Caspase inhibitors of the P35 family are more active when purified from yeast than bacteria

Ingo L. Brand, Srgjan Civciristov, Nicole L. Taylor, Gert H. Talbo, Delara Pantaki-Eimany, Vita Levina, Rollie J. Clem, Matthew A. Perugini, Marc Kvansakul, Christine J. Hawkins

Research output: Contribution to journalArticleResearchpeer-review

6 Citations (Scopus)

Abstract

Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a "reactive site loop" within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins) may underestimate their activity.

Original languageEnglish
Article numbere39248
Number of pages8
JournalPLoS ONE
Volume7
Issue number6
DOIs
Publication statusPublished - 14 Jun 2012
Externally publishedYes

Cite this

Brand, I. L., Civciristov, S., Taylor, N. L., Talbo, G. H., Pantaki-Eimany, D., Levina, V., ... Hawkins, C. J. (2012). Caspase inhibitors of the P35 family are more active when purified from yeast than bacteria. PLoS ONE, 7(6), [e39248]. https://doi.org/10.1371/journal.pone.0039248
Brand, Ingo L. ; Civciristov, Srgjan ; Taylor, Nicole L. ; Talbo, Gert H. ; Pantaki-Eimany, Delara ; Levina, Vita ; Clem, Rollie J. ; Perugini, Matthew A. ; Kvansakul, Marc ; Hawkins, Christine J. / Caspase inhibitors of the P35 family are more active when purified from yeast than bacteria. In: PLoS ONE. 2012 ; Vol. 7, No. 6.
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title = "Caspase inhibitors of the P35 family are more active when purified from yeast than bacteria",
abstract = "Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a {"}reactive site loop{"} within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins) may underestimate their activity.",
author = "Brand, {Ingo L.} and Srgjan Civciristov and Taylor, {Nicole L.} and Talbo, {Gert H.} and Delara Pantaki-Eimany and Vita Levina and Clem, {Rollie J.} and Perugini, {Matthew A.} and Marc Kvansakul and Hawkins, {Christine J.}",
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Brand, IL, Civciristov, S, Taylor, NL, Talbo, GH, Pantaki-Eimany, D, Levina, V, Clem, RJ, Perugini, MA, Kvansakul, M & Hawkins, CJ 2012, 'Caspase inhibitors of the P35 family are more active when purified from yeast than bacteria', PLoS ONE, vol. 7, no. 6, e39248. https://doi.org/10.1371/journal.pone.0039248

Caspase inhibitors of the P35 family are more active when purified from yeast than bacteria. / Brand, Ingo L.; Civciristov, Srgjan; Taylor, Nicole L.; Talbo, Gert H.; Pantaki-Eimany, Delara; Levina, Vita; Clem, Rollie J.; Perugini, Matthew A.; Kvansakul, Marc; Hawkins, Christine J.

In: PLoS ONE, Vol. 7, No. 6, e39248, 14.06.2012.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Caspase inhibitors of the P35 family are more active when purified from yeast than bacteria

AU - Brand, Ingo L.

AU - Civciristov, Srgjan

AU - Taylor, Nicole L.

AU - Talbo, Gert H.

AU - Pantaki-Eimany, Delara

AU - Levina, Vita

AU - Clem, Rollie J.

AU - Perugini, Matthew A.

AU - Kvansakul, Marc

AU - Hawkins, Christine J.

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N2 - Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a "reactive site loop" within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins) may underestimate their activity.

AB - Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a "reactive site loop" within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins) may underestimate their activity.

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Brand IL, Civciristov S, Taylor NL, Talbo GH, Pantaki-Eimany D, Levina V et al. Caspase inhibitors of the P35 family are more active when purified from yeast than bacteria. PLoS ONE. 2012 Jun 14;7(6). e39248. https://doi.org/10.1371/journal.pone.0039248