Deciphering the mechanism by which the relative Aβ42(43) to total Aβ ratio is regulated is central to understanding Alzheimer disease (AD) etiology; however, the mechanisms underlying changes in the Aβ42(43) ratio caused by familial mutations and γ-secretase modulators (GSMs) are unclear. Here, we show in vitro and in living cells that presenilin (PS)/γ-secretase cleaves Aβ42 into Aβ38, and Aβ43 into Aβ40 or Aβ38. Approximately 40% of Aβ38 is derived from Aβ43. Aβ42(43) cleavage is involved in the regulation of the Aβ42(43) ratio in living cells. GSMs increase the cleavage of PS/γ-secretase-bound Aβ42 (increase k(cat)) and slow its dissociation from the enzyme (decrease k(b)), whereas PS1 mutants and inverse GSMs show the opposite effects. Therefore, we suggest a concept to describe the Aβ42(43) production process and propose how GSMs act, and we suggest that a loss of PS/γ-secretase function to cleave Aβ42(43) may initiate AD and might represent a therapeutic target.
We have previously reported on cloning of the human gene encoding Bcl-2/adenovirus E1B 19 kDa-interacting protein 3-like protein (Bnip3L) and its growth inhibitory eect on cancer cells. Here we show that Bnip3L contains a motif similar to the BH3 domain which is conserved in Bcl-2 family proteins as well as containing a membrane-anchoring domain, and that Bnip3L interacts with Bcl-2 and Bcl-x L . Immuno¯uorescence microscopy revealed that Bnip3L was localized in the mitochondria, when in the presence of the membrane-anchoring domain. Transient expression of Bnip3L induced apoptosis of Rat-1 and HeLa cells and mutational analysis revealed that the BH3 domain and the membrane-anchoring domain were required for Bnip3L to induce cell death. Addition of recombinant Bnip3L to isolated mitochondria induced membrane potential loss and cytochrome c release both of which have been suggested to be prerequisite for apoptotic cell death. These results suggest that Bnip3L is one of the BH3-containing proapoptotic proteins and that it targets the mitochondria when inducing apoptosis.
Intramembrane proteolysis by presenilin-dependent ␥-secretase produces the Notch intracellular cytoplasmic domain (NCID) and Alzheimer disease-associated amyloid-. Here, we show that upon Notch signaling the intracellular domain of Notch-1 is cleaved into two distinct types of NICD species due to diversity in the site of S3 cleavage. Consistent with the N-end rule, the S3-V cleavage produces stable NICD with Val at the N terminus, whereas the S3-S/S3-L cleavage generates unstable NICD with Ser/Leu at the N terminus. Moreover, intracellular Notch signal transmission with unstable NICDs is much weaker than that with stable NICD. Importantly, the extent of endocytosis in target cells affects the relative production ratio of the two types of NICD, which changes in parallel with Notch signaling. Surprisingly, substantial amounts of unstable NICD species are generated from the Val3Gly and the Lys3Arg mutants, which have been reported to decrease S3 cleavage efficiency in cultured cells. Thus, we suggest that the existence of two distinct types of NICD points to a novel aspect of the intracellular signaling and that changes in the precision of S3 cleavage play an important role in the process of conversion from extracellular to intracellular Notch signaling.
The canonical pathway of Notch signaling is mediated by regulated intramembrane proteolysis (RIP). In the pathway, ligand binding results in sequential proteolysis of the Notch receptor, and presenilin (PS)-dependent intramembrane proteolysis at the interface between the membrane and cytosol liberates the Notch-1 intracellular domain (NICD), a transcription modifier. Because the degradation of the Notch-1 transmembrane domain is thought to require an additional cleavage near the middle of the transmembrane domain, extracellular small peptides (Notch-1 A-like peptide (N)) should be produced. Here we showed that N species are indeed secreted during the process of Notch signaling. We identified mainly two distinct molecular species of novel N, N21 and C-terminally elongated N25, which were produced in an ϳ5:1 ratio. This process is reminiscent of the production of Alzheimer disease-associated A. PS pathogenic mutants increased the production of the longer species of A (A42) from -amyloid protein precursor. We revealed that several Alzheimer disease mutants also cause a parallel increase in the secretion of the longer form of N. Strikingly, chemicals that modify the A42 level caused parallel changes in the N25 level. These results demonstrated that the characteristics of C-terminal elongation of N and A are almost identical. In addition, because many other type 1 membrane-bound receptors release intracellular domains by PS-dependent intramembrane proteolysis, we suspect that the release of A-or N-like peptides is a common feature of the proteolysis during RIP signaling. We anticipate that this study will open the door to searches for markers of RIP signaling and surrogate markers for A42 production.
