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Results for “Amyloid Precursor Protein Secretases”

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PubMed1996

Increased activity-regulating and neuroprotective efficacy of alpha-secretase-derived secreted amyloid precursor protein conferred by a C-terminal heparin-binding domain.

Proteolytic cleavage of beta-amyloid precursor protein (beta APP) by alpha-secretase results in release of one secreted form (sAPP) of APP (sAPP alpha), whereas cleavage by beta-secretase releases a C-terminally truncated sAPP (sAPP beta) plus amyloid beta-peptide (A beta). beta APP mutations linked to some inherited forms of Alzheimer's disease may alter its processing such that levels of sAPP alpha are reduced and levels of sAPP beta increased. sAPP alpha s may play important roles in neuronal plasticity and survival, whereas A beta can be neurotoxic. sAPP alpha was approximately 100-fold more potent than sAPP beta in protecting hippocampal neurons against excitotoxicity, A beta toxicity, and glucose deprivation. Whole-cell patch clamp and calcium imaging analyses showed that sAPP beta was less effective than sAPP alpha in suppressing synaptic activity, activating K+ channels, and attenuating calcium responses to glutamate. Using various truncated sAPP alpha and sAPP beta APP695 products generated by eukaryotic and prokaryotic expression systems, and synthetic sAPP peptides, the activity of sAPP alpha was localized to amino acids 591-612 at the C-terminus. Heparinases greatly reduced the actions of sAPP alpha s, indicating a role for a heparin-binding domain at the C-terminus of sAPP alpha in receptor activation. These findings indicate that alternative processing of beta APP has profound effects on the bioactivity of the resultant sAPP products and suggest that reduced levels of sAPP alpha could contribute to neuronal degeneration in Alzheimer's disease.

Alzheimer Disease↗
PubMed2003

gamma-Secretase can cleave amyloid precursor protein fragments independent of alpha- and beta-secretase pre-cutting.

Abeta is the major component of amyloid in the brain in Alzheimer's disease and is derived from an amyloid precursor protein (APP) by the sequential proteolytic processing of two putative proteases, called beta- and gamma-secretase. To clarify the mechanism of gamma-secretase processing, we created constructs contained the C-terminal domain of APP and analyzed the processing in COS-1 cells. We found that C-terminal fragments (CTFs) containing a short extra N-terminal region before the beta-secretase cleavage site were directly processed at gamma-secretase cleavage site. This suggests that gamma-secretase cleavage occurs independently and is not dependent on alpha- and beta-secretase cleavage.

Alzheimer Disease↗
PubMed2000

L-685,458, an aspartyl protease transition state mimic, is a potent inhibitor of amyloid beta-protein precursor gamma-secretase activity.

Progressive cerebral amyloid beta-protein (A beta) deposition is believed to play a central role in the pathogenesis of Alzheimer's disease (AD). Elevated levels of A beta(42) peptide formation have been linked to early-onset familial AD-causing gene mutations in the amyloid beta-protein precursor (A beta PP) and the presenilins. Sequential cleavage of A beta PP by the beta- and gamma-secretases generates the N- and C-termini of the A beta peptide, making both the beta- and gamma-secretase enzymes potential therapeutic targets for AD. The identity of the A beta PP gamma-secretase and the mechanism by which the C-termini of A beta are formed remain uncertain, although it has been suggested that the presenilins themselves are novel intramembrane-cleaving gamma-secretases of the aspartyl protease class [Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T., and Selkoe, D. J. (1999) Nature 398, 513-517]. In this study we report the identification of L-685,458 as a structurally novel inhibitor of A beta PP gamma-secretase activity, with a similar potency for inhibition of A beta(42) and A beta(40) peptides. This compound contains an hydroxyethylene dipeptide isostere which suggests that it could function as a transition state analogue mimic of an aspartyl protease. The preferred stereochemistry of the hydroxyethylene dipeptide isostere was found to be the opposite to that required for inhibition of the HIV-1 aspartyl protease, a factor which may contribute to the observed specificity of this compound. Specific and potent inhibitors of A beta PP gamma-secretase activity such as L-685,458 will enable important advances toward the identification and elucidation of the mechanism of action of this enigmatic protease.

