Identification of a Novel Aspartic Protease (Asp 2) as β-Secretase

doi:10.1006/mcne.1999.0811

Molecular and Cellular Neuroscience

Volume 14, Issue 6, December 1999, Pages 419-427

https://doi.org/10.1006/mcne.1999.0811 Get rights and content

Abstract

The Alzheimer's disease β-amyloid peptide (Aβ) is produced by excision from the type 1 integral membrane glycoprotein amyloid precursor protein (APP) by the sequential actions of β- and then γ-secretases. Here we report that Asp 2, a novel transmembrane aspartic protease, has the key activities expected of β-secretase. Transient expression of Asp 2 in cells expressing APP causes an increase in the secretion of the N-terminal fragment of APP and an increase in the cell-associated C-terminal β-secretase APP fragment. Mutation of either of the putative catalytic aspartyl residues in Asp 2 abrogates the production of the fragments characteristic of cleavage at the β-secretase site. The enzyme is present in normal and Alzheimer's disease (AD) brain and is also found in cell lines known to produce Aβ. Asp 2 localizes to the Golgi/endoplasmic reticulum in transfected cells and shows clear colocalization with APP in cells stably expressing the 751-amino-acid isoform of APP.

References (26)

N. Chevallier et al.
Cathepsin D displays in vitro β-secretase-like specificity
Brain. Res.
(1997)
G.G. Glenner et al.
Alzheimer's disease: Initial report of the purification and characterisation of a novel cerebrovascular amyloid protein
Biochem. Biophys. Res. Commun.
(1984)
A.B. Huber et al.
Metalloprotease MP100: A synaptic protease in rat brain
Brain Res.
(1999)
N. Ida et al.
Analysis of heterogeneous βA4 peptides in human cerebrospinal fluid and blood by a newly developed sensitive Western blot assay
J. Biol. Chem.
(1996)
S. Ishiura et al.
Putative N-terminal splitting enzyme of amyloid A4 peptides is the multicatalytic proteinase, ingensin, which is widely distributed in mammalian cells
FEBS Lett.
(1989)
U.S. Ladror et al.
Cleavage at the amino and carboxyl termini of Alzheimer's amyloid-β by cathepsin D
J. Biol. Chem.
(1994)
J.R. McDermott et al.
Human brain peptidase activity with the specificity to generate the N-terminus of the Alzheimer β-amyloid protein from its precursor
Biochem. Biophys. Res. Commun.
(1992)
S.R. Sahasrabudhe et al.
Enzymatic generation of the amino terminus of the β-amyloid peptide
J. Biol. Chem.
(1993)
M.J. Savage et al.
Cathepsin G: Localisation in human cerebral cortex and generation of amyloidogenic fragments from the β-amyloid precursor protein
Neuroscience
(1994)
A. Thompson et al.
Expression and characterisation of human β-secretase candidates metalloendopeptidase MP78 and cathepsin D in βAPP-overexpressing cells
Mol. Brain Res.
(1997)

C. Wild-Bode et al.

Intracellular generation and accumulation of amyloid β-peptide terminating at amino acid 42

J. Biol. Chem.

(1997)

D. Allsop et al.

Cited by (1017)

