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Pull-down of Calmodulin-binding Proteins
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Pull-down of Calmodulin-binding Proteins

Published on: January 23, 2012

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N-terminal and C-terminal domains of calmodulin mediate FADD and TRADD interaction

Giuliana Papoff1, Nadia Trivieri1, Sonia Marsilio1

  • 1National Research Council, Institute of Cell Biology and Neurobiology, Campus Adriano Buzzati-Traverso, Monterotondo, Rome, Italy.

Plos One
|February 3, 2015
PubMed

Insights

Calmodulin binding to FADD and TRADD proteins, crucial for cell fate, is regulated by calcium and methionine oxidation. Both N- and C-terminal domains of calmodulin are essential for these interactions.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Immunology

Background:

  • FADD (Fas-associated death domain) and TRADD (Tumor Necrosis Factor Receptor 1-associated death domain) proteins regulate mammalian cell fate.
  • These proteins are involved in death receptor-mediated signaling and linked to innate immunity via Toll-like receptors.

Purpose of the Study:

  • To identify and characterize calmodulin (CaM) binding sites on TRADD and FADD.
  • To investigate the role of calcium and CaM oxidation in regulating CaM-TRADD and CaM-FADD interactions.

Main Methods:

  • Database search analysis
  • Site-directed mutagenesis
  • Calmodulin pull-down assays
  • Binding assays with Met-to-Leu CaM mutants

Main Results:

  • A calcium-dependent CaM binding site was identified in the α-helices 1-2 of the TRADD death domain.
  • Oxidation of CaM methionines significantly reduced CaM affinity for both FADD and TRADD.
  • Both N- and C-terminal domains of CaM are critical for binding to FADD and TRADD.

Conclusions:

  • Calmodulin binding to TRADD and FADD is regulated by calcium and CaM oxidation.
  • These findings provide insights into the molecular mechanisms controlling cell death and innate immune signaling pathways.

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