Fig. 4: (R)−4-HNE increases the Aβ40/42 ratio via covalent adduction on residue Lys53 of C99.

a Levels of human-derived Aβ40 and Aβ42 in in vitro γ-secretase cleavage assay. The natural and 4-HNE-modified substrates (HNE-substrate) were, respectively from N2a-APPswe cells, a mouse neuronal cell line stably overexpressing human APPswe, treated with PBS or 10 μM (R)−4-HNE for 4 h. Natural and 4-HNE modified γ-secretase complexes (HNE-γ-secretase) were extracted from HEK293T cells pretreated with PBS or 10 μM (R)−4-HNE for 4 h. n = 3 biologically independent samples. b APP or 4-HNE-adducted APP were immunoprecipitated from N2a-APPswe cells pretreated with PBS or 10 μM (R)−4-HNE for 4 h and probed with 6E10 or 4-HNE antibody, respectively. c Levels of Aβ40 and Aβ42 generated from recombinant C99 (with a C-terminal His-tag, expressed in Escherichia coli from pET28a) in in vitro γ-secretase cleavage assay. 4-HNE-modified C99 (HNE-C99) was generated from recombinant C99 by reaction with 50-fold (mol/mol) (R)−4-HNE for 1 h at 37  °C. n = 3 biologically independent samples. d Sequence of C99. The secondary β-strand structural elements of C99, and possible 4-HNE-modified sites, are indicated. e An overall view of a portion of the C99 transmembrane fragment (C99∆) between the free stage (blue, PDB: 2LLM) and the γ-secretase-bound stage (pink, PDB: 6IYC) and a close-up view of the interaction of Met51−Lys53 fragment of C99 with PS1 through hydrogen bonds. f Potential reactions of the aldehyde and double bond groups in 4-HNE with proteins on –NH₂ group (Lys) via Michael addition and Schiff-base formation. g Representative MS/MS spectra of the peptide from C99 containing residue Lys53 modified by Schiff-base formation (Delta Mass, +138.10) with 4-HNE. Data are from deposited tandem mass-tag labeled proteomic data of amyloid plaques from postmortem brains with pathological AD and control (PXD005824). Data are presented as mean values ± SD. Statistical analysis was performed using a two-tailed Student’s t-test for two groups (c) and one-way ANOVA with LSD post-hoc test for multiple groups (a). Source data are provided as a Source Data file.