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Biochemical Characterization of a Novel Redox-Regulated Metacaspase in a Marine Diatom
Shiri Graff van Creveld1,2, Shifra Ben-Dor3, Avia Mizrachi1
1Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel.
Abstract:
Programmed cell death (PCD) in marine microalgae was suggested to be one of the mechanisms that facilitates bloom demise, yet its molecular components in phytoplankton are unknown. Phytoplankton are completely lacking any of the canonical components of PCD, such as caspases, but possess metacaspases. Metacaspases were shown to regulate PCD in plants and some protists, but their roles in algae and other organisms are still elusive. Here, we identified and biochemically characterized a type III metacaspase from the model diatom Phaeodactylum tricornutum, termed PtMCA-IIIc. Through expression of recombinant PtMCA-IIIc in E. coli, we revealed that PtMCA-IIIc exhibits a calcium-dependent protease activity, including auto-processing and cleavage after arginine. Similar metacaspase activity was detected in P. tricornutum cell extracts. PtMCA-IIIc overexpressing cells exhibited higher metacaspase activity, while CRISPR/Cas9-mediated knockout cells had decreased metacaspase activity compared to WT cells. Site-directed mutagenesis of cysteines that were predicted to form a disulfide bond decreased recombinant PtMCA-IIIc activity, suggesting its enhancement under oxidizing conditions. One of those cysteines was oxidized, detected in redox proteomics, specifically in response to lethal concentrations of hydrogen peroxide and a diatom derived aldehyde. Phylogenetic analysis revealed that this cysteine-pair is unique and widespread among diatom type III metacaspases. The characterization of a cell death associated protein in diatoms provides insights into the evolutionary origins of PCD and its ecological significance in algal bloom dynamics.
Insights
Marine microalgae programmed cell death (PCD) mechanisms remain unknown. Researchers identified a calcium-dependent metacaspase in diatoms, revealing its role in cell death and potential link to algal bloom dynamics.
Area of Science:
- Marine biology
- Molecular biology
- Biochemistry
Background:
- Programmed cell death (PCD) is crucial for marine microalgae bloom demise, but its molecular basis in phytoplankton is unclear.
- Phytoplankton lack canonical PCD components like caspases but possess metacaspases, whose roles in algae are largely unknown.
Purpose of the Study:
- To identify and biochemically characterize a type III metacaspase (PtMCA-IIIc) from the model diatom *Phaeodactylum tricornutum*.
- To elucidate the function and regulation of PtMCA-IIIc in diatom cell death.
Main Methods:
- Recombinant protein expression in *E. coli*
- Biochemical assays for protease activity
- CRISPR/Cas9 gene editing
- Site-directed mutagenesis
- Redox proteomics
- Phylogenetic analysis
Main Results:
- PtMCA-IIIc exhibits calcium-dependent protease activity, cleaving after arginine and undergoing auto-processing.
- Overexpression of PtMCA-IIIc increased activity, while knockout decreased it, confirming its role in metacaspase activity.
- Activity is enhanced under oxidizing conditions, with a unique cysteine pair found in diatom metacaspases.
- Oxidation of a specific cysteine was detected under stress from hydrogen peroxide and diatom-derived aldehydes.
Conclusions:
- PtMCA-IIIc is a key cell death-associated protein in diatoms.
- This finding provides insights into the evolution of PCD and its ecological role in algal bloom dynamics.
- Metacaspases represent a conserved mechanism for PCD regulation across diverse eukaryotic lineages.
