Negative Regulation of Cathepsins by β-Amyloid

Abstract

Genome wide association study (GWAS) uncovered Alzheimer's disease (AD) risk genes linked to the endo-lysosomal pathway. This pathway seems to be the gateway of protein aggregates, such as tau and α-synuclein, to the cytoplasm. Furthermore, we and others reported that the amyloid precursor protein (APP) C99 is predominantly processed by γ-secretase in the endo-lysosomal compartments, and β-amyloid (Aβ) peptides are enriched in the same subcellular loci. While the role(s) of APP/Aβ in the endo-lysosomal pathway has not been fully established, a recent study reported that Aβ, in particular Aβ42, inhibits cathepsin D (CTSD) activity. Here, we show using a cell-free in vitro assay that Aβ42 also blocks cathepsin B (CTSB) activity. Furthermore, we uncovered that the autocatalytic processing (i.e., conversion of single chain to heavy/light chains) of CTSB and CTSD is accelerated in APP-deficient cells compared with wild-type controls. Taken together, our findings further support the negative regulation of cathepsins by Aβ.

Keywords: Alzheimer's disease; cathepsin B; cathepsin D; endo-lysosomal pathway; β-amyloid.

Figure 1.

Aβ42 downregulates CTSB activity. AFC-conjugated CTSB substrate and recombinant CTSB were incubated with Aβ38, Aβ40, Aβ42, Aβ43, p3, or scramble Aβ peptides (1 µM). % Relative Inhibition (“no-peptide” and “no-protease” set as 0% and 100%, respectively) is shown. CTSB inhibitor Z-Phe-Phe-FMK (1 µM) was used to ensure the specificity of the in vitro activity assay. N = 3, *p < 0.05, ****p < 0.0001, one-way ANOVA. Figure 1-1 is supporting Figure 1.

Figure 2.

Abolished Aβ generation in APP/APLP2-deficient MEF cells. A, Aβ40 and (B) Aβ42 levels in the conditioned medium of APP/APLP2 dKO MEF cells are undetectable as opposed to WT controls. γ-Secretase inhibitor DAPT (1 µM) was used to ensure the specificity of ELISA. N = 4, **p < 0.01, ****p < 0.0001, one-way ANOVA.

Figure 3.

Accelerated CTSB autocatalysis in APP/APLP2-deficient cells. A, A representative Western blot image by detecting with a CTSB antibody, B shows that the ratio of CTSB heavy chain over single chain is significantly increased in APP/APLP2 knock-out compared with WT MEF cells. N = 3 independent experiments, *p < 0.05, Mann–Whitney U test. Figures 3-1 and 3-2 are supporting Figure 3.

Figure 4.

Increased CTSD autocatalysis in APP/APLP2-deficient cells. A, A representative Western blot image by detecting with a CTSD antibody. B, The ratio of CTSD heavy chain over single chain is significantly increased in APP/APLP2 dKO compared with WT MEF cells. N = 3 independent experiments, *p < 0.05, Mann–Whitney U test. Figure 4-1 is supporting Figure 4.