Bioassay-guided isolation of BACE1 inhibitors from Crataegus pinnatifida
2023, Phytomedicine Plus
Crataegus pinnatifida, functional food has been used in traditional Chinese medicine due to its significant pharmacological effects on various metabolic disorders, neurodegenerative diseases, as well as anti-cancer and anti-viral properties. Despite numerous studies reporting its anti-Alzheimer's disease (AD) activity, there is currently no research on the potential of its extract or constituents to inhibit β-site amyloid precursor protein cleaving enzyme 1 (BACE1).
The study aimed to evaluate the inhibitory effects of the methanolic extract of C. pinnatifida and its solvent-soluble fractions on cholinesterase and β-site amyloid precursor protein cleaving enzyme 1 (BACE1). Following a bioassay-guided isolation, we identified several compounds, including two triterpenoids [corosolic acid (1), euscaphic acid (2)], β-sitosterol glucoside (3), three flavonoids [astragalin (4), hyperoside (5), isovitexin (6)], and four phenolic acid derivatives [gentisic acid (7), protocatechuic acid (8), p-hydroxybenzoic acid (9), p-coumaric acid (10)] from EtOAc fraction. Additionally, two triterpenoids (3-epicorosolic acid (11) and ursolic acid (12)) and β-sitosterol (13) were isolated from the CH2Cl2 fraction, while isoorientin (14) and hymenoside X (15) were isolated from n-BuOH fraction. Notably, compounds 2, 7, 10, and 14 were reported for the first time from the fruit of C. pinnatifida.
We observed that 3-epicorosolic acid (11) exhibited promising inhibitory activity against BACE1 with IC₅₀ values of 40.12 ± 1.72 µM. Hyperoside (5) showed moderate activity with IC₅₀ values of 98.50 ± 4.20 µM, while corosolic acid (1) and astragalin (4) displayed mild BACE1 inhibitory activity, with IC₅₀ values of 179.55 ± 1.57 and 192.30 ± 5.14 µM, respectively. We also investigated the structure activity relationship of ursane-type triterpenoids based on in vitro and in silico results, which revealed that the hydroxyl groups position of the triterpenes have a significant impact on their potency. Our findings suggest that α‑hydroxyl group at the C-2 and C-3 positions and the absence of α‑hydroxyl group at the C-19 position are essential for BACE1 inhibitory activity.
This study highlights the significance of the configuration and position of hydroxyl groups and their impact on BACE1 inhibitory activity. The findings of this study could be leveraged to develop novel therapeutic approaches for Alzheimer's disease.
Amyloid-ß plaque formation and BACE1 accumulation in the brains of a 5xFAD Alzheimer's disease mouse model is associated with altered distribution and not proteolysis of BACE1 substrates Sez6 and Sez6L
2022, Mechanisms of Ageing and Development
The formation of amyloid-ß peptides (Aß), that accumulate in Alzheimer’s disease (AD) brains, involves proteolytic processing of the amyloid precursor protein (APP) firstly by ß-secretase (BACE1). Since BACE1 cleaves a plethora of other substrates, in this work we investigated whether the proteolysis and/or distribution of other BACE1 substrates, such as seizure protein 6 (Sez6) and seizure 6-like protein (Sez6L), is altered in AD. To test this we used 5xFAD mouse model brains that show an early accumulation of Aß plaques already at 2-months of age. Here we show for the first time that accumulation of BACE1 in peri-plaque regions and its enhanced levels in AD brains does not affect proteolysis of BACE1 substrates other than APP, such as Sez6 and Sez6L. We observed altered distribution of Sez6 and Sez6L in the area of Aß plaques in 5xFAD brains which is distinct to that of APP, BACE1 and/or LAMP1, suggesting different localization and/or function of these BACE1 substrates. While it is necessary to further elucidate the potential role that this may play in the course of AD, it is likely that Aß-targeted therapies may have beneficial effects against accumulation and/or altered distribution of BACE1 and its substrates, in addition to APP.
Protein interaction networks in neurodegenerative diseases: From physiological function to aggregation
2022, Journal of Biological Chemistry
The accumulation of protein inclusions is linked to many neurodegenerative diseases that typically develop in older individuals, due to a combination of genetic and environmental factors. In rare familial neurodegenerative disorders, genes encoding for aggregation-prone proteins are often mutated. While the underlying mechanism leading to these diseases still remains to be fully elucidated, efforts in the past 20 years revealed a vast network of protein–protein interactions that play a major role in regulating the aggregation of key proteins associated with neurodegeneration. Misfolded proteins that can oligomerize and form insoluble aggregates associate with molecular chaperones and other elements of the proteolytic machineries that are the frontline workers attempting to protect the cells by promoting clearance and preventing aggregation. Proteins that are normally bound to aggregation-prone proteins can become sequestered and mislocalized in protein inclusions, leading to their loss of function. In contrast, mutations, posttranslational modifications, or misfolding of aggregation-prone proteins can lead to gain of function by inducing novel or altered protein interactions, which in turn can impact numerous essential cellular processes and organelles, such as vesicle trafficking and the mitochondria. This review examines our current knowledge of protein–protein interactions involving several key aggregation-prone proteins that are associated with Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, or amyotrophic lateral sclerosis. We aim to provide an overview of the protein interaction networks that play a central role in driving or mitigating inclusion formation, while highlighting some of the key proteomic studies that helped to uncover the extent of these networks.
