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Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation
08:47

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

Published on: March 5, 2018

Nucleotide binding to CARD12 and its role in CARD12-mediated caspase-1 activation

Chafen Lu1, Anlai Wang, Lin Wang

  • 1Millennium Pharmaceuticals, Inc., 35 Landsdowne Street, Cambridge, MA 02139, USA. chafenlu@berkeley.edu

Biochemical and Biophysical Research Communications
|May 11, 2005
PubMed

Insights

The CARD12 protein

Area of Science:

  • Molecular Biology
  • Immunology
  • Protein Biochemistry

Background:

  • CARD12 (Ipaf/Clan) is a key regulator of caspase-1 activation, belonging to the nucleotide-binding site and leucine-rich repeat (NBS-LRR) protein family.
  • NBS-LRR proteins possess a nucleotide-binding site (NBS) domain with conserved motifs, but its specific nucleotide-binding properties and functional role in CARD12 remain uncharacterized.

Purpose of the Study:

  • To investigate the nucleotide-binding properties of the CARD12 NBS domain.
  • To determine the functional significance of nucleotide binding for CARD12-mediated caspase-1 activation.

Main Methods:

  • Development of a novel nucleotide-binding assay to study CARD12.
  • Site-directed mutagenesis of the P-loop motif (K175R) within the NBS domain.
  • Assessment of CARD12 self-association, procaspase-1 interaction, and pro-interleukin-1beta processing in transfected cells.

Main Results:

  • The NBS domain of CARD12 specifically binds ATP/dATP.
  • A P-loop mutation (K175R) abolished ATP/dATP binding to CARD12.
  • This mutation significantly impaired CARD12 self-association, procaspase-1 binding, and subsequent caspase-1 activation-dependent processing of pro-interleukin-1beta.

Conclusions:

  • CARD12's function in caspase-1 activation is critically dependent on its nucleotide-binding site.
  • The P-loop motif and its nucleotide-binding capability are essential for CARD12's molecular interactions and signaling.
  • These findings elucidate key molecular mechanisms underlying CARD12-mediated inflammatory responses.

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