title: The death enzyme CP14 is a unique papain-like cysteine proteinase with a pronounced S2 subsite selectivity.
abstract: The cysteine protease CP14 has been identified as a central component of a molecular module regulating programmed cell death in plant embryos. CP14 belongs to a distinct subfamily of papain-like cysteine proteinases of which no representative has been characterized thoroughly to date. However, it has been proposed that CP14 is a cathepsin H-like protease. We have now produced recombinant Nicotiana benthamiana CP14 (NbCP14) lacking the C-terminal granulin domain. As typical for papain-like cysteine proteinases, NbCP14 undergoes rapid autocatalytic activation when incubated at low pH. The mature protease is capable of hydrolysing several synthetic endopeptidase substrates, but cathepsin H-like aminopeptidase activity could not be detected. NbCP14 displays a strong preference for aliphatic over aromatic amino acids in the specificity-determining P2 position. This subsite selectivity was also observed upon digestion of proteome-derived peptide libraries. Notably, the specificity profile of NbCP14 differs from that of aleurain-like protease, the N. benthamiana orthologue of cathepsin H. We conclude that CP14 is a papain-like cysteine proteinase with unusual enzymatic properties which may prove of central importance for the execution of programmed cell death during plant development.
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Melanie
authors.initials: M
authors.lastname: Paireder
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Mehofer
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Tholen
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Porodko
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Philipp
authors.initials: P
authors.lastname: Schähs
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Daniel
authors.initials: D
authors.lastname: Maresch
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, Germany.
authors.forename: Martin L
authors.initials: ML
authors.lastname: Biniossek
authors.affiliation: The Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, United Kingdom.
authors.forename: Renier A L
authors.initials: RA
authors.lastname: van der Hoorn
authors.affiliation: Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia.
authors.forename: Brigita
authors.initials: B
authors.lastname: Lenarcic
authors.affiliation: Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia.
authors.forename: Marko
authors.initials: M
authors.lastname: Novinec
authors.affiliation: Institute of Molecular Medicine and Cell Research, University of Freiburg, Germany; BIOSS Centre for Biological Signaling Studies, University of Freiburg, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Schilling
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria. Electronic address: lukas.mach@boku.ac.at.
authors.forename: Lukas
authors.initials: L
authors.lastname: Mach
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D056655:Cathepsin H / Q000737:chemistry; D002403:Cathepsins / Q000737:chemistry; D057056:Cysteine Proteases / Q000737:chemistry*; D006868:Hydrolysis; D007313:Insecta; D013058:Mass Spectrometry; D010206:Papain / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D011485:Protein Binding; D040901:Proteomics; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D014026:Nicotiana
chemical_list: D000911:Antibodies, Monoclonal; D010455:Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D002403:Cathepsins; D057056:Cysteine Proteases; D056655:Cathepsin H; D010206:Papain
keywords: Cathepsin; Cell death; Cysteine protease; Degradomics; Plant