acid sequence analysis indicates that isolated atrial amyloid is derived from atrial natriuretic peptide

Isolated atrial amyloid, the most frequent senile cardiac amyloid type, was chemically analysed. Amyloid fibrils obtained from a patient (NIP) were extracted and the predominant lowmolecular-weight polypeptide (approximately 3.5 kDa, designated ASc₂ NIP) was isolated by size exclusion high performance liquid chromatography in 60% formic acid. N-Terminal amino acid sequence analysis of this polypeptide was identical to that of the atrial natriuretic peptide α-hANP for the first 12 residues determined.     

N-terminal amino acid sequence analysis indicates that isolated atrial amyloid is derived from atrial natriuretic peptide

Isolated atrial amyloid, the most frequent senile cardiac amyloid type, was chemically analysed. Amyloid fibrils obtained from a patient (NIP) were extracted and the predominant low-molecular-weight polypeptide (approximately 3.5 kDa, designated ASc2 NIP) was isolated by size exclusion high performance liquid chromatography in 60% formic acid. N-Terminal amino acid sequence analysis of this polypeptide was identical to that of the atrial natriuretic peptide alpha-hANP for the first 12 residues determined.     

Fibrillar islet amyloid polypeptide (amylin) is internalised by macrophages but resists proteolytic degradation

Pancreatic islet amyloid, formed from islet amyloid polypeptide, is found in 96% of Type II (non-insulin-dependent) diabetic patients. Islet amyloidosis is progressive and apparently irreversible. Fibrils immunoreactive for islet amyloid polypeptide are found in macrophages associated with amyloid, suggesting that deposits can be phagocytosed. To determine the mechanism for the recognition and internalisation of fibrils, mouse peritoneal macrophages were cultured with fibrillar synthetic human islet amyloid polypeptide. Fibrils did not exert a cytotoxic effect over 72 h of culture. The uptake and degradation of fibrils was analysed by quantitative light-and electron-microscopic immunocytochemistry and immunoreactivity was detectable in 86±3% cells within 6 h of culture. Neither polyinosinic acid (200 µg/ml) nor nocodazole (10 µg/ml) inhibited fibril uptake, suggesting that internalisation is not blocked by poly-ions and is independent of microtubule assembly. Inhibition of pseudopodia formation by cytochalasin B blocked fibriI uptake. Fibril aggregates became condensed in lysosomes to form protofilaments and were resistant to intracellular proteolysis. Fibrils can be phagocytosed by macrophages in vitro but amyloid-associated factors may block the recognition of fibrils in vivo preventing the removal of islet amyloid in diabetes.     

Regulated intramembrane proteolysis--lessons from amyloid precursor protein processing

Regulated intramembrane proteolysis (RIP) controls the communication between cells and the extracellular environment. RIP is essential in the nervous system, but also in other tissues. In the RIP process, a membrane protein typically undergoes two consecutive cleavages. The first one results in the shedding of its ectodomain. The second one occurs within its transmembrane domain, resulting in secretion of a small peptide and the release of the intracellular domain into the cytosol. The proteolytic cleavage fragments act as versatile signaling molecules or are further degraded. An increasing number of membrane proteins undergo RIP. These include growth factors, cytokines, cell adhesion proteins, receptors, viral proteins and signal peptides. A dysregulation of RIP is found in diseases, such as leukemia and Alzheimer's disease. One of the first RIP substrates discovered was the amyloid precursor protein (APP). RIP processing of APP controls the generation of the amyloid β-peptide, which is believed to cause Alzheimer's disease. Focusing on APP as the best-studied RIP substrate, this review describes the function and mechanism of the APP RIP proteases with the goal to elucidate cellular mechanisms and common principles of the RIP process in general.     

Amyloid precursor-like protein 1 influences endocytosis and proteolytic processing of the amyloid precursor protein

Ectodomain shedding of the amyloid precursor protein (APP) is a key regulatory step in the generation of the Alzheimer disease amyloid beta peptide (Abeta). The molecular mechanisms underlying the control of APP shedding remain little understood but are in part dependent on the low density lipoprotein receptor-related protein (LRP), which is involved in APP endocytosis. Here, we show that the APP homolog APLP1 (amyloid precursor-like protein 1) influences APP shedding. In human embryonic kidney 293 cells expression of APLP1 strongly activated APP shedding by alpha-secretase and slightly reduced beta-secretase cleavage. As revealed by domain deletion analysis, the increase in APP shedding required the NPTY amino acid motif within the cytoplasmic domain of APLP1. This motif is conserved in APP and is essential for the endocytosis of APP and APLP1. Unrelated membrane proteins containing similar endocytic motifs did not affect APP shedding, showing that the increase in APP shedding was specific to APLP1. In LRP-deficient cells APLP1 no longer induced APP shedding, suggesting that in wild-type cells APLP1 interferes with the LRP-dependent endocytosis of APP and there by increases APP alpha-cleavage. In fact, an antibody uptake assay revealed that expression of APLP1 reduced the rate of APP endocytosis. In summary, our study provides a novel mechanism for APP shedding, in which APLP1 affects the endocytosis of APP and makes more APP available for alpha-secretase cleavage.     

