Caspase enzymology and activation mechanisms

Methods Enzymol. 2014;544:161-78. doi: 10.1016/B978-0-12-417158-9.00007-8.

Abstract

Apical caspases 8, 9, and 10 are only active as dimers. These dimers are unstable, and to characterize their activity they need to be maintained in vitro in a dimeric state. We provide updated methods for those looking to characterize various aspects of caspase function. We describe full methods for those looking to activate caspases in vitro using kosmotropic reagents, an essential step in characterizing upstream (apical) caspases. We detail methods for fusion of caspase domains to engineered dimerization domains as an alternative method to trigger regulated dimerization of caspases. We also describe methods to determine caspase activity profiles in cells and provide methods for studying the ability of SMAC-mimetic reagents to release inhibition of caspases by IAPs.

Keywords: Dimerization; IAP; Kosmotrope; Substrate.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Caspases / chemistry
  • Caspases / genetics
  • Caspases / metabolism*
  • Enzyme Activation*
  • Enzyme Assays / methods
  • Humans
  • Indicators and Reagents
  • Inhibitor of Apoptosis Proteins / metabolism
  • Mutation
  • Protein Multimerization*
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism

Substances

  • Indicators and Reagents
  • Inhibitor of Apoptosis Proteins
  • Recombinant Proteins
  • Caspases