Analysis of the minimal specificity of caspase-2 and identification of Ac-VDTTD-AFC as a caspase-2-selective peptide substrate
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....
Main Authors: | , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Portland Press, Biochemical Society
2014-03-01
|
Series: | Bioscience Reports |
Subjects: | |
Online Access: | http://www.bioscirep.org/bsr/034/e100/bsr034e100.htm |
id |
doaj-5763080905e04fe09ff27f85b823805f |
---|---|
record_format |
Article |
spelling |
doaj-5763080905e04fe09ff27f85b823805f2020-11-25T00:32:14ZengPortland Press, Biochemical SocietyBioscience Reports1573-49352014-03-01342e0010010.1042/BSR20140025BSR20140025Analysis of the minimal specificity of caspase-2 and identification of Ac-VDTTD-AFC as a caspase-2-selective peptide substrateTanja Kitevska0Sarah J. Roberts1Delara Pantaki‑Eimany2Sarah E. Boyd3Fiona L. Scott4Christine J. Hawkins5 Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3086, Victoria, Australia Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3086, Victoria, Australia Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3086, Victoria, Australia Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3086, Victoria, Australia Receptos, 10835 Road to the Cure, Suite 205, San Diego, CA 92121, U.S.A. Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3086, Victoria, Australia 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. http://www.bioscirep.org/bsr/034/e100/bsr034e100.htmapoptosiscaspasecleavageproteaseRunx1substrate |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Tanja Kitevska Sarah J. Roberts Delara Pantaki‑Eimany Sarah E. Boyd Fiona L. Scott Christine J. Hawkins |
spellingShingle |
Tanja Kitevska Sarah J. Roberts Delara Pantaki‑Eimany Sarah E. Boyd Fiona L. Scott Christine J. Hawkins Analysis of the minimal specificity of caspase-2 and identification of Ac-VDTTD-AFC as a caspase-2-selective peptide substrate Bioscience Reports apoptosis caspase cleavage protease Runx1 substrate |
author_facet |
Tanja Kitevska Sarah J. Roberts Delara Pantaki‑Eimany Sarah E. Boyd Fiona L. Scott Christine J. Hawkins |
author_sort |
Tanja Kitevska |
title |
Analysis of the minimal specificity of caspase-2 and identification of Ac-VDTTD-AFC as a caspase-2-selective peptide substrate |
title_short |
Analysis of the minimal specificity of caspase-2 and identification of Ac-VDTTD-AFC as a caspase-2-selective peptide substrate |
title_full |
Analysis of the minimal specificity of caspase-2 and identification of Ac-VDTTD-AFC as a caspase-2-selective peptide substrate |
title_fullStr |
Analysis of the minimal specificity of caspase-2 and identification of Ac-VDTTD-AFC as a caspase-2-selective peptide substrate |
title_full_unstemmed |
Analysis of the minimal specificity of caspase-2 and identification of Ac-VDTTD-AFC as a caspase-2-selective peptide substrate |
title_sort |
analysis of the minimal specificity of caspase-2 and identification of ac-vdttd-afc as a caspase-2-selective peptide substrate |
publisher |
Portland Press, Biochemical Society |
series |
Bioscience Reports |
issn |
1573-4935 |
publishDate |
2014-03-01 |
description |
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.
|
topic |
apoptosis caspase cleavage protease Runx1 substrate |
url |
http://www.bioscirep.org/bsr/034/e100/bsr034e100.htm |
work_keys_str_mv |
AT tanjakitevska analysisoftheminimalspecificityofcaspase2andidentificationofacvdttdafcasacaspase2selectivepeptidesubstrate AT sarahjroberts analysisoftheminimalspecificityofcaspase2andidentificationofacvdttdafcasacaspase2selectivepeptidesubstrate AT delarapantakieimany analysisoftheminimalspecificityofcaspase2andidentificationofacvdttdafcasacaspase2selectivepeptidesubstrate AT saraheboyd analysisoftheminimalspecificityofcaspase2andidentificationofacvdttdafcasacaspase2selectivepeptidesubstrate AT fionalscott analysisoftheminimalspecificityofcaspase2andidentificationofacvdttdafcasacaspase2selectivepeptidesubstrate AT christinejhawkins analysisoftheminimalspecificityofcaspase2andidentificationofacvdttdafcasacaspase2selectivepeptidesubstrate |
_version_ |
1725320143106473984 |