Amino Acid Sequence↗
PubMed2000

Role of presenilin in gamma-secretase cleavage of amyloid precursor protein.

Cerebral accumulation of the amyloid beta-protein (Abeta) is an early and invariant event in the pathogenesis of Alzheimer's disease (AD). Mutations in the presenilin (PS) 1 and 2 genes that increase production of the highly amyloidogenic Abeta(42) are the most common cause of familial AD. Deletion of PS1 in mice reduces Abeta generation, indicating that PS1 mediates the last step in the generation of Abeta from beta-amyloid precursor protein (APP) by the unidentified gamma-secretase. Mutating either of two conserved transmembrane aspartates in PS1 significantly reduced Abeta production and increased the APP C-terminal fragments that are gamma-secretase substrates. These results indicate that PS1 is either a unique diaspartyl cofactor for gamma-secretase or is itself gamma-secretase. Furthermore, studies on the gamma-secretase-like proteolytic processing of Notch and Ire1 suggest a common mechanism for the involvement of PS1 in intramembrane proteolysis of membrane proteins.

Alzheimer Disease↗
PubMed1997

Proteasome contributes to the alpha-secretase pathway of amyloid precursor protein in human cells.

A major histopathological hallmark in Alzheimer's disease consists of the extracellular deposition of the amyloid beta-peptide (A beta) that is proteolytically derived from the beta-amyloid precursor protein (beta APP). An alternative, nonamyloidogenic cleavage, elicited by a protease called alpha-secretase, occurs inside the A beta sequence and gives rise to APP alpha, a major secreted C-terminal-truncated form of beta APP. Here, we demonstrate that human embryonic kidney 293 (HK293) cells contain a chymotryptic-like activity that can be ascribed to the proteasome and that selective inhibitors of this enzyme reduce the phorbol 12,13-dibutyrate-sensitive APP alpha secretion by these cells. Furthermore, we establish that a specific proteasome blocker, lactacystin, also induces increased secretion of A beta peptide in stably transfected HK293 cells overexpressing wild-type beta APP751. Altogether, this study represents the first identification of a proteolytic activity, namely, the proteasome, contributing likely through yet unknown intracellular relays, to the alpha-secretase pathway in human cells.

Amyloid Precursor Protein Secretases↗
PubMed2006

An antibody to the beta-secretase cleavage site on amyloid-beta-protein precursor inhibits amyloid-beta production.

Proteolytic cleavage of amyloid-beta-protein precursor (AbetaPP) by beta- and gamma-secretases results in production of the amyloid-beta peptide (Abeta) that accumulates in the brains of sufferers of Alzheimer's disease (AD). We have developed a monoclonal antibody, 2B12, which binds in the vicinity of the beta-secretase cleavage site on AbetaPP but does not bind within the Abeta region. We hypothesised that this antibody, directed against the substrate rather than the enzyme, could inhibit cleavage of AbetaPP by beta-secretase via steric hindrance and thus reduce downstream production of Abeta. The antibody would enter cells by binding to AbetaPP when it is at the cell surface and then be internalised with the protein. We subsequently demonstrated that, after addition of 2B12 to standard growth media, this antibody was indeed capable of inhibiting Abeta40 production in neuroblastoma and astrocytoma cells expressing native AbetaPP, as measured by an ELISA. This inhibition was both concentration- and time-dependent and was specific to 2B12. We were only able to inhibit approximately 50% of Abeta40 production suggesting that not all AbetaPP is trafficked to the cell surface. We propose that this antibody could be used as a novel, putative therapy for the treatment of AD.

Amyloid Precursor Protein Secretases↗
PubMed1997

Novel beta-secretase cleavage of beta-amyloid precursor protein in the endoplasmic reticulum/intermediate compartment of NT2N cells.