Plasmalogen synthesis is regulated via alkyl-dihydroxyacetonephosphate-synthase by amyloid precursor protein processing and is affected in Alzheimer's disease

Lipids play an important role as risk or protective factors in Alzheimer's disease, which is characterized by amyloid plaques composed of aggregated amyloid-beta. Plasmalogens are major brain lipids and controversially discussed to be altered in Alzheimer's disease (AD) and whether changes in plasmalogens are cause or consequence of AD pathology. Here, we reveal a new physiological function of the amyloid precursor protein (APP) in plasmalogen metabolism. The APP intracellular domain was found in vivo and in vitro to increase the expression of the alkyl-dihydroxyacetonephosphate-synthase (AGPS), a rate limiting enzyme in plasmalogen synthesis. Alterations in APP dependent changes of AGPS expression result in reduced protein and plasmalogen levels. Under the pathological situation of AD, increased amyloid-beta level lead to increased reactive oxidative species production, reduced AGPS protein and plasmalogen level. Accordingly, phosphatidylethanol plasmalogen was decreased in the frontal cortex of AD compared to age matched controls. Our findings elucidate that plasmalogens are decreased as a consequence of AD and regulated by APP processing under physiological conditions.     

Oxidation of cholesterol by amyloid precursor protein and beta-amyloid peptide

Alzheimer's disease (AD) is characterized by accumulation of the neurotoxic peptide beta-amyloid, which is produced by proteolysis of amyloid precursor protein (APP). APP is a large membrane-bound copper-binding protein that is essential in maintaining synaptic function and may play a role in synaptogenesis. beta-Amyloid has been shown to contribute to the oxidative stress that accompanies AD. Later stages of AD are characterized by neuronal apoptosis. However, the biochemical function of APP and the mechanism of the toxicity of beta-amyloid are still unclear. In this study, we show that both beta-amyloid and APP can oxidize cholesterol to form 7beta-hydroxycholesterol, a proapoptotic oxysterol that was neurotoxic at nanomolar concentrations. 7beta-Hydroxycholesterol inhibited secretion of soluble APP from cultured rat hippocampal H19-7/IGF-IR neuronal cells and inhibited tumor necrosis factor-alpha-converting enzyme alpha-secretase activity but had no effect on beta-site APP-cleaving enzyme 1 activity. 7beta-Hydroxycholesterol was also a potent inhibitor of alpha-protein kinase C, with a K(i) of approximately 0.2 nm. The rate of reaction between cholesterol and beta-amyloid was comparable to the rates of cholesterol-metabolizing enzymes (k(cat) = 0.211 min(-)1). The rate of production of 7beta-hydroxycholesterol by APP was approximately 200 times lower than by beta-amyloid. Oxidation of cholesterol was accompanied by stoichiometric production of hydrogen peroxide and required divalent copper. The results suggest that a function of APP may be to produce low levels of 7-hydroxycholesterol. Higher levels produced by beta-amyloid could contribute to the oxidative stress and cell loss observed in Alzheimer's disease.     

Cysteine 73 in bleomycin hydrolase is critical for amyloid precursor protein processing

Human bleomycin hydrolase (hBH) is a neutral cysteine protease that may regulate the secretion of soluble amyloid precursor protein (APP) and amyloid beta (A(beta)), which is a major constituent of the Alzheimer's disease-associated amyloid plaques. We have now determined that APP interacts with hBH by using yeast two hybrid methods and in vitro binding studies revealed that APP interacted with a 68 amino acid region that includes the catalytic domain of hBH. Ectopic expression of hBH increased the secretion of A(beta) but not of a second secreted protein, apolipoprotein A-I. Expression of hBH in which the catalytic cysteine 73 was mutated to serine failed to increase A(beta) secretion. These results indicate a critical role for cysteine 73 of hBH in mediating APP processing.     

