Abstract
Backgroundγ-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.MethodsFor 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.ResultThese 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.ConclusionCollectively, 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.
Highlights
Background γ-Secretase was originally identified as the protease that carried out the final cleavage to release the amyloid beta (Aβ) from the amyloid β precursor protein (APP) and was an early target for pharmacologic inhibition of amyloid β peptide (Aβ) by γ-secretase inhibitors (GSIs) in Alzheimer’s disease (AD) [1, 2]. γ-Secretase cleaves hundreds of type I transmembrane proteins, typically following ectodomain shedding, though it cleaves some full-length proteins as well [3]. γ-Secretase mediates signaling events by cleavage within the substrate’s transmembrane domain (TMD)
γ-Secretase modulator (GSM) action is restricted to APP whereas Inverse γ-secretase modulator (iGSM) alter cleavage of other substrates GSMs have been shown to alter APP processing but not mouse Notch1; Wanngren et al reported that a second generation GSM did modulate human Nβ [27]
None of the three GSMs used in this study, GSM1, Compound 2, and cholestenoic acid (CA), altered the γ-secretase cleavage of rNotch1sub (Fig. 1a, b)
Summary
Background γSecretase was originally identified as the protease that carried out the final cleavage to release the amyloid beta (Aβ) from the amyloid β precursor protein (APP) and was an early target for pharmacologic inhibition of Aβ by γ-secretase inhibitors (GSIs) in Alzheimer’s disease (AD) [1, 2]. γ-Secretase cleaves hundreds of type I transmembrane proteins, typically following ectodomain shedding, though it cleaves some full-length proteins as well [3]. γ-Secretase mediates signaling events by cleavage within the substrate’s transmembrane domain (TMD). Γ-Secretase mediates signaling events by cleavage within the substrate’s transmembrane domain (TMD). This cleavage untethers the intracellular domain (ICD) from the membrane, allowing these domains to transduce signals by distributing to other sites within the cell [4]. In other cases, this cleavage can terminate a signaling event. After ectodomain cleavage by a sheddase, the transmembrane carboxyl terminal fragments (CTFs) of Notch and APP are processed by γ-secretase in a stepwise manner [2]. After ectodomain cleavage by a sheddase, the transmembrane carboxyl terminal fragments (CTFs) of Notch and APP are processed by γ-secretase in a stepwise manner [2]. γ-Secretase initially cleaves CTFs at a site near the cytoplasmic face of the membrane (S3 or ε-cleavage) and that cleavage is followed by a 3 to 5 sequential di, tri, or tetra-peptide cleavages (S4 or γ -cleavage) [2, 4,5,6,7]
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