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Complex relationships between substrate sequence and sensitivity to alterations in ?-secretase processivity induced by ?-secretase modulators.

ABSTRACT: ?-Secretase catalyzes the final cleavage of the amyloid precursor protein (APP), resulting in the production of amyloid-? (A?) peptides with different carboxyl termini. Presenilin (PSEN) and amyloid precursor protein (APP) mutations linked to early onset familial Alzheimer's disease modify the profile of A? isoforms generated, by altering both the initial ?-secretase cleavage site and subsequent processivity in a manner that leads to increased levels of the more amyloidogenic A?42 and in some circumstances A?43. Compounds termed ?-secretase modulators (GSMs) and inverse GSMs (iGSMs) can decrease and increase levels of A?42, respectively. As GSMs lower the level of production of pathogenic forms of long A? isoforms, they are of great interest as potential Alzheimer's disease therapeutics. The factors that regulate GSM modulation are not fully understood; however, there is a growing body of evidence that supports the hypothesis that GSM activity is influenced by the amino acid sequence of the ?-secretase substrate. We have evaluated whether mutations near the luminal border of the transmembrane domain (TMD) of APP alter the ability of both acidic, nonsteroidal anti-inflammatory drug-derived carboxylate and nonacidic, phenylimidazole-derived classes of GSMs and iGSMs to modulate ?-secretase cleavage. Our data show that point mutations can dramatically reduce the sensitivity to modulation of cleavage by GSMs but have weaker effects on iGSM activity. These studies support the concept that the effect of GSMs may be substrate selective; for APP, it is dependent on the amino acid sequence of the substrate near the junction of the extracellular domain and luminal segment of the TMD.

SUBMITTER: Jung JI

PROVIDER: S-EPMC3985764 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Complex relationships between substrate sequence and sensitivity to alterations in γ-secretase processivity induced by γ-secretase modulators.

Jung Joo In JI Ran Yong Y Cruz Pedro E PE Rosario Awilda M AM Ladd Thomas B TB Kukar Thomas L TL Koo Edward H EH Felsenstein Kevin M KM Golde Todd E TE

Biochemistry 20140320 12

γ-Secretase catalyzes the final cleavage of the amyloid precursor protein (APP), resulting in the production of amyloid-β (Aβ) peptides with different carboxyl termini. Presenilin (PSEN) and amyloid precursor protein (APP) mutations linked to early onset familial Alzheimer's disease modify the profile of Aβ isoforms generated, by altering both the initial γ-secretase cleavage site and subsequent processivity in a manner that leads to increased levels of the more amyloidogenic Aβ42 and in some ci ...[more]

PMID: 24620716

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Project description:BACKGROUND:γ-Secretase is a multiprotein protease that cleaves amyloid protein precursor (APP) and other type I transmembrane proteins. It has two catalytic subunits, presenilins 1 and 2 (PS1 and 2). In our previous report, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates and slightly different potencies of PS1 versus PS2 inhibition for select γ-secretase inhibitors (GSIs) on various substrates. In this study, we investigated whether γ-secretase modulators (GSMs) and inverse γ-secretase modulators (iGSMs) modulate γ-secretase processivity using multiple different substrates. We next used HEK 293T cell lines in which PSEN1 or PSEN2 was selectively knocked out to investigate processivity and response to GSMs and iGSMs. METHODS:For cell-free γ-secretase cleavage assay, recombinant substrates were incubated with CHAPSO-solubilized CHO or HEK 293T cell membrane with GSMs or iGSMs in suitable buffer. For cell-based assay, cDNA encoding substrates were transfected into HEK 293T cells. Cells were then treated with GSMs or iGSMs, and conditioned media were collected. Aβ and Aβ-like peptide production from cell-free and cell-based assay were measured by ELISA and mass spectrometry. RESULT:These studies demonstrated that GSMs are highly selective for effects on APP, whereas iGSMs have a more promiscuous effect on many substrates. Surprisingly, iGSMs actually appear to act as like GSIs on select substrates. The data with PSEN1 or PSEN2 knocked out HEK 293T reveal that PS1 has higher processivity and response to GSMs than PS2, but PS2 has higher response to iGSM. CONCLUSION:Collectively, these data indicate that GSMs are likely to have limited target-based toxicity. In addition, they show that iGSMs may act as substrate-selective GSIs providing a potential new route to identify leads for substrate-selective inhibitors of certain γ-secretase-mediated signaling events. With growing concerns that long-term β-secretase inhibitor is limited by target-based toxicities, such data supports continued development of GSMs as AD prophylactics.

Dataset Information

Complex relationships between substrate sequence and sensitivity to alterations in ?-secretase processivity induced by ?-secretase modulators.

Publications

Complex relationships between substrate sequence and sensitivity to alterations in γ-secretase processivity induced by γ-secretase modulators.

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