title: Substrate determinants in the C99 juxtamembrane domains differentially affect γ-secretase cleavage specificity and modulator pharmacology.
abstract: 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.
authors.affiliation: Global Research and Early Development, Merck Serono SA, Geneva, Switzerland.
authors.forename: Solenne
authors.initials: S
authors.lastname: Ousson
authors.forename: Arman
authors.initials: A
authors.lastname: Saric
authors.forename: Aurelie
authors.initials: A
authors.lastname: Baguet
authors.forename: Christophe
authors.initials: C
authors.lastname: Losberger
authors.forename: Stephane
authors.initials: S
authors.lastname: Genoud
authors.forename: Francis
authors.initials: F
authors.lastname: Vilbois
authors.forename: Bruno
authors.initials: B
authors.lastname: Permanne
authors.forename: Ishrut
authors.initials: I
authors.lastname: Hussain
authors.forename: Dirk
authors.initials: D
authors.lastname: Beher
mesh_terms: D000544:Alzheimer Disease / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D053829:Amyloid Precursor Protein Secretases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004789:Enzyme Activation; D057809:HEK293 Cells; D006801:Humans; D007930:Leucine / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: C545487:APP protein, human; D016564:Amyloid beta-Protein Precursor; D008565:Membrane Proteins; D053829:Amyloid Precursor Protein Secretases; D007930:Leucine; D008239:Lysine