Previous studies have demonstrated that NT2N neurons derived from a human embryonal carcinoma cell line (NT2) constitutively process the endogenous wild-type beta-amyloid precursor protein (APP) to amyloid beta peptide in an intracellular compartment. These studies indicate that other proteolytic fragments generated by intracellular processing must also be present in these cells. Here we show that the NH2-terminal fragment of APP generated by beta-secretase cleavage (APPbeta) is indeed produced from the endogenous full length APP (APPFL). Pulse-chase studies demonstrated a precursor-product relationship between APPFL and APPbeta as well as intracellular and secreted APPbeta fragments. In addition, trypsin digestion of intact NT2N cells at 4 degrees C did not abolish APPbeta recovered from the cell lysates. Furthermore, the production of intracellular APPbeta from wild-type APP appears to be a unique characteristic of postmitotic neurons, since intracellular APPbeta was not detected in several non-neuronal cell lines. Significantly, production of APPbeta occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15 degrees C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.

Amyloid Precursor Protein Secretases↗
PubMed2002

Presenilin 1 mutations activate gamma 42-secretase but reciprocally inhibit epsilon-secretase cleavage of amyloid precursor protein (APP) and S3-cleavage of notch.

The presenilin 1 (PS1) and presenilin 2 (PS2) proteins are necessary for proteolytic cleavage of the amyloid precursor protein (APP) within its transmembrane domain. One of these cleavage events (termed gamma-secretase) generates the C-terminal end of the Abeta-peptide by proteolysis near residue 710 or 712 of APP(770). Another event (termed gamma-like or epsilon-secretase cleavage) cleaves near residue 721 at approximately 2-5 residues inside the cytoplasmic membrane boundary to generate a series of stable, C-terminal APP fragments. This latter cleavage is analogous to S3-cleavage of Notch. We report here that specific mutations in the N terminus, loop, or C terminus of PS1 all increase the production of Abeta(42) but cause inhibition of both epsilon-secretase cleavage of APP and S3-cleavage of Notch. These data support the hypothesis that epsilon-cleavage of APP and S3-cleavage of Notch are similar events. They also argue that, although both the gamma-site and the epsilon-site cleavage of APP are presenilin-dependent, they are likely to be independent catalytic events.

Amino Acid Sequence↗
PubMed1998

Caveolae, plasma membrane microdomains for alpha-secretase-mediated processing of the amyloid precursor protein.

Caveolae are plasma membrane invaginations where key signaling elements are concentrated. In this report, both biochemical and histochemical analyses demonstrate that the amyloid precursor protein (APP), a source of Abeta amyloid peptide, is enriched within caveolae. Caveolin-1, a principal component of caveolae, is physically associated with APP, and the cytoplasmic domain of APP directly participates in this binding. The characteristic C-terminal fragment that results from APP processing by alpha-secretase, an as yet unidentified enzyme that cleaves APP within the Abeta amyloid sequence, was also localized within these caveolae-enriched fractions. Further analysis by cell surface biotinylation revealed that this cleavage event occurs at the cell surface. Importantly, alpha-secretase processing was significantly promoted by recombinant overexpression of caveolin in intact cells, resulting in increased secretion of the soluble extracellular domain of APP. Conversely, caveolin depletion using antisense oligonucletotides prevented this cleavage event. Our current results indicate that caveolae and caveolins may play a pivotal role in the alpha-secretase-mediated proteolysis of APP in vivo.

Amyloid Precursor Protein Secretases↗
PubMed2006

ADAM10 activation is required for green tea (-)-epigallocatechin-3-gallate-induced alpha-secretase cleavage of amyloid precursor protein.

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.

Alzheimer Disease↗
PubMed2004

Human BACE forms dimers and colocalizes with APP.

