Skip to main content
. 2021 Dec 14;21(1):e13531. doi: 10.1111/acel.13531

FIGURE 6.

FIGURE 6

Cathepsin L (CTSL) is the main protease in degradation of lamin B1. (a) Recombinant human (rh) LB1 (1 µg) was incubated with equal amount of (50 ng) rhCasp6, rhCTSL, and rhCTSB at pH 7.4 for the indicated time. Western blot comparing the protease activity of CTSL, CTSB, and CASP6 in processing of LB1, in a cell‐free condition. The 21 kDa fragment of LB1, similar to the one seen in Aβ42 treatment and AD mouse and human samples was only produced by rhCTSL and inhibited by its specific inhibitor. Data are representative of 4 independent experiments. CASP6 produced a ~46 kDa fragment that was inhibited by z‐VEID‐FMK. rhCTSB did not have any effect on rhLB1. (b) Enzymatic activity and Western blotting confirming that CASP6 is degraded by CTSL. rhCasp6 (1 µg) was incubated with rhCTSL and rhCTSB (50 ng each) without/with their specific inhibitors for 1 h at 37°C. Only rhCTSL degraded rhCasp6. These results were further validated using rhCasp6 enzymatic activity using Ac‐VEID‐amc as substrate (bar graph). (c) In vitro reaction mixture of rhLB1 and rhCTSL was subjected to mass spectrometry, and CTSL‐mediated cleavage sites on LB1 were determined by analyzing the small peptides produced in mass spectrometry. The abundance of the peptides was established by considering peptide spectrum match (PSM) 7 or more. The cleavage sites are indicated with blue arrow heads. Peptides are listed in Table S1. (d) Genetic inhibition of CTSL prevents invagination of LB1. Representative 3D confocal microscopy depicting the effect of Aβ42 on LB1 was assessed in WT, Ctsl−/− , and Ctsb−/− MEF cells after treatment with 5 µM of Aβ42 for 16 h. Depletion of CTSL−/− effectively prevented LB1 damage. n = 3 independent experiments, *p < 0.05, and ***p < 0.001, respectively (one‐way ANOVA/Tukey's post hoc), Scale bar: 10 µm. (e) Western blotting confirmed that overexpression of CTSL (TghCTSL+/+) is sufficient to induce LB1 cleavage in normal conditions, however, pre‐treatment with z‐FY‐CHO effectively prevented LB1 damage. WT, Ctsl−/− , and Ctsb−/− indicate wilt type, CTSL, and CTSB knock out, respectively