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Caspases01:24

Caspases

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Caspase, a family of cysteine proteases, serve as effectors in apoptosis. The ced3 gene in C.elegans was first identified to be involved in apoptosis. This gene encodes the ced-3 caspase that is similar to the interleukin-1-beta converting enzyme or ICE in mammals. In addition to apoptosis, caspases also function in the inflammatory response. Inflammatory caspases are essential in activating pro-inflammatory cytokines that recruit immune cells and block the replication of pathogens inside...
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Activity-based probes trap early active intermediates during metacaspase activation.

Vida Štrancar1, Katarina P van Midden1, Daniel Krahn2,3

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Calcium binding activates essential metacaspases (cysteine proteases) in plants and fungi. New probes reveal active, unprocessed metacaspase intermediates form upon calcium binding before precursor processing.

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Cell biologyFunctional aspects of cell biologyMethodology in biological sciences

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protease research

Background:

  • Metacaspases are crucial cysteine proteases in plants, fungi, and protists.
  • Their regulation by calcium and proteolytic maturation mechanisms remain largely unknown.

Purpose of the Study:

  • To develop and validate activity-based probes for metacaspase types.
  • To investigate calcium-mediated activation of metacaspases from precursors in vitro.

Main Methods:

  • Designed substrate-inspired tetrapeptide probes with an acyloxymethylketone (AOMK) reactive group.
  • Utilized purified type-I, type-II, and type-III metacaspases for in vitro studies.
  • Analyzed calcium-dependent labeling of metacaspases and their proteoforms.

Main Results:

  • Metacaspase labeling by probes was strictly dependent on calcium presence.
  • A high-affinity probe labeled mature and higher molecular weight metacaspase proteoforms.
  • Labeling of unprocessed metacaspase intermediates occurred only with calcium.

Conclusions:

  • Metacaspase activation is calcium-dependent.
  • Calcium binding triggers the formation of previously unidentified active metacaspase intermediates.
  • These intermediates precede the final precursor processing in metacaspase activation.