Assessment of metacaspase activity in phytoplankton

Dina Spungin, Ilana Berman-Frank

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

Programmed cell death (PCD) is an irreversible, genetically-controlled form of cell suicide in which an endogenous biochemical pathway leads to morphological changes and ultimately, cellular demise. PCD is accompanied by de-novo protein synthesis of a family of proteases-"caspases"that are often used as a diagnostic marker of PCD. Although phytoplankton do not contain true caspases, caspase-like activity (hypothetical proteins with analogous activity) has been traditionally used as a diagnostic marker of PCD in marine phytoplankton. Increased caspase-like proteolytic activity was demonstrated when synthetic fluorogenic activity substrates specific for caspases (with an Asp at the P1 position) were applied upon PCD induction. Metacaspases, cysteine proteases, share structural properties with those of caspases, yet they are highly specific for Arg and Lys cleavage site at the P1 position implying that caspase specific substrates are not indicative of metacaspase catalytic activity. This method specifically tests direct metacaspase activity in phytoplankton by the cleavage of the fluorogenic metacaspase substrate Ac-VRPR-AMC. Metacaspase activity was tested by the addition of a metacaspase specific peptide that is conjugated to the fluorescent reporter molecule. The cleavage of the peptide by the metacaspase releases the fluorochrome that, when excited by light, emits fluorescence. The level of metacaspase enzymatic activity in the cell lysate is directly proportional to the fluorescence signal detected. The use of specific standards in this test enables the quantification of the fluorescence results. This assay directly allows monitoring the metacaspase cleavage products and thereby tracing evidence for programmed cell death.

Original languageEnglish
Article numbere3341
JournalBio-protocol
Volume9
Issue number16
DOIs
StatePublished - 2019
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by the United States-Israel Binational Science Foundation (BSF) grant 2008048 to IBF and KDB, a collaborative grant from MOST Israel and the High Council for Science and Technology (HCST)-France to IBF, and GIF funding No. 1133/2011 to IBF. This protocol was adapted and modified from our previous studies (Spungin et al., 2018 and 2019).

Publisher Copyright:
Copyright © 2019 The Authors; exclusive licensee Bio-protocol LLC.

Keywords

  • Cell extracts
  • Cyanobacteria
  • Fluorescence
  • Metacaspase activity
  • Phytoplankton
  • Programmed cell death

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