Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in Arabidopsis

Y Ge,E A Mckenzie,L Mach,H Mclellan,P Gallois,A Danon,V Rotari,Y-M Cai,L Bonneau

doi:10.1038/cdd.2016.34

Abstract

Programmed cell death (PCD) is used by plants for development and survival to biotic and abiotic stresses. The role of caspases in PCD is well established in animal cells. Over the past 15 years, the importance of caspase-3-like enzymatic activity for plant PCD completion has been widely documented despite the absence of caspase orthologues. In particular, caspase-3 inhibitors blocked nearly all plant PCD tested. Here, we affinity-purified a plant caspase-3-like activity using a biotin-labelled caspase-3 inhibitor and identified Arabidopsis thaliana cathepsin B3 (AtCathB3) by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Consistent with this, recombinant AtCathB3 was found to have caspase-3-like activity and to be inhibited by caspase-3 inhibitors. AtCathepsin B triple-mutant lines showed reduced caspase-3-like enzymatic activity and reduced labelling with activity-based caspase-3 probes. Importantly, AtCathepsin B triple mutants showed a strong reduction in the PCD induced by ultraviolet (UV), oxidative stress (H2O2, methyl viologen) or endoplasmic reticulum stress. Our observations contribute to explain why caspase-3 inhibitors inhibit plant PCD and provide new tools to further plant PCD research. The fact that cathepsin B does regulate PCD in both animal and plant cells suggests that this protease may be part of an ancestral PCD pathway pre-existing the plant/animal divergence that needs further characterisation.

Highlights

Programmed cell death (PCD) is relevant to many aspects of an organism’s life and plants are no exception.[1]
We have reported previously that UV-C radiation induced a caspase-3-like activity in Arabidopsis with an optimum around pH 5 and that UV-C-induced PCD could be totally blocked by the addition of the caspase-3 inhibitor Ac-DEVDCHO.[14]
LC-MS/MS analysis of the band identified only two peptides corresponding to one protein: Arabidopsis cathepsin B3 (AtCathB3, At4g01610) (Figure 1d), one of the three Arabidopsis cathepsin B paralogues (Figure 1e): AtCathB1 (At1g02300), AtCathB2 (At1g02305) and AtCathB3 (At4g01610)

Summary

Introduction

Programmed cell death (PCD) is relevant to many aspects of an organism’s life and plants are no exception (reviewed in Drury and Gallois).[1]. We propose here that caspase-3 inhibitors reduce PCD in plants by targeted cathepsin B since a cathB1-2-3 triple mutant has a reduced PCD after treatment using ultraviolet (UV), H2O2, methyl viologen (MV) and tunicamycin. These findings are reminiscent of the involvement of cathepsin B in mammalian PCD (reviewed in Rozman-Pungercar et al.[13])

Methods

Results

Conclusion