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.

Dimerization of Bacterial Diaminopimelate Decarboxylase Is Essential for Catalysis

“…Recombinant proteins were also subjected to trypsin digestion and the resulting tryptic fragments were sequenced by tandem mass spectrometry as reported previously (17).…”

Effect of baculovirus P35 protein on apoptosis in brain tissue of rats with acute cerebral infarction

“…It can inhibit cell apoptosis in mammalian, insect, and nemathelminth species, and is a widely effective inhibitor of apoptosis. Research has shown that P35 protein can combine the conserved constituent caspase in the apoptosis pathway and interact with it so as to inhibit its activity (Guy and Friesen, 2008;Brand et al, 2012). In vitro study results have shown that the P35 protein can form a complex that has steric stability with respect to its target molecules and can inhibit the activity of caspase in a competitive binding mode.…”

Identification of a dimeric KDG aldolase from Agrobacterium tumefaciens

“…Matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) was conducted using an Ultraflex III MALDI‐TOF/TOF mass spectrometer controlled by FlexControl software v2.0 (Bruker Daltonics) . The mass spectrum was acquired in linear positive mode and the data were calibrated externally with Bruker's MALDI mass calibration protein standards in the range of 5734–66431 Da (Supporting Information Figure S2).…”