title: Alanine substitutions in the GXXXG motif alter C99 cleavage by γ-secretase but not its dimerization.
abstract: The amyloid β (Aβ) protein is a major component of senile plaques, one of the neuropathological hallmarks of Alzheimer's disease. Amyloidogenic processing of amyloid precursor protein (APP) by β- and γ-secretases leads to production of Aβ. APP contains tandem triple repeats of the GXXXG motif in its extracellular juxtamembrane and transmembrane regions. It is reported that the GXXXG motif is related to protein-protein interactions, but it remains controversial whether the GXXXG motif in APP is involved in substrate dimerization and whether dimerization affects γ-secretase-dependent cleavage. Therefore, the relationship between the GXXXG motifs, substrate dimerization, and γ-secretase-dependent cleavage sites remains unclear. Here, we applied blue native poly acrylamide gel electrophoresis to examine the effect of alanine substitutions within the GXXXG motifs of APP carboxyl terminal fragment (C99) on its dimerization and Aβ production. Surprisingly, alanine substitutions in the motif failed to alter C99 dimerization in detergent soluble state. Cell-based and solubilized γ-secretase assays demonstrated that increasing alanine substitutions in the motif tended to decrease long Aβ species such as Aβ42 and Aβ43 and to increase in short Aβ species concomitantly. Our data suggest that the GXXXG motif is crucial for Aβ production, but not for C99 dimerization.
authors.affiliation: Laboratory of Cognition and Aging, Graduate School of Brain Science, Doshisha University, Kyoto, Japan.
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Higashide
authors.affiliation: Department of Neuropathology, Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan.
authors.forename: Seiko
authors.initials: S
authors.lastname: Ishihara
authors.affiliation: Department of Neuropathology, Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan.
authors.forename: Mika
authors.initials: M
authors.lastname: Nobuhara
authors.affiliation: Laboratory of Cognition and Aging, Graduate School of Brain Science, Doshisha University, Kyoto, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ihara
authors.affiliation: Department of Neuropathology, Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan.
authors.forename: Satoru
authors.initials: S
authors.lastname: Funamoto
mesh_terms: D000409:Alanine / Q000235:genetics* / Q000378:metabolism*; D020816:Amino Acid Motifs / Q000502:physiology; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000502:physiology*; D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000235:genetics / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007313:Insecta; D010446:Peptide Fragments / Q000378:metabolism*; D055503:Protein Multimerization / Q000502:physiology*
chemical_list: D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D010446:Peptide Fragments; C543304:amyloid beta-protein precursor C-terminal fragment beta, human; D053829:Amyloid Precursor Protein Secretases; D000409:Alanine
keywords: Alzheimer disease; amyloid-β; dimerization; enzyme processing; gamma-secretase; transmembrane domain