Analysis of the minimal specificity of caspase-2 and identification of Ac-VDTTD-AFC as a caspase-2-selective peptide substrate

Biosci Rep. 2014 Apr 1;34(2):e00100. doi: 10.1042/BSR20140025. Print 2014 Apr 1.

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.

Keywords: Runx1; apoptosis; caspase; cleavage; protease; substrate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Caspase 2 / chemistry*
  • Caspase 2 / genetics
  • Caspase 2 / metabolism*
  • Caspase 3 / chemistry
  • Caspase 3 / genetics
  • Caspase 3 / metabolism
  • Core Binding Factor Alpha 2 Subunit / chemistry*
  • Core Binding Factor Alpha 2 Subunit / genetics
  • Core Binding Factor Alpha 2 Subunit / metabolism*
  • Cysteine Endopeptidases / chemistry*
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / metabolism*
  • Enzyme Activation
  • HEK293 Cells
  • Humans
  • Peptides / chemistry*
  • Peptides / genetics
  • Peptides / metabolism*

Substances

  • Core Binding Factor Alpha 2 Subunit
  • Peptides
  • RUNX1 protein, human
  • CASP2 protein, human
  • CASP3 protein, human
  • Caspase 2
  • Caspase 3
  • Cysteine Endopeptidases