Modulation of amyloid precursor protein processing by synthetic ceramide analogues

Previous studies suggest that membrane lipids may regulate proteolytic processing of the amyloid precursor protein (APP) to generate amyloid-beta peptide (Abeta). In the present study, we have assessed the capacity for a series of structurally related synthetic ceramide analogues to modulate APP processing in vitro. The compounds tested are established glucosylceramide synthase (GS) inhibitors based on the d-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) structure. PDMP and related compounds PPMP and EtDO-P4 inhibited Abeta secretion from Chinese hamster ovary cells expressing human APP (CHO-APP) with approximate IC₅₀ values of 15, 5, and 1 μM, respectively. A trend for reduced secretion of the APP alpha-secretase product, sAPPalpha, was also observed in PDMP-treated cells but not in PPMP- or ETDO-P4-treated cells, whereas levels of the cellular beta-secretase product APP C-terminal fragment, CTFbeta, were increased by both PDMP and PPMP but unaltered with EtDO-P4 treatment. Our data also revealed that EtDO-P4 inhibits endogenous Abeta production by human neurons. In conclusion, this study provides novel information regarding the regulation of APP processing by synthetic ceramide analogues and reveals that the most potent of these compounds is EtDO-P4.     

IGF-1-induced processing of the amyloid precursor protein family is mediated by different signaling pathways

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.     

An antimicrobial peptide is produced by extracellular processing of a protein from Propionibacterium jensenii

A protease-activated antimicrobial peptide (PAMP) and its inactive precursor were purified from the culture supernatant of Propionibacterium jensenii LMG 3032 and characterized at the molecular level. PAMP is a 64-amino-acid cationic peptide of 6,383 Da with physicochemical features similar to those of bacteriocins from gram-positive bacteria. This peptide displayed bactericidal activity against several propionibacteria and lactobacilli. DNA sequencing indicated that the PAMP-encoding gene (pamA) is translated as a proprotein of 198 amino acids with an N-terminal signal peptide of 27 amino acids and that PAMP constitutes the C-terminal part of this precursor. The amino acid sequence of pro-PAMP showed no similarity to those of other known proteins. By using activity tests and mass spectrometry, we showed that PAMP was formed upon protease treatment of the precursor protein. The propionibacteria produced the PAMP precursor constitutively during growth up to a level of approximately 4 mg/liter, but the producing bacteria were unable to activate the precursor. The requirement for an external protease represents a novel strategy for generating antimicrobial peptides.     

A 15-kDa interferon-induced protein is derived by COOH-terminal processing of a 17-kDa precursor

An interferon-induced 15-kDa protein is synthesized from a precursor of higher molecular weight; the precursor contains 165 amino acids (17 kDa), whereas the stable product (15 kDa) contains 156 amino acids. The stable 15-kDa form is derived from the precursor 17-kDa form by the removal of eight amino acids from the COOH terminus and the methionine from the NH2 terminus. The existence of the precursor 17-kDa protein can be demonstrated after brief periods of in vivo labeling with [35S]methionine and by translation of mRNA in vitro.     

Lactic acid induces aberrant amyloid precursor protein processing by promoting its interaction with endoplasmic reticulum chaperone proteins

Background

Lactic acid, a natural by-product of glycolysis, is produced at excess levels in response to impaired mitochondrial function, high-energy demand, and low oxygen availability. The enzyme involved in the production of β-amyloid peptide (Aβ) of Alzheimer''s disease, BACE1, functions optimally at lower pH, which led us to investigate a potential role of lactic acid in the processing of amyloid precursor protein (APP).

Methodology/Principal Findings

Lactic acid increased levels of Aβ40 and 42, as measured by ELISA, in culture medium of human neuroblastoma cells (SH-SY5Y), whereas it decreased APP metabolites, such as sAPPα. In cell lysates, APP levels were increased and APP was found to interact with ER-chaperones in a perinuclear region, as determined by co-immunoprecipitation and fluorescence microscopy studies. Lactic acid had only a very modest effect on cellular pH, did increase the levels of ER chaperones Grp78 and Grp94 and led to APP aggregate formation reminiscent of aggresomes.

Conclusions/Significance

These findings suggest that sustained elevations in lactic acid levels could be a risk factor in amyloidogenesis related to Alzheimer''s disease through enhanced APP interaction with ER chaperone proteins and aberrant APP processing leading to increased generation of amyloid peptides and APP aggregates.     

Cell-type-specific processing of the amyloid precursor protein by Presenilin during Drosophila development

The cleavage of proteins within their transmembrane domain by Presenilin (PS) has an important role in different signalling pathways and in Alzheimer's disease. Nevertheless, not much is known about the regulation of PS activity. It has been suggested that substrate recognition by the PS complex depends only on the size of the extracellular domain independent of the amino-acid sequence and that PS activity is constitutive in all cells that express the minimal components of the complex. We report here the development of an in vivo reporter system that allowed us to analyse the processing of human amyloid precursor protein (APP) and the Notch receptor tissue specifically during Drosophila development in the living organism. Using this system, we demonstrate differences between APP and Notch processing and show that PS-mediated cleavage of APP can be regulated in different cell types independent of the size of the extracellular domain.