Presenilin Proteins Undergo Heterogeneous Endoproteolysis between Thr291and Ala299and Occur as Stable N- and C-Terminal Fragments in Normal and Alzheimer Brain Tissue

doi:10.1006/nbdi.1997.0129

Neurobiology of Disease

Volume 3, Issue 4, 1997, Pages 325-337

https://doi.org/10.1006/nbdi.1997.0129 Get rights and content

Abstract

Humans inheriting missense mutations in thepresenilin(PS)1 and -2 genes undergo progressive cerebral deposition of the amyloid β-protein at an early age and develop a clinically and pathologically severe form of familial Alzheimer's disease (FAD). Because PS1 mutations cause the most aggressive known form of AD, it is important to elucidate the structure and function of this multitransmembrane protein in the brain. Using a panel of region-specific PS antibodies, we characterized the presenilin polypeptides in mammalian tissues, including brains of normal, AD, and PS1-linked FAD subjects, and in transfected and nontransfected cell lines. Very little full-length PS1 or -2 was detected in brain and untransfected cells; instead the protein occurred as a heterogeneous array of stable N- and C-terminal proteolytic fragments that differed subtly among cell types and mammalian tissues. Sequencing of the major C-terminal fragment from PS1-transfected human 293 cells showed that the principal endoproteolytic cleavage occurs at and near Met₂₉₈in the proximal portion of the large hydrophilic loop. Full-length PS1 in these cells is quickly turned over (T_1/2 ≈ 60 min), in part to the two major fragments. The sizes and amounts of the PS fragments were not significantly altered in four FAD brains with the Cys410Tyr PS1 missense mutation. Our results indicate that presenilins are rapidly processed to N- and C-terminal fragments in both neural and nonneural cells and that interference with this processing is not an obligatory feature of FAD-causing mutations.

References (27)

A. Doan et al.
Protein topology of presenilin 1
Neuron
(1996)
C.A. Lemere et al.
Sequence of deposition of heterogeneous amyloid β-peptides and Apo E in Down syndrome: Implications for initial events in amyloid plaque formation
Neurobiol. Disease
(1996)
M. Mercken et al.
Characterization of human presenilin 1 using N-terminal specific monoclonal antibodies: Evidence that Alzheimer mutations affect proteolytic processing
FEBS Lett.
(1996)
M.B. Podlisny et al.
Aggregation of secreted amyloid β-protein into SDS-stable oligomers in cell culture
J. Biol. Chem.
(1995)
T. Suzuki et al.
Regional and cellular presenilin 1 gene expression in human and rat tissues
Biochem. Biophys. Res. Commun.
(1996)
R.E. Tanzi et al.
The gene defects responsible for familial Alzheimer's disease
Neurobiol. Dis.
(1996)
G. Thinakaran et al.
Endoprotreolysis of presenilin 1 and accumulation of processed derivatives in vivo
Neuron
(1996)
D.H. Cribbs et al.
Widespread neuronal expression of the presenilin-1 early onset Alzheimer's disease gene in the murine brain
Am. J. Pathol.
(1996)
A. Goate et al.
Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease
Nature
(1991)
C. Haass et al.
Amyloid β-peptide is produced by cultured cells during normal metabolism
Nature
(1992)

E. Harlow et al.

Antibodies: A Laboratory Manual

(1988)

D.M. Hartley et al.

Glutamate receptor-induced 45Ca²⁺

J. Neurosci.

(1993)

T. Iwatsubo et al.

Amyloid β protein (Aβ) deposition: Aβ42(43) precedes Aβ40 in Down syndrome

Ann. Neurol.

(1995)

