Fig. 1
From: Development of selective deconjugases for membrane-anchored LC3A/B in post-mitotic neurons
The effect of mutagenesis that partially disrupts the α3-helix structure of the CAD of RavZ on selective enzymatic activity. (A) Schematic diagram of 3xFlag-fused deconjugase_Fy and 2xFy-CAD(α3 helix Alanine mutants) constructs. (B) Confocal images showing autophagosome formation of eGFP-LC3B and mRFP-GABARAPL1 co-expressed with RavZ proteins or LIR(Fy)-containing α3 helix Alanine mutants of catalytic domain in HKO HeLa cells upon autophagy induction (100 nM bafilomycin A1 [BafA1]) in Earle’s Balanced Salts solution (EBSS) for 2 h, (+ EBSS, BafA1). Scale bar: 10 μm. (C) Bar graphs illustrate the each eGFP-LC3B- or mRFP-GABARAPL1-positive autophagosome spot number for each cell. Values are presented as means + SEM (n ≥ 20 for each group). ns. not significant; ***P < 0.001 compared with 2xFy-CAD-expressing cells with one-way ANOVA in conjunction with the Newman–Keuls multiple comparison test. (D) Western blot showing the delipidation of endogenous LC3B and GABARAPL1 in mouse cortical neuron lysates expressing RavZ proteins or LIR(Fy)-containing α3 helix Alanine mutants of catalytic domain upon autophagy inhibition (50µM CQ for 2 h). (E) Bar graphs illustrate the ratios of LC3B-II/LC3B-I and GABARAPL1-II/GABARAPL1-I. Values are presented as means ± SEM (n = 4). ns. not significant; *P < 0.033, **P < 0.002, **P < 0.001 compared with RavZ-expressing cells using mixed-effects analysis in conjunction with the uncorrected Fisher’s LSD test. (F) Western blot showing the delipidation of endogenous LC3B and GABARAPL1 in mouse cortical neuron lysates expressing RavZ proteins or LIR(Fy)-containing α3 helix Alanine mutants of catalytic domain upon autophagy induction and inhibition (Trahalose 100mM/24 h, bafilomycin A1 10 nM/24 h). (G) Bar graphs illustrate the ratios of LC3B-II/LC3B-I and GABARAPL1-II/GABARAPL1-I. Values are presented as means ± SEM (n = 3). ns. not significant; *P < 0.033, **P < 0.002 compared with RavZ expressing cells with Friedman test in conjunction with the Dunn’s multiple comparisons test. (H-I) confocal images showing formation of LC3B (H) and GABARAPL1 (I)-containing autophagosomes in Tuj-1 positive mouse cortical neurons transiently expressing RavZ proteins or 2xFy-CAD(YFKGAAA) upon autophagy inhibition (100nM BafA1 for 2 h). Scale bar: 10 μm