PS1 N- and C-terminal fragments form a complex that functions in APP processing and Notch signaling

Levitan, Diane; Lee, Julie; Song, Lixin; Manning, Ron; Wong, Gwen; Parker, Eric M.; Zhang, Lili

doi:10.1073/pnas.211321898

Cited by 76 publications

(66 citation statements)

References 35 publications

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“…2E). These data are consistent with the previous reports (12,33,34) on the in vitro ␥-secretase assays using different types of recombinant C100 with or without C-terminal tags.…”

Section: Resultssupporting

confidence: 93%

Sulindac Sulfide Is a Noncompetitive γ-Secretase Inhibitor That Preferentially Reduces Aβ42 Generation

Takahashi

Hayashi

Tominari

et al. 2003

Journal of Biological Chemistry

185

189

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Nonsteroidal anti-inflammatory drugs (NSAIDs) have been known to reduce risk for Alzheimer's disease. In addition to the anti-inflammatory effects of NSAIDs to block cylooxygenase, it has been shown recently that a subset of NSAIDs selectively inhibits the secretion of highly amyloidogenic A␤42 from cultured cells, although the molecular target(s) of NSAIDs in reducing the activity of ␥-secretase for A␤42 generation (␥ 42 -secretase) still remain unknown. Here we show that sulindac sulfide (SSide) directly acts on ␥-secretase and preferentially inhibits the ␥ 42 -secretase activity derived from the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate-solubilized membrane fractions of HeLa cells, in an in vitro ␥-secretase assay using recombinant amyloid ␤ precursor protein C100 as a substrate. SSide also inhibits activities for the generation of A␤40 as well as for Notch intracellular domain at higher concentrations. Notably, SSide displayed linear noncompetitive inhibition profiles for ␥ 42 -secretase in vitro. Our data suggest that SSide is a direct inhibitor of ␥-secretase that preferentially affects the ␥ 42 -secretase activity. Alzheimer's disease (AD)1 is a dementing neurodegenerative disorder of the elderly characterized pathologically by neuronal loss in the cerebral cortex accompanied by massive deposition of amyloid ␤ peptides (A␤) as senile plaques (1). A␤ is produced by sequential proteolytic cleavages of the amyloid ␤ precursor protein (␤APP) by a set of membrane-bound proteases termed ␤-and ␥-secretases. The C-terminal length of A␤ generated by ␥-secretase is heterogeneous; A␤42 is a relatively minor molecular species of the A␤ secreted from cells, but it has a much higher propensity to aggregate and form amyloid compared with other A␤ species. These findings provide strong support for the hypothesis that the deposition of A␤42 is closely related to the pathogenesis of AD, implicating ␥-secretase as an important therapeutic target.Mutations in PS1 or PS2 genes account for the majority of early onset familial AD, and these mutations cause an increase in the ratio or levels of production of A␤42 (1). It is known that PS is essential for the ␥-secretase-mediated intramembranous cleavage not only for ␤APP but for other type I transmembrane proteins (e.g. Notch, ErbB4, E-cadherin, low density lipoprotein receptor-related protein, and CD44) (2). PS proteins undergo endoproteolysis to generate N-and C-terminal fragments and interact with other proteins (i.e. nicastrin, APH-1, and PEN-2) to form a high molecular weight (HMW) protein complex (3). The functional role of PS complex in ␥-secretase activity still remains unknown. However, aspartyl protease transition state analogue inhibitors of ␥-secretase, which harbors a hydroxyl ethylene isostere or a difluoro alcohol moiety, directly label PS fragments (4 -6). In addition, a systematic analysis using a variety of PS mutants revealed that HMW complex formation of PS as well as conserved aspartyl residues within the transmembrane domain are es...

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“…2E). These data are consistent with the previous reports (12,33,34) on the in vitro ␥-secretase assays using different types of recombinant C100 with or without C-terminal tags.…”

Section: Resultssupporting

confidence: 93%

Sulindac Sulfide Is a Noncompetitive γ-Secretase Inhibitor That Preferentially Reduces Aβ42 Generation

Takahashi

Hayashi

Tominari

et al. 2003

Journal of Biological Chemistry

185

189

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“…3C), respectively. The significant increase in the ratio of Aβ42∶Aβ40 peptides in the PS1ΔE9-L166P or PS1ΔE9-G384A double mutants compared with PS1ΔE9 indicates that either mutant combined with ΔE9 has an additive effect on γ-secretase activity, findings that are consistent with observations made in in vivo settings (33,34). Moreover, the elevated ratio of Aβ42∶Aβ40 elicited by the double mutants in this reconstitution system indicates that both mutations directly affect the active site of γ-secretase itself rather than other cellular processes.…”

Section: Resultssupporting

confidence: 88%

“…To further examine the effects of FAD-linked PS1 variants on the production of Aβ40 and Aβ42, we engineered PS1ΔE9-MBP constructs to include the well-characterized L166P and G384A (31, 32) mutations. The rationale for this experiment is that expression of PS1 harboring two FAD mutations leads to a considerable elevation in the Aβ42∶Aβ40 ratio compared to the ratio obtained by expression of PS1 with single mutations (33,34). The PS1ΔE9-MBP fusion proteins were reconstituted into liposomes, and proteoliposmes were treated by thrombin and isolated (Fig.…”

