Caspase Inhibitors of the P35 Family Are More Active When Purified from Yeast than Bacteria

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

MaviP35 and AcP35 are more active when purified from yeast than bacteria.

<p>(<b>A</b>) FLAG-tagged MaviP35 or AcP35 were purified from bacteria or yeast. The indicated concentrations of inhibitors were assayed for their ability to inhibit cleavage of 100 µM Ac-DEVD-AFC by 30 nM caspase 3. Error bars represent S.E.M. from three independent replicates. (<b>B</b>) A competitive model was used to determine the caspase 3 inhibition constants for the P35 proteins purified from yeast <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039248#pone.0039248-Brand1" target="_blank">[22]</a> and bacteria.</p

MaviP35 proteins purified from bacteria and yeast have similar secondary structures.

<p>FLAG-tagged MaviP35 proteins were purified from yeast and bacteria and then subjected to circular dichroism (CD) analyses. (<b>A</b>) 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>B</b>) Table of secondary structure proportions and apparent melting temperature for MaviP35 purified from bacterial and yeast. (<b>C</b>) 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.</p

Bacterially produced MaviP35 is more prone to aggregation <i>in vitro</i> than yeast-expressed MaviP35.

<p>FLAG-tagged MaviP35 proteins were purified from bacteria or yeast and then treated with 0 or 5 µM of the crosslinker BMH prior to analysis by SDS-PAGE and anti-FLAG immunoblotting. Arrows on the right indicate the expected positions of various oligomeric species based on the migrations of the molecular weight markers. The expected molecular weight of octameric MaviP35 (286 kDa) would be greater than that of the largest marker used, so its migration is difficult to accurately estimate. A representative immunoblot is shown from four separate experiments.</p

Analytical ultracentrifugation of MaviP35 purified from bacteria and yeast.

<p>(<b>A</b>) MaviP35-FLAG proteins were purified from bacteria (gray line) and yeast (black line) and then subjected to gel filtration. (<b>B–F</b>) Analytical ultracentrifugation of the fractions indicated in gray shading yielded the sedimentation profiles shown. The molecular weights estimated for each peak are stated. Numbers in parentheses indicate numbers of MaviP35-FLAG monomers that could comprise the major oligomeric species resolved by analytical ultracentrifugation.</p

Caspase 3 can cleave MaviP35 purified from yeast but not bacteria.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039248#pone-0039248-g005" target="_blank">Figure 5</a>) and the ∼72 kDa fraction of the yeast-purified sample (“y3” in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039248#pone-0039248-g005" target="_blank">Figure 5</a>) 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.</p

Expression in bacteria or yeast does not substantially alter the primary structural features of MaviP35 and AcP35.

<p>FLAG-tagged MaviP35 and AcP35 were purified from bacteria or yeast. (<b>A</b>) Each sample was analyzed using Matrix Assisted Laser Desorption Ionisation-Mass Spectrometry. (<b>B</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.</p

MaviP35 purified from yeast and bacteria differ in their mobility during size exclusion chromatography.

<p>FLAG-tagged MaviP35 proteins were purified from bacteria (<b>A</b>) or yeast (<b>B</b>) and then subjected to gel filtration. (<b>C</b>) Selected fractions were analyzed by SDS-PAGE and anti-FLAG immunoblotting. (<b>D</b>) Proteins from each selected gel filtration fraction or buffer (−) were mixed with caspase 3 and then maximal rate of cleavage of Ac-DEVD-AFC was monitored fluorometrically. (<b>C, D</b>) Because the yield of the yeast-purified protein was lower than the bacterial sample, the bacterial fractions were diluted 8-fold prior to these analyses.</p