Modeling Metazoan Apoptotic Pathways in Yeast.

This chapter describes techniques for characterizing metazoan apoptotic pathways using Saccharomyces cerevisiae. Active forms of the major apoptotic effectors-caspases, Bax and Bak-are all lethal to yeast. Using this lethality as a readout of caspase/Bax/Bak activity, proteins and small molecules that directly or indirectly regulate the activity of these effectors can be investigated in yeast, and apoptotic inhibitors can be identified using functional yeast-based screens. Caspase activity can also be monitored in yeast by cleavage-dependent liberation of a transcription factor from the plasma membrane, enabling it to activate the lacZ reporter gene. This system can be used to define the sequences that can be efficiently cleaved by particular caspases.

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.

Protein disaggregation by the AAA+ chaperone ClpB involves partial threading of looped polypeptide segments.

The ring-forming AAA+ chaperone ClpB cooperates with the DnaK chaperone system to reactivate aggregated proteins. With the assistance of DnaK, ClpB extracts unfolded polypeptides from aggregates via substrate threading through its central channel. Here we analyze the processing of mixed aggregates consisting of protein fusions of misfolded and native domains. ClpB-DnaK reactivated all aggregated fusion proteins with similar efficiency, without unfolding native domains, demonstrating that partial threading of the misfolded moiety is sufficient to solubilize aggregates. Reactivation by ClpB-DnaK occurred even when two stably folded domains flanked the aggregated moiety, indicating threading of internal substrate segments. In contrast with the related AAA+ chaperone ClpC, ClpB lacks a robust unfolding activity, enabling it to sense the conformational state of substrates. ClpB rings are highly unstable, which may facilitate dissociation from trapped substrates during threading.