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

Brand IL, Civciristov S, Taylor NL, Talbo GH, Pantaki-Eimany D, Levina V, Clem RJ, Perugini MA, Kvansakul M, Hawkins CJ - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia.

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.

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Caspase 3 can cleave MaviP35 purified from yeast but not bacteria.FLAG-tagged MaviP35 was purified from bacteria or yeast by affinity chromotography and gel filtration. The ∼94 kDa gel filtration fraction of the bacterial preparation (“b4” in Figure 5) and the ∼72 kDa fraction of the yeast-purified sample (“y3” in Figure 5) were incubated with 0, 10, 100 or 1000 ng/ml caspase 3 for 1 hour, and then subjected to SDS-PAGE and anti-FLAG immunoblotting.
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pone-0039248-g007: Caspase 3 can cleave MaviP35 purified from yeast but not bacteria.FLAG-tagged MaviP35 was purified from bacteria or yeast by affinity chromotography and gel filtration. The ∼94 kDa gel filtration fraction of the bacterial preparation (“b4” in Figure 5) and the ∼72 kDa fraction of the yeast-purified sample (“y3” in Figure 5) were incubated with 0, 10, 100 or 1000 ng/ml caspase 3 for 1 hour, and then subjected to SDS-PAGE and anti-FLAG immunoblotting.

Mentions: The pseudo-substrate mechanism of inhibition employed by P35 proteins requires their cleavage by caspases. We reasoned that the conformation adopted by MaviP35 expressed in bacteria may obscure the reactive site loop and restrict its accessibility to caspases. Gel filtration fractions from the yeast and bacterial MaviP35 purifications were incubated with caspase 3 and their sensitivities to cleavage were monitored by immunoblotting. Although caspase 3 was able to cleave MaviP35 isolated from yeast to produce the expected 25.7 kDa carboxyl terminal FLAG-tagged product, it failed to cleave the bacterially-expressed protein (Figure 7).


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

Brand IL, Civciristov S, Taylor NL, Talbo GH, Pantaki-Eimany D, Levina V, Clem RJ, Perugini MA, Kvansakul M, Hawkins CJ - PLoS ONE (2012)

Caspase 3 can cleave MaviP35 purified from yeast but not bacteria.FLAG-tagged MaviP35 was purified from bacteria or yeast by affinity chromotography and gel filtration. The ∼94 kDa gel filtration fraction of the bacterial preparation (“b4” in Figure 5) and the ∼72 kDa fraction of the yeast-purified sample (“y3” in Figure 5) were incubated with 0, 10, 100 or 1000 ng/ml caspase 3 for 1 hour, and then subjected to SDS-PAGE and anti-FLAG immunoblotting.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375223&req=5

pone-0039248-g007: Caspase 3 can cleave MaviP35 purified from yeast but not bacteria.FLAG-tagged MaviP35 was purified from bacteria or yeast by affinity chromotography and gel filtration. The ∼94 kDa gel filtration fraction of the bacterial preparation (“b4” in Figure 5) and the ∼72 kDa fraction of the yeast-purified sample (“y3” in Figure 5) were incubated with 0, 10, 100 or 1000 ng/ml caspase 3 for 1 hour, and then subjected to SDS-PAGE and anti-FLAG immunoblotting.
Mentions: The pseudo-substrate mechanism of inhibition employed by P35 proteins requires their cleavage by caspases. We reasoned that the conformation adopted by MaviP35 expressed in bacteria may obscure the reactive site loop and restrict its accessibility to caspases. Gel filtration fractions from the yeast and bacterial MaviP35 purifications were incubated with caspase 3 and their sensitivities to cleavage were monitored by immunoblotting. Although caspase 3 was able to cleave MaviP35 isolated from yeast to produce the expected 25.7 kDa carboxyl terminal FLAG-tagged product, it failed to cleave the bacterially-expressed protein (Figure 7).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia.

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.

Show MeSH
Related in: MedlinePlus