Cleavage of Kv2.1 by BACE1 decreases potassium current and reduces neuronal apoptosis
2022, Neurochemistry International
As an aspartic protease, β-site APP cleaving enzyme 1 (BACE1) can efficiently cleave amyloid precursor protein (APP) to produce amyloid beta (Aβ), a chief constituent of senile plaques in Alzheimer's disease. Thus, BACE1 inhibitor is identified as a therapeutic candidate for AD. However, recent failures of clinical trials using BACE1 inhibitors emphasized that comprehensively understanding of BACE1 function is particularly important. Kv2.1, a potassium channel, modulates potassium current in cortical neurons and potassium efflux is a requisite event in the process of cell apoptosis. Previously we showed that BACE2 cleaves Kv2.1 and reduces neuronal apoptosis. Our study here showed that BACE1 cleaves Kv2.1, and results in decreased I_k of Kv2.1. Furthermore, we demonstrated that the BACE1-cleaved Kv2.1 reduces neuronal apoptosis and BACE1 inhibitor markedly increases neuronal apoptosis. Our work indicates that BACE1 plays a neuroprotective role to reduce potassium efflux by cleavage of Kv2.1, implying inhibition of BACE1 may be neurotoxic.
The Swedish dilemma - the almost exclusive use of APPswe-based mouse models impedes adequate evaluation of alternative β-secretases
2022, Biochimica et Biophysica Acta - Molecular Cell Research
Citation Excerpt :
The first identified β-secretase, which is in the field considered as the prevalent one, is β-site APP cleaving enzyme 1 (BACE1) [19,22,23,28]. BACE1 is a type I membrane-bound aspartyl protease of the pepsin family [20]. The highest expression levels of BACE1 are found in the brain, mainly in neurons, however, it also shows almost ubiquitous expression in peripheral tissues [20,21,23,29].
Alzheimer's disease (AD) is the most common form of dementia, however incurable so far. It is widely accepted that aggregated amyloid β (Aβ) peptides play a crucial role for the pathogenesis of AD, as they cause neurotoxicity and deposit as so-called Aβ plaques in AD patient brains. Aβ peptides derive from the amyloid precursor protein (APP) upon consecutive cleavage at the β- and γ-secretase site. Hence, mutations in the APP gene are often associated with autosomal dominant inherited AD. Almost thirty years ago, two mutations at the β-secretase site were observed in two Swedish families (termed Swedish APP (APPswe) mutations), which led to early-onset AD. Consequently, APPswe was established in almost every common AD mouse model, as it contributes to early Aβ plaque formation and cognitive impairments. Analyzing these APPswe-based mouse models, the aspartyl protease BACE1 has been evolving as the prominent β-secretase responsible for Aβ release in AD and as the most important therapeutic target for AD treatment. However, with respect to β-secretase processing, the very rare occurring APPswe variant substantially differs from wild-type APP. BACE1 dominates APPswe processing resulting in the release of Aβ1-x, whereas N-terminally truncated Aβ forms are scarcely generated. However, these N-terminally truncated Aβ species such as Aβ2-x, Aβ3-x and Aβ4-x are elevated in AD patient brains and exhibit an increased potential to aggregate compared to Aβ1-x peptides. Proteases such as meprin β, cathepsin B and ADAMTS4 were identified as alternative β-secretases being capable of generating these N-terminally truncated Aβ species from wild-type APP. However, neither meprin β nor cathepsin B are capable of generating N-terminally truncated Aβ peptides from APPswe. Hence, the role of BACE1 for the Aβ formation during AD might be overrepresented through the excessive use of APPswe mouse models. In this review we critically discuss the consideration of BACE1 as the most promising therapeutic target. Shifting the focus of AD research towards alternative β secretases might unveil promising alternatives to BACE1 inhibitors constantly failing in clinical trials due to ineffectiveness and harmful side effects.
Aspartic Proteases of Alzheimer's Disease: β- and γ--Secretases
2022, Encyclopedia of Cell Biology: Volume 1-6, Second Edition
Two aspartic proteases, β- and γ-secretases, sequentially cut the amyloid β-protein precursor (APP) to produce the amyloid β-peptide of Alzheimer’s disease (AD). β-Secretase is membrane tethered and sheds the APP ectodomain, while γ-secretase is a membrane-embedded complex that cleaves within the transmembrane domain of APP. These proteases are important therapeutic targets for AD. However, both enzymes have other substrates besides APP, and blocking some of these processes can have toxic consequences. The role of these proteases in AD, their mechanisms and regulation, and the prospects of developing safe inhibitors or modulators will be discussed.

