Further characterization of metacaspase expression and activity in marine phytoplankton

Abstract

Metacaspases are considered to be ancestral cysteinyl aspartate specific proteases involved in the initiation and execution of apoptotic programmed cell death (PCD). The widespread presence of metacaspases genes among a variety of phytoplankton, suggests that they play a fundamental role in cell turnover, aquatic food webs, and biogeochemical cycles. Yet, there are still fundamental questions that exist about these enzymes: How does metacaspase diversity relate to different evolutionary plastid lineages? What is their relationship to cell physiology? Are metacaspases associated with caspase activity? Using hydrolysis of a fluorogenic canonical tetrapeptide substrate and western blot analysis, we report on the induction of caspase-like activity and metacaspases-like protein expression from four ecologically and evolutionarily diverse phytoplankton species, including a chlorophyte (Dunaliella tertiolecta), a haptophyte (Isochrysis galbana), a diatom (Thalassiosira weissflogii), and a dinoflagellate (Amphidinium carterae). These derived eukaryotic lineages represented phytoplankton from primary (D. tertiolecta), secondary (I. galbana and T. weissflogii), and tertiary endosymbiotic (A. carterae) events. Immunohybridization to polyclonal antisera raised against a coccolithophore metacaspase indicated high conservation of caspase-like proteins and an accumulation of metacaspase complexity with evolutionary complexity. An E. huxleyi CCMP1516 (Ehux1516) and EhV86 host-virus model system was used to link dramatic increases in caspase specific activity with protein signatures via a genome-enabled, proteomic approach. Both the host and virus have sequenced genomes and EhV86 strongly triggered caspase activity. Caspase activation was measured through in vitro cleavage of fluorogenic peptide substrates with up to ~170-fold increase during infection. Subsets of partially purified proteins were associated with enhanced caspase specific activity and displayed hybridization to metacaspase antibodies. Pooled subsets of caspase active fractions from size exclusion chromatography were subjected to both 1D SDS-PAGE and 2D gel electrophoresis (GE) followed by mass spectrometry analysis. SYPRO-Ruby staining of 2D gels yielded approximately 10-17 clearly definable protein spots for caspase active fractions. Seventeen proteins hits, including four from the EhV86 proteome, were homologues to proteases or death related proteins, suggesting these proteins may be responsible for observed caspase activities and some may be virally derived. Further work should focus on validating the activity and function of these gene products.