Investigating apoptosis: Characterization and analysis of Trichoplusia ni-caspase-1 through overexpression and RNAi mediated silencing

Abstract

In both mammals and invertebrates, caspases play a critical role in apoptosis. Although Lepidopteron caspases have been widely studied in Spodoptera frugiperda cells, this is not the case for Trichoplusia ni cells, despite their widespread use for the production of recombinant protein and differences in baculovirus infectivity between the two species. We have cloned, expressed, purified and characterized Tn-caspase-1 in several situations: in its overexpression, in silencing via RNA interference (RNAi), during baculovirus infection, and in interactions with baculovirus protein p35. Overexpression can transiently increase caspase activity in T. ni (High Five™) cells, while silencing results in a greater than 6-fold decrease. The reduction in caspase activity resulted in a reduction in the level of apoptosis, demonstrating the ability to affect apoptosis by modulating Tn-caspase-1. During baculovirus infection, caspase activity remains low until approximately 5 days post infection, at which point it increases dramatically, though not in those cells treated with dsRNA. Our results demonstrate that Tn-caspase-1 is presumably the principal effector caspase present in High Five cells, and that it is inhibited by baculovirus protein p35. Finally, our results indicate differences between RNAi and p35 as effector molecules for modulating caspase activity and apoptosis during cell growth and baculovirus infection.