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Related Concept Videos

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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Verification of a putative caspase substrate.

Gavin P McStay1, Douglas R Green2

  • 1Department of Life Sciences, New York Institute of Technology, Old Westbury, New York 11568;

Cold Spring Harbor Protocols
|August 3, 2014
PubMed
Summary
This summary is machine-generated.

This study validates caspase substrates identified through proteomic screening. A novel method uses radiolabeled substrates to confirm genuine caspase targets, ensuring accurate apoptosis pathway analysis.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Proteomic approaches are used to identify caspase substrates during apoptosis.
  • These methods offer insights into apoptosis-related pathways but require substrate validation.
  • Distinguishing true caspase targets from artifacts is crucial for reliable conclusions.

Purpose of the Study:

  • To describe a validation method for putative caspase substrates identified by proteomic screening.
  • To ensure the accuracy of identified substrates and the reliability of apoptosis pathway analysis.

Main Methods:

  • Utilizing in vitro transcription/translation to synthesize radiolabeled putative caspase substrates.
  • Incubating these labeled substrates with purified caspases.
  • Assessing substrate cleavage by caspases to confirm genuine targets.

Main Results:

  • The described method allows for the direct assessment of caspase activity on specific substrates.
  • Validation confirms whether identified proteins are indeed cleaved by caspases in vitro.
  • This protocol provides a means to filter out false positives from proteomic screens.

Conclusions:

  • The described validation protocol is essential for confirming bona fide caspase substrates.
  • Accurate substrate identification strengthens the understanding of caspase-mediated apoptosis pathways.
  • This method enhances the reliability of data derived from large-scale proteomic screens.