␥-Secretase is a multisubunit protease complex that is responsible for generating amyloid- peptides, which are associated with Alzheimer disease. The catalytic subunit of ␥-secretase is presenilin 1 (PS1), which contains an initial substrate-binding site that is distinct from the catalytic site. Processive cleavage is suggested in the intramembrane-cleaving mechanism of ␥-secretase. However, it largely remains unknown as to how ␥-secretase recognizes its substrate during proteolysis. Here, we identified that the ␣-helical structural region of hydrophilic loop 1 (HL1) and the C-terminal region of human PS1 are distinct substrate-binding sites. Mutational analyses revealed that substrate binding to the HL1 region is critical for both -and ␥-cleavage, whereas binding to the C-terminal region hampers ␥-cleavage. Moreover, we propose that substrate binding triggers conformational changes in PS1, rendering it suitable for catalysis. Our data provide new insights into the complicated catalytic mechanism of PS1.
γ-Secretase is an intramembrane cleaving protease that is responsible for the generation of amyloid-β peptides, which are linked to the pathogenesis of Alzheimer disease. Recently, γ-secretase modulators (GSMs) have been shown to specifically decrease production of the aggregation-prone and toxic longer Aβ species, and concomitantly increase the levels of shorter Aβ. We previously found that phenylimidazole-type GSMs bind to presenilin 1 (PS1), the catalytic subunit of the γ-secretase, and allosterically modulate γ-secretase activity. However, the precise conformational alterations in PS1 remained unclear. Here we mapped the amino acid residues in PS1 that is crucial for the binding and pharmacological actions of E2012, a phenylimidazole-type GSM, using photoaffinity labeling and the substituted cysteine accessibility method. We also demonstrated that a piston-like vertical motion of transmembrane domain (TMD) 1 occurs during modulation of Aβ production. Taking these results together, we propose a model for the molecular mechanism of phenylimidazole-type GSMs, in which the trimming activity of γ-secretase is modulated by the position of the TMD1 of PS1 in the lipid bilayer. Reduction of the toxic longer amyloid-β peptide is one of the therapeutic approaches for Alzheimer disease. A subset of small compounds called γ-secretase modulators specifically decreases the longer amyloid-β production, although its mechanistic action remains unclear. Here we found that the modulator compound E2012 targets to the hydrophilic loop 1 of presenilin 1, which is a catalytic subunit of the γ-secretase. Moreover, E2012 triggers the piston movement of the transmembrane domain 1 of presenilin 1, which impacts on the γ-secretase activity. These results illuminate how γ-secretase modulators allosterically affect the proteolytic activity, and highlight the importance of the structural dynamics of presenilin 1 in the complexed process of the intramembrane cleavage.
Mutations in the presenilin genes (PS1, PS2) have been linked to the majority of familial Alzheimer's disease (AD). Although great efforts have been made to investigate pathogenic PS mutations, which ultimately cause an increase in the toxic form of β-amyloid (Aβ), the intrinsic physiological functions of PS in human neurons remain to be determined. In this study, to investigate the physiological roles of PS in human neurons, we generated PS1 conditional knockout induced pluripotent stem cells (iPSCs), in which PS1 can be selectively abrogated under Cre transduction with or without additional PS2 knockout. We showed that iPSC-derived neural progenitor cells do not confer a maintenance ability in the absence of both PS1 and PS2, showing the essential role of PS in Notch signaling. We then generated PS-null human cortical neurons, where PS1 was intact until full neuronal differentiation occurred. Aβ40 production was reduced exclusively in human PS1/PS2-null neurons along with a concomitant accumulation of APP-CTFs, whereas Aβ42 was decreased in neurons devoid of PS2. Unlike previous studies in mice, in which APP cleavage is largely attributable to PS1, γ-secretase activity seemed to be comparable between PS1 and PS2. In contrast, cleavage of another substrate, N-cadherin, was impaired only in neurons devoid of PS1. Moreover, PS2/γ-secretase exists largely in late endosomes/lysosomes, as measured by specific antibody against the γ-secretase complex, in which Aβ42 species are supposedly produced. Using this novel stem cell-based platform, we assessed important physiological PS1/PS2 functions in mature human neurons, the dysfunction of which could underlie AD pathogenesis. Significance Statement Presenilins are crucial catalytic subunits of γ-secretase, an intramembranous protease complex, whose mutations underlie AD pathogenesis via the dysregulation of Aβ generation. The γ-secretase complex exhibits heterogeneity via the assembly of PS1 or PS2, but the correlation of γ-secretase heterogeneity with substrate processing remains to be established in human neurons. Here, using a novel iPSC-derived cellular model carrying PS1 and/or PS2 conditional knockout alleles, we uncovered the unique processing of three substrates, Notch, APP and N-cadherin, by PS1 or PS2 in human neural cell contexts. Furthermore, the intrinsic subcellular localization of γ-secretase depends on PS1 or PS2, leading to putative differences in the processing of substrates. This novel 3 platform will help ensure the correlation of γ-secretase/substrates in human neurons.
␥-Secretase is an intramembrane-cleaving protease that produces amyloid- peptide 42 (A 42 ), which is the toxic and aggregation-prone species of A that causes Alzheimer's disease. Here, we used the substituted cysteine accessibility method to analyze the structure of transmembrane domains (TMDs) 4 and 5 of human presenilin 1 (PS1), a catalytic subunit of ␥-secretase. We revealed that TMD4 and TMD5 face the intramembranous hydrophilic milieu together with TMD1, TMD6, TMD7, and TMD9 of PS1 to form the catalytic pore structure. Notably, we found a correlation in the distance between the cytosolic sides of TMD4/TMD7 and A 42 production levels, suggesting that allosteric conformational changes of the cytosolic side of TMD4 affect A 42 -generating ␥-secretase activity. Our results provide new insights into the relationship between the structure and activity of human PS1.
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