title: Analysis of the minimal specificity of caspase-2 and identification of Ac-VDTTD-AFC as a caspase-2-selective peptide substrate.
abstract: Caspase-2 is an evolutionarily conserved but enigmatic protease whose biological role remains poorly understood. To date, research into the functions of caspase-2 has been hampered by an absence of reagents that can distinguish its activity from that of the downstream apoptotic caspase, caspase-3. Identification of protein substrates of caspase-2 that are efficiently cleaved within cells may also provide clues to the role of this protease. We used a yeast-based transcriptional reporter system to define the minimal substrate specificity of caspase-2. The resulting profile enabled the identification of candidate novel caspase-2 substrates. Caspase-2 cleaved one of these proteins, the cancer-associated transcription factor Runx1, although with relatively low efficiency. A fluorogenic peptide was derived from the sequence most efficiently cleaved in the context of the transcriptional reporter. This peptide, Ac-VDTTD-AFC, was efficiently cleaved by purified caspase-2 and auto-activating caspase-2 in mammalian cells, and exhibited better selectivity for caspase-2 relative to caspase-3 than reagents that are currently available. We suggest that this reagent, used in parallel with the traditional caspase-3 substrate Ac-DEVD-AFC, will enable researchers to monitor caspase-2 activity in cell lysates and may assist in the determination of stimuli that activate caspase-2 in vivo.
authors.affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3086, Victoria, Australia.
authors.forename: Tanja
authors.initials: T
authors.lastname: Kitevska
authors.affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3086, Victoria, Australia.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Roberts
authors.affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3086, Victoria, Australia.
authors.forename: Delara
authors.initials: D
authors.lastname: Pantaki-Eimany
authors.affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3086, Victoria, Australia.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Boyd
authors.affiliation: Receptos, 10835 Road to the Cure, Suite 205, San Diego, CA 92121, U.S.A.
authors.forename: Fiona L
authors.initials: FL
authors.lastname: Scott
authors.affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3086, Victoria, Australia c.hawkins@latrobe.edu.au.
authors.forename: Christine J
authors.initials: CJ
authors.lastname: Hawkins
mesh_terms: D053143:Caspase 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053148:Caspase 3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050676:Core Binding Factor Alpha 2 Subunit / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D057809:HEK293 Cells; D006801:Humans; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D050676:Core Binding Factor Alpha 2 Subunit; D010455:Peptides; C493728:RUNX1 protein, human; C490360:CASP2 protein, human; C505407:CASP3 protein, human; D053143:Caspase 2; D053148:Caspase 3; D003546:Cysteine Endopeptidases
keywords: Runx1; apoptosis; caspase; cleavage; protease; substrate