title: Serpin1 of Arabidopsis thaliana is a suicide inhibitor for metacaspase 9.
abstract: Metacaspases are distant relatives of animal caspases found in plants, fungi and protozoa. We demonstrated previously that two type II metacaspases of Arabidopsis thaliana, AtMC4 and AtMC9 are Arg/Lys-specific cysteine-dependent proteases. We screened a combinatorial tetrapeptide library of 130,321 substrates with AtMC9. Here, we show that AtMC9 is a strict Arg/Lys-specific protease. Based on the position-specific scoring matrix derived from the substrate library results, the tetrapeptide Val-Arg-Pro-Arg was identified as an optimized substrate. AtMC9 had a kcat/KM of 4.6x10(5) M-1 s-1 for Ac-Val-Arg-Pro-Arg-amido-4-methyl-coumarin, representing a more than 10-fold improvement over existing fluorogenic substrates. A yeast two-hybrid screen with catalytically inactive AtMC9 as bait identified a serine protease inhibitor, designated AtSerpin1, which was found to be a potent inhibitor of AtMC9 activity in vitro through cleavage of its reactive center loop and covalent binding to AtMC9. On the basis of the substrate profiling of AtMC9 and confirmation through site-directed mutagenesis, the inhibitory P4-P1 cleavage site of AtSerpin1 was determined to be Ile-Lys-Leu-Arg351. Further mutagenesis of the AtSerpin1 inhibitory cleavage site modulated AtMC9 inhibition positively or negatively. Both AtMC9 and AtSerpin1 were localized in the extracellular space, suggesting an in vivo interaction as well. To our knowledge, this is the first report of plant protease inhibition by a plant serpin.
authors.affiliation: Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, B-9052 Gent, Belgium.
authors.forename: Dominique
authors.initials: D
authors.lastname: Vercammen
authors.forename: Beatrice
authors.initials: B
authors.lastname: Belenghi
authors.forename: Brigitte
authors.initials: B
authors.lastname: van de Cotte
authors.forename: Tine
authors.initials: T
authors.lastname: Beunens
authors.forename: Julie-Ann
authors.initials: JA
authors.lastname: Gavigan
authors.forename: Riet
authors.initials: R
authors.lastname: De Rycke
authors.forename: Anouk
authors.initials: A
authors.lastname: Brackenier
authors.forename: Dirk
authors.initials: D
authors.lastname: Inzé
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Harris
authors.forename: Frank
authors.initials: F
authors.lastname: Van Breusegem
mesh_terms: D029681:Arabidopsis Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D019151:Peptide Library; D021381:Protein Transport; D015843:Serpins / Q000378:metabolism / Q000502:physiology*; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques
chemical_list: D029681:Arabidopsis Proteins; D061945:Caspase Inhibitors; D009842:Oligopeptides; D019151:Peptide Library; D015843:Serpins; D020169:Caspases; C516211:metacaspase 9, Arabidopsis