Alzheimer´s disease (AD) is a devastating neurodegenerative disease of the elderly population. Genetic evidence strongly suggests that aberrant generation and/or clearance of the neurotoxic amyloid-β peptide (Aβ) is triggering the disease. Aβ is generated from the amyloid precursor protein (APP) by the sequential cleavages of β- and γ-secretase. The latter cleavage by γ-secretase, a unique and fascinating four-component protease complex, occurs in the APP transmembrane domain thereby releasing Aβ species of 37-43 amino acids in length including the longer, highly pathogenic peptides Aβ42 and Aβ43. The lack of a precise understanding of Aβ generation as well as of the functions of other γ-secretase substrates has been one factor underlying the disappointing failure of γ-secretase inhibitors in clinical trials, but on the other side also been a major driving force for structural and in depth mechanistic studies on this key AD drug target in the past few years. Here we review recent breakthroughs in our understanding of how the γ-secretase complex recognizes substrates, of how it binds and processes β-secretase cleaved APP into different Aβ species, as well as the progress made on a question of outstanding interest, namely how clinical AD mutations in the catalytic subunit presenilin and the γ-secretase cleavage region of APP lead to relative increases of Aβ42/43. Finally, we discuss how the knowledge emerging from these studies could be used to therapeutically target this enzyme in a safe way.
Surrogate markers for the Alzheimer disease (AD)-associated 42-amino acid form of amyloid-β (Aβ42) have been sought because they may aid in the diagnosis of AD and for clarification of disease pathogenesis. Here, we demonstrate that human cerebrospinal fluid (CSF) contains three APLP1-derived Aβ-like peptides (APL1β) that are generated by β- and γ-cleavages at a concentration of ∼4.5 nM. These novel peptides, APL1β25, APL1β27 and APL1β28, were not deposited in AD brains. Interestingly, most γ-secretase modulators (GSMs) and familial AD-associated presenilin1 mutants that up-regulate the relative production of Aβ42 cause a parallel increase in the production of APL1β28 in cultured cells. Moreover, in CSF from patients with pathological mutations in presenilin1 gene, the relative APL1β28 levels are higher than in non-AD controls, while the relative Aβ42 levels are unchanged or lower. Most strikingly, the relative APL1β28 levels are higher in CSF from sporadic AD patients (regardless of whether they are at mild cognitive impairment or AD stage), than those of non-AD controls. Based on these results, we propose the relative level of APL1β28 in the CSF as a candidate surrogate marker for the relative level of Aβ42 production in the brain.
The presenilin (PS)/gamma-secretase complex, which contains not only PS but also Aph-1, PEN-2, and nicastrin, mediates proteolysis of the transmembrane domain of beta-amyloid protein precursor (betaAPP). Intramembrane proteolysis occurs at the interface between the membrane and cytosol (epsilon-site) and near the middle of the transmembrane domain (gamma-site), generating the betaAPP intracellular domain (AICD) and Alzheimer disease-associated Abeta, respectively. Both cleavage sites exhibit some diversity. Changes in the precision of gamma-cleavage, which potentially results in secretion of pathogenic Abeta42, have been intensively studied, while those of epsilon-cleavage have not. Although a number of PS-associated factors have been identified, it is unclear whether any of them physiologically regulate the precision of cleavage by PS/gamma-secretase. Moreover, there is currently no clear evidence of whether PS/gamma-secretase function differs according to the subcellular site. Here, we show that endocytosis affects the precision of PS-dependent epsilon-cleavage in cell culture. Relative production of longer AICDepsilon49 increases on the plasma membrane, whereas that of shorter AICDepsilon51 increases on endosomes; however, this occurs without a concomitant major change in the precision of cleavage at gamma-sites. Moreover, very similar changes in the precision of epsilon-cleavage are induced by alteration of the pH. Our findings demonstrate that the precision of epsilon-cleavage by PS/gamma-secretase changes depending upon the conditions and the subcellular location. These results suggest that the precision of cleavage by the PS/gamma-secretase complex may be physiologically regulated by the subcellular location and conditions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.