Beta-site APP-cleaving enzyme (BACE) is a membrane-bound aspartyl protease with no strict primary preference for cleavage. The molecular mechanisms that link the gamma-secretase multicomponent amyloid precursor protein (APP) processing complex to biochemical properties of BACE generating the N terminus of the amyloid beta-peptide have not, as yet, been identified. We found that in human brain tissue, BACE occurred as a dimer. The overall stability of the BACE homodimer was based on intermolecular interactions that were not affected by high salt, nonionic detergents or reducing conditions. BACE homodimers could only partially be separated even under strong denaturing conditions and revealed dramatic differences in the surface charge distribution compared with the monomer. In contrast, the soluble ectodomain of truncated BACE revealed a seemingly lower avidity to the prototypic aspartate protease inhibitor pepstatin and exclusively occurred in the monomeric form. Immunocytochemical studies colocalized APP and BACE in the plasma membrane of cells expressing endogenous levels of BACE and overexpressing APP. In cells that were cotransfected with APP and a putative active site D289A mutant of BACE, colocalization persisted. Remaining enzyme activity was found to be attributable to the mutant protease. Accordingly, inactivation of the carboxyl-terminal active site motif of BACE without an impairment of overall enzyme activity suggests that the enzyme may act as a dimer. Thus, homodimerization of BACE may help the enzyme to acquire specific mechanisms to associate with its substrates to exert catalytic activity.

Amyloid Precursor Protein Secretases↗
PubMed2000

Regulation of APP cleavage by alpha-, beta- and gamma-secretases.

Proteolytic cleavage of the amyloid protein from the amyloid protein precursor (APP) by APP secretases is a key event in Alzheimer's disease (AD) pathogenesis. alpha-Secretases cleave APP within the amyloid sequences, whereas beta- and gamma-secretases cleave on the N- and C-terminal ends respectively. The transmembrane aspartyl protease BACE has been identified as beta-secretase and several proteases (ADAM-10, TACE, PC7) may be alpha-secretases. A number of studies have suggested that presenilins could be gamma-secretases, although this remains to be demonstrated conclusively. Inhibition of beta- and gamma-secretase, or stimulation of alpha-secretase, is a rational strategy for therapeutic intervention in AD.

Amino Acid Sequence↗
PubMed1994

Identification of the multicatalytic enzyme as a possible gamma-secretase for the amyloid precursor protein.

One of the main components of the senile plaques in brain tissue from patients with Alzheimer's disease is the beta-amyloid peptide. This peptide is proteolytically cleaved from the amyloid precursor protein by the action of at least two proteases, a beta-secretase which generates the N-terminus and a gamma-secretase which generates the C-terminus. Neither of these proteases have been identified. To identify proteases that are candidates for the gamma-secretase we synthesized a small fluorescent peptide substrate containing the amino acids comprising the C-terminus of the longest beta-amyloid peptide identified. This substrate is hydrolyzed by a single activity in homogenates from both cells and brain tissue and we have demonstrated that this activity is the multicatalytic enzyme or proteasome. Furthermore, using specific inhibitors, we have demonstrated that the fluorescent substrate is hydrolyzed by the chymotrypsin-like activity of the multicatalytic enzyme.

Alzheimer Disease↗
PubMed1999

Purification and cloning of amyloid precursor protein beta-secretase from human brain.

Proteolytic processing of the amyloid precursor protein (APP) generates amyloid beta (Abeta) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by beta-secretase at the amino terminus of the Abeta peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of beta-cleaved soluble APP, and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by gamma-secretase(s) leads to the formation of Abeta. The pathogenic mutation K670M671-->N670L671 at the beta-secretase cleavage site in APP, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased beta-secretase cleavage of the mutant substrate. Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the beta-secretase cleavage site, and find it to be the predominant beta-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for beta-secretase. Cloning and expression of the enzyme reveals that human brain beta-secretase is a new membrane-bound aspartic proteinase.

Amino Acid Sequence↗
PubMed1995

Iron levels modulate alpha-secretase cleavage of amyloid precursor protein.

