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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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MaviP35 proteins purified from bacteria and yeast have similar secondary structures.FLAG-tagged MaviP35 proteins were purified from yeast and bacteria and then subjected to circular dichroism (CD) analyses. (A) Wavelength scans were performed at 20°C. The final spectra is the average result of three scans (open circles). The CONTINLL algorithm calculated the nonlinear least squares best fit (solid line) against the SP29 protein database with r.m.s.d. values≤0.073. (B) Table of secondary structure proportions and apparent melting temperature for MaviP35 purified from bacterial and yeast. (C) Ellipticity at 216 nm was measured between 20 and 90°C (open circles). The nonlinear regression analysis (dashed lines) fitted the curves to a one step transition between folded and unfolded confirmations.
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pone-0039248-g003: MaviP35 proteins purified from bacteria and yeast have similar secondary structures.FLAG-tagged MaviP35 proteins were purified from yeast and bacteria and then subjected to circular dichroism (CD) analyses. (A) Wavelength scans were performed at 20°C. The final spectra is the average result of three scans (open circles). The CONTINLL algorithm calculated the nonlinear least squares best fit (solid line) against the SP29 protein database with r.m.s.d. values≤0.073. (B) Table of secondary structure proportions and apparent melting temperature for MaviP35 purified from bacterial and yeast. (C) Ellipticity at 216 nm was measured between 20 and 90°C (open circles). The nonlinear regression analysis (dashed lines) fitted the curves to a one step transition between folded and unfolded confirmations.

Mentions: The above-mentioned mass spectrometry analyses suggested that the P35 proteins expressed in bacteria and yeast had similar primary structures. Circular dichroism (CD) spectroscopy was used to explore the possibility that differences in secondary structure may underlie the substantially greater activity exhibited by yeast-purified MaviP35, relative to bacterial MaviP35. Wavelength scans of each sample were performed to estimate the fractions of various secondary structural elements adopted by bacterially and yeast-expressed MaviP35 in aqueous solution. The resulting spectra each displayed a single minimum at approximately 216 nm (Figure 3A, open circles), which is indicative of a protein sample with predominantly β structure [34]. The CD spectra were fitted by nonlinear least squares regression against relevant protein databases [35], [36]. The best fit for the MaviP35 samples (Figure 3A, solid line) was obtained by applying the CONTINLL algorithm against the SP29 database. This predicted that both samples had large proportions of β-structure (30–32%) and unordered secondary structure (29–30%) in combination with significant proportions of β-turn (22%) and α-helical (17–18%) structure (Figure 3B). These secondary structure proportions correlate well with structural predictions for MaviP35 [22] based on the structure of AcP35 determined by X-ray crystallography [18]. To assess thermal stability of the samples, CD thermal denaturation studies were performed at 216 nm between 20°C and 90°C (Figure 3C, open symbols). Nonlinear regression analysis of the data to a one step transition model resulted in a fit with R2 = 0.9843 and 0.9737 for the bacterial and yeast samples respectively (Figure 3C, dashed lines). These fits revealed the apparent melting temperatures (Tmeltapp) for bacterially-produced MaviP35 to be 72.9±0.1°C and yeast-isolated MaviP35 to be 69.7±0.3°C, suggesting that the bacterially-purified form of MaviP35 was slightly more thermostable than the yeast-purified form.


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)

MaviP35 proteins purified from bacteria and yeast have similar secondary structures.FLAG-tagged MaviP35 proteins were purified from yeast and bacteria and then subjected to circular dichroism (CD) analyses. (A) Wavelength scans were performed at 20°C. The final spectra is the average result of three scans (open circles). The CONTINLL algorithm calculated the nonlinear least squares best fit (solid line) against the SP29 protein database with r.m.s.d. values≤0.073. (B) Table of secondary structure proportions and apparent melting temperature for MaviP35 purified from bacterial and yeast. (C) Ellipticity at 216 nm was measured between 20 and 90°C (open circles). The nonlinear regression analysis (dashed lines) fitted the curves to a one step transition between folded and unfolded confirmations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375223&req=5

pone-0039248-g003: MaviP35 proteins purified from bacteria and yeast have similar secondary structures.FLAG-tagged MaviP35 proteins were purified from yeast and bacteria and then subjected to circular dichroism (CD) analyses. (A) Wavelength scans were performed at 20°C. The final spectra is the average result of three scans (open circles). The CONTINLL algorithm calculated the nonlinear least squares best fit (solid line) against the SP29 protein database with r.m.s.d. values≤0.073. (B) Table of secondary structure proportions and apparent melting temperature for MaviP35 purified from bacterial and yeast. (C) Ellipticity at 216 nm was measured between 20 and 90°C (open circles). The nonlinear regression analysis (dashed lines) fitted the curves to a one step transition between folded and unfolded confirmations.
Mentions: The above-mentioned mass spectrometry analyses suggested that the P35 proteins expressed in bacteria and yeast had similar primary structures. Circular dichroism (CD) spectroscopy was used to explore the possibility that differences in secondary structure may underlie the substantially greater activity exhibited by yeast-purified MaviP35, relative to bacterial MaviP35. Wavelength scans of each sample were performed to estimate the fractions of various secondary structural elements adopted by bacterially and yeast-expressed MaviP35 in aqueous solution. The resulting spectra each displayed a single minimum at approximately 216 nm (Figure 3A, open circles), which is indicative of a protein sample with predominantly β structure [34]. The CD spectra were fitted by nonlinear least squares regression against relevant protein databases [35], [36]. The best fit for the MaviP35 samples (Figure 3A, solid line) was obtained by applying the CONTINLL algorithm against the SP29 database. This predicted that both samples had large proportions of β-structure (30–32%) and unordered secondary structure (29–30%) in combination with significant proportions of β-turn (22%) and α-helical (17–18%) structure (Figure 3B). These secondary structure proportions correlate well with structural predictions for MaviP35 [22] based on the structure of AcP35 determined by X-ray crystallography [18]. To assess thermal stability of the samples, CD thermal denaturation studies were performed at 216 nm between 20°C and 90°C (Figure 3C, open symbols). Nonlinear regression analysis of the data to a one step transition model resulted in a fit with R2 = 0.9843 and 0.9737 for the bacterial and yeast samples respectively (Figure 3C, dashed lines). These fits revealed the apparent melting temperatures (Tmeltapp) for bacterially-produced MaviP35 to be 72.9±0.1°C and yeast-isolated MaviP35 to be 69.7±0.3°C, suggesting that the bacterially-purified form of MaviP35 was slightly more thermostable than the yeast-purified form.

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