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The γ-secretase complex catalyzes the intramembrane cleavage of C99, a carboxy-terminal fragment of the amyloid precursor protein. Two paralogs of its catalytic subunit presenilin (PS1 and PS2) are expressed which are autocatalytically cleaved into an N-terminal and a C-terminal fragment during maturation of γ-secretase. In this study, we compared the efficiency and specificity of C99 cleavage by PS1- and PS2-containing γ-secretases. Mass spectrometric analysis of cleavage products obtained in cell-free and cell-based assays revealed that the previously described lower amyloid-β (Aβ)38 generation by PS2 is accompanied by a reciprocal increase in Aβ37 production. We further found PS1 and PS2 to show different preferences in the choice of the initial cleavage site of C99. However, the differences in Aβ38 and Aβ37 generation appear to mainly result from altered subsequent stepwise cleavage of Aβ peptides. Apart from these differences in cleavage specificity, we confirmed a lower efficiency of initial C99 cleavage by PS2 using a detergent-solubilized γ-secretase system. By investigating chimeric PS1/2 molecules, we show that the membrane-embedded, nonconserved residues of the N-terminal fragment mainly account for the differential cleavage efficiency and specificity of both presenilins. At the level of individual transmembrane domains (TMDs), TMD3 was identified as a major modulator of initial cleavage site specificity. The efficiency of endoproteolysis strongly depends on nonconserved TMD6 residues at the interface to TMD2, i.e., at a putative gate of substrate entry. Taken together, our results highlight the role of individual presenilin TMDs in the cleavage of C99 and the generation of Aβ peptides.
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Alzheimer’s disease (AD) stands out as a major disease without any form of preventative or disease modifying therapy. This is not for lack of trying. 33 phase 3 clinical trials of drugs targeting amyloid beta (Aβ) have failed to slow cognitive decline in AD. The field is at a cross-roads about whether to continue anti-Aβ therapy or more actively pursue alternative targets. With the burden of this disease to patients, families, and healthcare budgets growing yearly, the need for disease modifying AD therapies has become one of the highest priorities in all of medicine. While pathology, genetic and biochemical data offer a popular narrative for the causative role of Aβ, there are alternative explanations, and dissenting findings that, now more than ever, warrant thorough reanalysis. This review questions the major assumptions about Aβ on which therapies for AD were premised, and invites renewed interrogation into AD pathogenesis.
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The rising prevalence of Alzheimer’s disease (AD), together with the lack of effective treatments, portray it as one of the major health challenges of our times. Untangling AD implies advancing the knowledge of the biology that gets disrupted during the disease while deciphering the molecular and cellular mechanisms leading to AD-related neurodegeneration. In fact, a solid mechanistic understanding of the disease processes stands as an essential prerequisite for the development of safe and effective treatments.
Genetics has provided invaluable clues to the genesis of the disease by revealing deterministic genes - Presenilins (PSENs) and the Amyloid Precursor Protein (APP) - that, when affected, lead in an autosomal dominant manner to early-onset, familial AD (FAD). PSEN is the catalytic subunit of the membrane-embedded γ-secretase complexes, which act as proteolytic switches regulating key cell signalling cascades. Importantly, these intramembrane proteases are responsible for the production of Amyloid β (Aβ) peptides from APP. The convergence of pathogenic mutations on one functional pathway, the amyloidogenic cleavage of APP, strongly supports the significance of this process in AD pathogenesis.
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Citation Excerpt :
NICD release and translocation was soon after found to be presenilin-dependent [39,40]. Further clues to the biochemical function of presenilin were provided by the findings that presenilin itself undergoes endoproteolytic processing into an N-terminal fragment (NTF) and C-terminal fragment (CTF), that the formation of these fragments is tightly regulated by limiting cellular factors, and that NTF and CTF are stable, remain associated, and assemble into a high-molecular-weight complex [41–48]. These findings together suggested that the presenilin holoprotein is a precursor and that the functional form of presenilin is the associated NTF and CTF.
γ-Secretase was initially defined as a proteolytic activity that cleaves within the transmembrane of the amyloid precursor protein (APP) to produce the amyloid β-peptide of Alzheimer’s disease. The discovery of mutations in APP and the presenilins associated with familial Alzheimer’s disease and their effects on APP processing dovetailed with pharmacological studies on γ-secretase, leading to the revelation that presenilins are unprecedented membrane-embedded aspartyl proteases. Other members of what became known as the γ-secretase complex were subsequently identified. In parallel with these advances, connections between presenilins and Notch receptors essential to metazoan development became evident, resulting in the concurrent realization that γ-secretase also carries out intramembrane proteolysis of Notch as part of its signaling mechanism. Substantial progress has been made toward elucidating how γ-secretase carries out complex processing of transmembrane domains, how it goes awry in familial Alzheimer’s disease, the scope of its substrates, and the atomic details of its structure. Critical questions remain for future study, toward further unraveling the complexity of this unique membrane-embedded proteolytic machine and its roles in biology and disease.

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Regular ArticlePresenilin Proteins Undergo Heterogeneous Endoproteolysis between Thr291and Ala299and Occur as Stable N- and C-Terminal Fragments in Normal and Alzheimer Brain Tissue☆

Abstract

Neuron

Neurobiol. Disease

FEBS Lett.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Neurobiol. Dis.

Neuron

Widespread neuronal expression of the presenilin-1 early onset Alzheimer's disease gene in the murine brain

Am. J. Pathol.

Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease

Nature

Amyloid β-peptide is produced by cultured cells during normal metabolism