Substrate determinants in the C99 juxtamembrane domains differentially affect γ–secretase cleavage specificity and modulator pharmacology

Abstract

The molecular mechanisms governing c-secretase cleavage specificity are not fully understood. Herein, we demonstrate that extending the transmembrane domain of the amyloid precursor protein-derived C99 substrate in proximity to the cytosolic face strongly influences c-secretase cleavage specificity. Sequential insertion of leucines or replacement of membrane-anchoring lysines by leucines elevated the production of Ab42, whilst lowering production of Ab40. A single insertion or replacement was sufficient to produce this phenotype, suggesting that the helical length distal to the e-site is a critical determinant of c-secretase cleavage specificity. Replacing the lysine at the luminal membrane border (K28) with glutamic acid (K28E) increased Ab37 and reduced Ab42 production. Maintaining a positive charge with an arginine replacement, however, did not alter cleavage specificity. Using two potent and structurally distinct c-secretase modulators (GSMs), we elucidated the contribution of K28 to the modulatory mechanism. Surprisingly, whilst lowering the potency of the non-steroidal anti-inflammatory drug-type GSM, the K28E mutation converted a heteroaryl-type GSM to an inverse GSM. This result implies the proximal lysine is critical for the GSM mechanism and pharmacology. This region is likely a major determinant for substrate binding and we speculate that modulation of substrate binding is the fundamental mechanism by which GSMs exert their action.

Rare variants in β-Amyloid precursor protein (APP) and Parkinson’s disease

“…This could possibly be due to an alteration in the site at which APP interacts with γ-secretases, a mechanism recently postulated for increased Aβ37 production in response to an artificial APP variant (c.2095A4G (p.(K699E))) 10 amino acids N-terminal of our variant. 21 None of the other APP variants showed an altered Aβ spectrum. However, further studies are necessary to exclude that these variants could affect the structure and, accordingly, the aggregation potential of generated Aβ as has been demonstrated for some AD-linked variants (reviewed in Haass et al 22 ).…”

Complex Relationships between Substrate Sequence and Sensitivity to Alterations in γ-Secretase Processivity Induced by γ-Secretase Modulators

“…Notably, as we were preparing these data for publication, Ousson et al reported similar effects of the K624E mutation on GSM activity. 41 In their study using a signal peptide CTFβ construct (SP-C99) containing the K28E mutation, they found a diminished responsiveness to GSM-1 for decreasing the level of Aβ42 and a shift in activity for a nonacidic heteroaryl-type GSM (E2012) to an iGSM in the presence of the mutation.…”

“…20,41 The K624E mutant generates more of the shorter Aβ species such as Aβ33 and Aβ37. As explained above for the 3xK mutant, because the phosphate group has a negative charge, the K624E residue may sit with the phosphate group driving the Aβ production more like the noncharged mutant allowing the construct to sink a bit further down than the WT (single lysine).…”

“…Thus, the collective data suggesting substrate specificity and the lack of effect on ε-cleavage reinforce the potential intrinsic safety of GSMs as prophylactic therapeutics for the treatment of AD. 16,20,33,37,40,41,72,73 …”

Transmembrane Substrate Determinants for γ-Secretase Processing of APP CTFβ

“…59, 60 Moreover, a previous study of systematic insertions at the APP C-terminus demonstrated that Aβ42/40 was increased no matter how many residues were added to the C-terminal end, and thus shifts in product lines did not correlate with the number of C-terminal residues added. 61 However, all of these studies were performed in cells, where the expression and trafficking of mutants can be issues that affect Aβ production. Our examination of CTFβ C-terminal deletions provides important direct in vitro validation of these previous studies.…”