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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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Expression in bacteria or yeast does not substantially alter the primary structural features of MaviP35 and AcP35.FLAG-tagged MaviP35 and AcP35 were purified from bacteria or yeast. (A) Each sample was analyzed using Matrix Assisted Laser Desorption Ionisation-Mass Spectrometry. (B) Reduced and alkylated proteins were subjected to trypsin digestion, then the peptides were analyzed by LC-ESI-MS. The intensity of peaks of masses corresponding to the predicted peptide mass of unmodified or modified amino terminal tryptic peptides were measured. Based on the assumption that the peak intensities are proportional to the peptide amount, the relative amount of each peptide was calculated. The integrities of the peptides were confirmed by MS/MS analysis.
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pone-0039248-g002: Expression in bacteria or yeast does not substantially alter the primary structural features of MaviP35 and AcP35.FLAG-tagged MaviP35 and AcP35 were purified from bacteria or yeast. (A) Each sample was analyzed using Matrix Assisted Laser Desorption Ionisation-Mass Spectrometry. (B) Reduced and alkylated proteins were subjected to trypsin digestion, then the peptides were analyzed by LC-ESI-MS. The intensity of peaks of masses corresponding to the predicted peptide mass of unmodified or modified amino terminal tryptic peptides were measured. Based on the assumption that the peak intensities are proportional to the peptide amount, the relative amount of each peptide was calculated. The integrities of the peptides were confirmed by MS/MS analysis.

Mentions: A number of possible explanations were tested to explore the differential activities of P35 family proteins isolated from bacteria and yeast. Mass spectrometry methods were employed to examine the primary structures of AcP35 and MaviP35 purified from the different sources, to determine whether post-translational modifications differed depending on the source of recombinant protein. The proteins from both sources had molecular weights consistent with each protein lacking its initiating methionine residue (Figure 2A). This excluded the possibility that prokaryotic proteases cleaved and incapacitated the bacterially-purified inhibitors, or that large post-translational modifications which modulated function occurred in one species but not the other. Analysis of the amino terminal tryptic peptides generated from each protein confirmed the absence of the initiating methionine (Figure 2B). Similar proportions of bacterially purified AcP35 and MaviP35 were N-acetylated (21% and 24% respectively). In contrast, only 13% of AcP35 and only 12% of MaviP35 proteins were N-acetylated when expressed in yeast. This difference of around 10% between the proteins purified from bacteria and yeast seems unlikely to account for the 280-fold superiority of yeast-purified MaviP35 with respect to caspase inhibition (Figure 1B).


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)

Expression in bacteria or yeast does not substantially alter the primary structural features of MaviP35 and AcP35.FLAG-tagged MaviP35 and AcP35 were purified from bacteria or yeast. (A) Each sample was analyzed using Matrix Assisted Laser Desorption Ionisation-Mass Spectrometry. (B) Reduced and alkylated proteins were subjected to trypsin digestion, then the peptides were analyzed by LC-ESI-MS. The intensity of peaks of masses corresponding to the predicted peptide mass of unmodified or modified amino terminal tryptic peptides were measured. Based on the assumption that the peak intensities are proportional to the peptide amount, the relative amount of each peptide was calculated. The integrities of the peptides were confirmed by MS/MS analysis.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0039248-g002: Expression in bacteria or yeast does not substantially alter the primary structural features of MaviP35 and AcP35.FLAG-tagged MaviP35 and AcP35 were purified from bacteria or yeast. (A) Each sample was analyzed using Matrix Assisted Laser Desorption Ionisation-Mass Spectrometry. (B) Reduced and alkylated proteins were subjected to trypsin digestion, then the peptides were analyzed by LC-ESI-MS. The intensity of peaks of masses corresponding to the predicted peptide mass of unmodified or modified amino terminal tryptic peptides were measured. Based on the assumption that the peak intensities are proportional to the peptide amount, the relative amount of each peptide was calculated. The integrities of the peptides were confirmed by MS/MS analysis.
Mentions: A number of possible explanations were tested to explore the differential activities of P35 family proteins isolated from bacteria and yeast. Mass spectrometry methods were employed to examine the primary structures of AcP35 and MaviP35 purified from the different sources, to determine whether post-translational modifications differed depending on the source of recombinant protein. The proteins from both sources had molecular weights consistent with each protein lacking its initiating methionine residue (Figure 2A). This excluded the possibility that prokaryotic proteases cleaved and incapacitated the bacterially-purified inhibitors, or that large post-translational modifications which modulated function occurred in one species but not the other. Analysis of the amino terminal tryptic peptides generated from each protein confirmed the absence of the initiating methionine (Figure 2B). Similar proportions of bacterially purified AcP35 and MaviP35 were N-acetylated (21% and 24% respectively). In contrast, only 13% of AcP35 and only 12% of MaviP35 proteins were N-acetylated when expressed in yeast. This difference of around 10% between the proteins purified from bacteria and yeast seems unlikely to account for the 280-fold superiority of yeast-purified MaviP35 with respect to caspase inhibition (Figure 1B).

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