View all citing articles on Scopus

¹: Joint first authors.

²: To whom correspondence should be addressed. E-mail: [email protected].

View full text

Article preview

Molecular and Cellular Neuroscience

Abstract

References (26)

Cathepsin D displays in vitro β-secretase-like specificity

Brain. Res.

Alzheimer's disease: Initial report of the purification and characterisation of a novel cerebrovascular amyloid protein

Biochem. Biophys. Res. Commun.

Metalloprotease MP100: A synaptic protease in rat brain

Brain Res.

Analysis of heterogeneous βA4 peptides in human cerebrospinal fluid and blood by a newly developed sensitive Western blot assay

J. Biol. Chem.

Putative N-terminal splitting enzyme of amyloid A4 peptides is the multicatalytic proteinase, ingensin, which is widely distributed in mammalian cells

FEBS Lett.

Cleavage at the amino and carboxyl termini of Alzheimer's amyloid-β by cathepsin D

J. Biol. Chem.

Human brain peptidase activity with the specificity to generate the N-terminus of the Alzheimer β-amyloid protein from its precursor

Biochem. Biophys. Res. Commun.

Enzymatic generation of the amino terminus of the β-amyloid peptide

J. Biol. Chem.

Cathepsin G: Localisation in human cerebral cortex and generation of amyloidogenic fragments from the β-amyloid precursor protein

Neuroscience

Expression and characterisation of human β-secretase candidates metalloendopeptidase MP78 and cathepsin D in βAPP-overexpressing cells

Mol. Brain Res.

Intracellular generation and accumulation of amyloid β-peptide terminating at amino acid 42

J. Biol. Chem.

Cited by (1017)

Bioassay-guided isolation of BACE1 inhibitors from Crataegus pinnatifida

Amyloid-ß plaque formation and BACE1 accumulation in the brains of a 5xFAD Alzheimer's disease mouse model is associated with altered distribution and not proteolysis of BACE1 substrates Sez6 and Sez6L

Protein interaction networks in neurodegenerative diseases: From physiological function to aggregation

Cleavage of Kv2.1 by BACE1 decreases potassium current and reduces neuronal apoptosis

The Swedish dilemma - the almost exclusive use of APPswe-based mouse models impedes adequate evaluation of alternative β-secretases

Aspartic Proteases of Alzheimer's Disease: β- and γ--Secretases

Regular ArticleIdentification of a Novel Aspartic Protease (Asp 2) as β-Secretase

Abstract

Brain. Res.

Biochem. Biophys. Res. Commun.

Brain Res.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Neuroscience

Mol. Brain Res.

J. Biol. Chem.

Regular Article
Identification of a Novel Aspartic Protease (Asp 2) as β-Secretase