The amyloid precursor protein (APP) is a membrane-spanning glycoprotein that is the source of beta A4 peptides, which aggregate in Alzheimer's disease to form senile plaques. APP is cleaved within the beta A4 sequence to release a soluble N-terminal derivative (APPsol), which has a wide range of trophic and protective functions. In the current study we have examined the hypothesis that iron availability may modulate expression or processing of APP, whose mRNA contains, based on sequence homology, a putative iron response element (IRE). Radiolabeled APP and its catabolites were precipitated from lysates and conditioned medium of stably transfected HEK 293 cells using antibodies selective for C-terminal, beta A4, and N-terminal domains. The relative abundance of the different APP catabolites under different conditions of iron availability was determined by quantitative densitometry after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data show a specific effect on the production of APPsol. Using standard conditions previously established for IRE studies, it was found that iron chelation reduces APPsol production, whereas iron level elevation augments it. No changes were observed in levels of immature and mature APP holoprotein or in the C-terminal alpha-secretase derivative C83, beta A4, and p3 peptides. The specificity for modulatory changes in APPsol suggests that iron acts at the level of alpha-secretase activity. In addition to its modulatory effects, iron at very high levels was found to inhibit maturation of APP and production of its downstream catabolites without blocking formation of immature APP. The data establish a potential physiological role for iron in controlling the processing of APP.(ABSTRACT TRUNCATED AT 250 WORDS)

Amyloid Precursor Protein Secretases↗
PubMed2006

Role of amyloid precursor protein, amyloid-beta and gamma-secretase in cholesterol maintenance.

Lipids play an important part as risk factors for Alzheimer's disease. This article summarizes the current understanding of the molecular mechanism by which amyloid-beta (Abeta) peptides regulate cholesterol and sphingomyelin metabolism, and how in return cholesterol and sphingomyelin regulate Abeta peptide production. An understanding of the physiological function of amyloid precursor protein processing and Abeta function is critical for the development of future therapeutic approaches, e.g. statin treatment.

Alzheimer Disease↗
PubMed2003

ADAMs family members as amyloid precursor protein alpha-secretases.

In the non-amyloidogenic pathway, the Alzheimer's amyloid precursor protein (APP) is cleaved within the amyloid-beta domain by alpha-secretase precluding deposition of intact amyloid-beta peptide. The large ectodomain released from the cell surface by the action of alpha-secretase has several neuroprotective properties. Studies with protease inhibitors have shown that alpha-secretase is a zinc metalloproteinase, and several members of the adamalysin family of proteins, tumour necrosis factor-alpha convertase (TACE, ADAM17), ADAM10, and ADAM9, all fulfil some of the criteria required of alpha-secretase. We review the evidence for each of these ADAMs acting as the alpha-secretase. What seems to be emerging from numerous studies, including those with mice in which each of the ADAMs has been knocked out, is that there is a team of zinc metalloproteinases able to cleave APP at the alpha-secretase site. We also discuss how upregulation of alpha-secretase activity by muscarinic agonists, cholesterol-lowering drugs, steroid hormones, non-steroidal anti-inflammatory drugs, and metal ions may explain some of the therapeutic actions of these agents in Alzheimer's disease.

ADAM Proteins↗
PubMed1996

Intracellular generation of amyloid beta-protein from amyloid beta-protein precursor fragment by direct cleavage with beta- and gamma-secretase.

Two amyloid beta protein precursor (beta APP) fragments involving Met and 103 amino acids of C-terminus of beta APP (delta NOR-beta) and its KM-NL substitution (delta NL-beta) were expressed in COS-7 cells to clarify the proteolytic cleavages to generate amyloid beta protein (A beta). The 4.5-kD protein, A beta with additional N-terminal amino acids, and 4-kD A beta were directly produced and released from 12.5-kD expression proteins without any production of 11.4-kD C-terminal fragment starting at N-terminus of A beta and 3-kD "p3" A beta derivative. Intracellular 4-kD A beta was also detected. The substitution of KM-NL of beta APP found in Swedish familial Alzheimer's disease (AD) promoted the production of intracellular A beta and its release with no increase in level of 11.4-kD C-terminal fragment. These results suggested the presence of a distinct pathway in which A beta is directly cleaved at both N- and C-termini from beta APP fragment intracellularly to release A beta. Since KM-NL substitution enhanced intracellular A beta generation, this pathway may be associated with amyloidogenesis in AD.

Alzheimer Disease↗