A comprehensive panel of turn-on caspase biosensors for investigating caspase specificity and caspase activation pathways

Sujan S. Shekhawat, Sean T. Campbell, Indraneel Ghosh

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Caspases play a central role in apoptosis, differentiation, and proliferation, and represent important therapeutic targets for treating cancer and inflammatory disorders. Toward the goal of developing new tools to probe caspase substrate cleavage specificity as well as to systematically interrogate caspase activation pathways, we have constructed and investigated a comprehensive panel of caspase biosensors with a split-luciferase enabled bioluminescent read out. We first interrogated the panel of caspase biosensors for substrate cleavage specificity of caspase 1-10 in widely utilized in vitro translation systems, namely, rabbit reticulocyte lysate (RRL) and wheat germ extract (WGE). Commercial RRL was found to be unsuitable for investigating caspase specificity, owing to surprising levels of endogenous caspase activity, while specificity profiles of the caspase sensors in WGE agree very well with traditional peptide probes. The full panel of biosensors was utilized for studying caspase activation and inhibition in several mammalian cytosolic extracts, clearly demonstrating that they can be utilized to directly monitor activation or inhibition of procaspase 3/7. Furthermore, the complete panel of caspase biosensors also provided new insights into caspase activation pathways wherein we surprisingly discovered the activation of procaspase 3/7 by caspase 4/5.

Original languageEnglish (US)
Pages (from-to)2353-2364
Number of pages12
JournalChemBioChem
Volume12
Issue number15
DOIs
StatePublished - Oct 17 2011

Fingerprint

Biosensing Techniques
Caspases
Biosensors
Chemical activation
Caspase 7
Reticulocytes
Caspase 3
Substrate Specificity
Triticum
Caspase 10
Caspase 1
Rabbits
Substrates
Luciferases
Apoptosis
Peptides

Keywords

  • Apoptosis
  • Biosensors
  • Caspases
  • Coiled coils
  • Proteases

ASJC Scopus subject areas

  • Biochemistry
  • Organic Chemistry
  • Molecular Medicine
  • Molecular Biology

Cite this

A comprehensive panel of turn-on caspase biosensors for investigating caspase specificity and caspase activation pathways. / Shekhawat, Sujan S.; Campbell, Sean T.; Ghosh, Indraneel.

In: ChemBioChem, Vol. 12, No. 15, 17.10.2011, p. 2353-2364.

Research output: Contribution to journalArticle

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