Fig. 5: (R)−4-HNE adducted on Lys53 by Schiff-base formation caused positional deviation of C99, leading to increased Aβ40/42 ratio.

a The Schiff-base formation reaction of Ac-Leu–Lys–Lys–Lys–Gln (residues 52-56 of C99) with (R)−4-HNE. Matrix-assisted laser desorption/ionization-time of flight MS (MALDI-TOF MS) detection of natural peptide Ac-Leu–Lys–Lys–Lys–Gln (left, upper) and 4-HNE-modified peptides (right, upper). Representative MS/MS spectra of natural peptide Ac-Leu–Lys–Lys–Lys–Gln (left, lower) and modified peptide (right, lower) with 4-HNE adduction on the first Lys residue (Lys53 of C99) via Schiff-base formation, the main product of this reaction. b WB verification of recombinant C99, Lys53Arg mutant C99 (C99^Lys53Arg), and 4-HNE-modified HNE-C99 and HNE-C99^Lys53Arg. C99 was detected by C1/6.1 antibody. 4-HNE-modified proteins were detected by 4-HNE antibody. c Levels of generated Aβ40 and Aβ42 in in vitro γ-secretase cleavage assays from recombinant C99, C99^Lys53Arg, and their 4-HNE-modified forms, determined by ELISA assay. n = 3 biologically independent samples. d The predicted docking structures of human γ-secretase with peptide Val46–Lys55 of C99 (purple) or with modified peptide Val46–Lys53(HNE)–Lys55 (green) by ZDOCK and sequence alignment was done in PyMOL. The structures of PS1 (the main γ-secretase component) and natural Val46–Lys55 peptide were derived from the Cryo-EM structure (PDB: 6IYC). The 4-HNE modification on Lys53 (after Schiff-base formation) was manually added in PyMOL based on the natural peptide structure from 6IYC. The dashed lines and the numbers represent the distance between Asp385 or Asp257 of PS1 and –NH or C=O groups of residue Leu49 of C99 [purple, natural Val46-Lys53; green, modified Val46-Lys53(HNE)-Lys55]. Data are presented as mean values ± SD. Statistical analysis was performed using one-way ANOVA with LSD post-hoc test for multiple groups. Source data are provided as a Source Data file.