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Ousson S.,Global Research and Early Development Merck Serono SA | Ousson S.,Asceneuron SA | Saric A.,Global Research and Early Development Merck Serono SA | Baguet A.,Global Research and Early Development Merck Serono SA | And 8 more authors.
Journal of Neurochemistry

The molecular mechanisms governing γ-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 γ-secretase cleavage specificity. Sequential insertion of leucines or replacement of membrane-anchoring lysines by leucines elevated the production of Aβ42, whilst lowering production of Aβ40. A single insertion or replacement was sufficient to produce this phenotype, suggesting that the helical length distal to the ε-site is a critical determinant of γ-secretase cleavage specificity. Replacing the lysine at the luminal membrane border (K28) with glutamic acid (K28E) increased Aβ37 and reduced Aβ42 production. Maintaining a positive charge with an arginine replacement, however, did not alter cleavage specificity. Using two potent and structurally distinct γ-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. Mutagenesis studies were conducted to explore γ-secretase cleavage specificity and modulation. Elongation of the distal juxtamembrane domain changes cleavage specificity. The K28E mutation at the proximal juxtamembrane domain changes γ-secretase cleavage specificity and modulator pharmacology. Thus, we have identified critical determinants in the proximal and distal juxtamembrane domains of the APP C99 substrate which differentially affect γ-secretase cleavage specificity and modulator pharmacology. Modulation of substrate binding could be a potential mechanism of action for γ-secretase modulators. © 2012 International Society for Neurochemistry. Source

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