Section: Resultsmentioning

confidence: 99%

Activation and intrinsic γ-secretase activity of presenilin 1

Ahn

Shelton

Tian

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

133

158

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A complex composed of presenilin (PS), nicastrin, PEN-2, and APH-1 is absolutely required for γ-secretase activity in vivo. Evidence has emerged to suggest a role for PS as the catalytic subunit of γ-secretase, but it has not been established that PS is catalytically active in the absence of associated subunits. We now report that bacterially synthesized, recombinant PS (rPS) reconstituted into liposomes exhibits γ-secretase activity. Moreover, an rPS mutant that lacks a catalytic aspartate residue neither exhibits reconstituted γ-secretase activity nor interacts with a transition-state γ-secretase inhibitor. Importantly, we demonstrate that rPS harboring mutations that cause early onset familial Alzheimer's disease (FAD) lead to elevations in the ratio of Aβ42 to Aβ40 peptides produced from a wild-type APP substrate and that rPS enhances the Aβ42∕ Aβ40 peptide ratio from FAD-linked mutant APP substrates, findings that are entirely consistent with the results obtained in in vivo settings. Thus, γ-secretase cleavage specificity is an inherent property of the polypeptide. Finally, we demonstrate that PEN2 is sufficient to promote the endoproteolysis of PS1 to generate the active form of γ-secretase. Thus, we conclusively establish that activated PS is catalytically competent and the bimolecular interaction of PS1 and PEN2 can convert the PS1 zymogen to an active protease.intermembrane-cleaving proteases | notch | presenilinase | reconstitution

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“…When this set of experiments was performed in PS-1/2ϩ/ϩ cells, all PS-1 mutants that were defective in cleaving Notch in PS-1/2Ϫ/Ϫ MEFs generated NICD regardless of the PS-1 mutant type (data not shown), supporting the notion that PS-1/2Ϫ/Ϫ MEFs should be used to investigate the effect of exogenous PS-1 constructs. There are controversial reports debating whether the defects of D257A and D385A mutants are the result of a change in their catalytic domains or a lack of endoproteolytic activity (12)(13)(14)(15)(16)(17)(18). To demonstrate directly that the defects of PS-1 D257A and D385A mutants are caused by changes in their catalytic sites and also to analyze the different effects of the PS-1 NTF and CTF, we constructed normal and mutant NTFs and CTFs tagged with GFP at the N terminus as follows: wt NTF (1-298 amino acids), wt CTF (299 -467 amino acids), D257A NTF (1-298 amino acids in which the aspartate in residue 257 is point mutated to alanine), D385A CTF (299 -467 amino acids in which the aspartate in residue 385 is point mutated to alanine).…”

Section: Notch Cleavage Depends On the Presence Of Endogenous Ps-1 Anmentioning

confidence: 99%

“…Studies with artificial and spontaneously occurring PS-1 mutants reveal that the activity of ␥-secretase is critically dependent on the presence of two intact aspartate residues, Asp-257 and Asp-385, and on the endoproteolysis of PS-1. However, it is still unclear whether the defective PS-1 mutants, D257A and D385A, result from the changes in the catalytic domain or from the lack of their own endoproteolysis (12)(13)(14)(15)(16)(17)(18).…”

mentioning

confidence: 99%

Presenilin-1 D257A and D385A Mutants Fail to Cleave Notch in Their Endoproteolyzed Forms, but Only Presenilin-1 D385A Mutant Can Restore Its γ-Secretase Activity with the Compensatory Overexpression of Normal C-terminal Fragment

Kim¹,

Ki²,

Park

et al. 2005

Journal of Biological Chemistry

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The enzyme ␥-secretase is involved in the cleavage of several type I membrane proteins, such as Notch 1 and amyloid precursor protein. Presenilin-1 (PS-1) is one of the critical integral membrane protein components of the ␥-secretase complex and is processed endoproteolytically, generating N-and C-terminal fragments (NTF and CTF, respectively). PS-1 is also known to incorporate into a high molecular weight complex by binding to other ␥-secretase components such as Nicastrin, Aph-1, and Pen-2. Mutations on PS-1 can alter the effects of ␥-secretase on its many substrates to different extents. Here, we showed that PS-1 mutants have a different activity for Notch cleavage, which depended on the PS-1 mutation site. We demonstrated that defective PS-1 mutants located in CTF, i.e. D385A and C410Y, could restore their ␥-secretase activities with the compensatory overexpression of wild type CTF or of minimal deleted CTF (amino acids 349 -467). However, the defective PS-1 D257A mutant could not restore their ␥-secretase activities with the compensatory overexpression of wild type NTF. In comparison, both D257A NTF and D385A CTF could abolish the ␥-secretase activity of wild type and pathogenic PS-1 mutants. We also showed that PS-1 NTF but not CTF forms strong high molecular weight aggregates in SDS-PAGE. Taken together, results have shown that NTF and CTF integrate differently into high molecular weight aggregates and that PS-1 Asp-257 and Asp-385 have different accessibilities in their unendoproteolyzed conformation.

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PS1 N- and C-terminal fragments form a complex that functions in APP processing and Notch signaling

Cited by 76 publications

References 35 publications

Sulindac Sulfide Is a Noncompetitive γ-Secretase Inhibitor That Preferentially Reduces Aβ42 Generation

Sulindac Sulfide Is a Noncompetitive γ-Secretase Inhibitor That Preferentially Reduces Aβ42 Generation

Activation and intrinsic γ-secretase activity of presenilin 1

Presenilin-1 D257A and D385A Mutants Fail to Cleave Notch in Their Endoproteolyzed Forms, but Only Presenilin-1 D385A Mutant Can Restore Its γ-Secretase Activity with the Compensatory Overexpression of Normal C-terminal Fragment

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