title: Inhibition of cathepsin B by caspase-3 inhibitors blocks programmed cell death in Arabidopsis.
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.
authors.affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.|College of Marine Life Science, Ocean University of China, No. 5 Yushan Road, Qingdao, China.
authors.forename: Y
authors.initials: Y
authors.lastname: Ge
authors.affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.
authors.forename: Y-M
authors.initials: YM
authors.lastname: Cai
authors.affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.
authors.forename: L
authors.initials: L
authors.lastname: Bonneau
authors.affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.
authors.forename: V
authors.initials: V
authors.lastname: Rotari
authors.forename: A
authors.initials: A
authors.lastname: Danon
authors.affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.
authors.forename: E A
authors.initials: EA
authors.lastname: McKenzie
authors.affiliation: Division of Plant Science, The James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK.
authors.forename: H
authors.identifier: 0000-0003-4490-7053
authors.initials: H
authors.lastname: McLellan
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: L
authors.initials: L
authors.lastname: Mach
authors.affiliation: Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.
authors.forename: P
authors.identifier: 0000-0002-6305-2807
authors.initials: P
authors.lastname: Gallois
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D017360:Arabidopsis / Q000201:enzymology* / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D061945:Caspase Inhibitors / Q000494:pharmacology*; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000145:classification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000633:toxicity; D018384:Oxidative Stress / Q000187:drug effects; D010269:Paraquat / Q000633:toxicity; D010802:Phylogeny; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000254:growth & development; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D036226:Seedlings / Q000187:drug effects / Q000254:growth & development / Q000528:radiation effects; D053719:Tandem Mass Spectrometry; D014466:Ultraviolet Rays
chemical_list: D029681:Arabidopsis Proteins; D061945:Caspase Inhibitors; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D002401:Cathepsin B; D010269:Paraquat