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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
11:36

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Published on: July 25, 2019

Ubiquitin regulates caspase recruitment domain-mediated signaling by nucleotide-binding oligomerization

Aaron M Ver Heul1, C Andrew Fowler, S Ramaswamy

  • 1Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52246, USA.

The Journal of Biological Chemistry
|January 10, 2013
PubMed

Insights

Nucleotide-binding oligomerization domain-containing proteins (NOD1 and NOD2) activate immune responses. This study reveals that ubiquitin binding to NOD1 and NOD2 CARDs acts as a negative feedback mechanism, regulating RIP2 signaling.

Area of Science:

  • Immunology
  • Molecular Biology
  • Cell Biology

Background:

  • NOD1 and NOD2 are intracellular pattern recognition receptors crucial for innate immunity.
  • These receptors initiate inflammatory and autophagic pathways via their caspase recruitment domains (CARDs).
  • CARDs function as protein interaction hubs regulating immune signaling cascades.

Purpose of the Study:

  • To investigate the interaction of NOD1 CARD with ubiquitin (Ub).
  • To elucidate the role of Ub binding in regulating NOD1 and NOD2 signaling.
  • To understand the mechanism of negative feedback in NOD-mediated immune responses.

Main Methods:

  • Nuclear Magnetic Resonance (NMR) spectroscopy to identify the Ub binding site on NOD1 CARD.
  • Structure-guided mutagenesis to create NOD1 and NOD2 mutants with altered Ub binding.
  • In vitro and in vivo assays to assess the impact of Ub binding on receptor activity and downstream signaling.

Main Results:

  • NOD1 CARD directly binds ubiquitin (Ub) via a specific hydrophobic surface.
  • Ub competes with receptor-interacting protein kinase 2 (RIP2) for binding to NOD1 CARD.
  • Mutants of NOD1 and NOD2 lacking Ub binding exhibit enhanced ligand-stimulated signaling, indicating increased activity.

Conclusions:

  • Ubiquitin binding to NOD1 and NOD2 CARDs acts as a negative feedback regulator.
  • This Ub-mediated feedback mechanism modulates the activation of RIP2 and downstream immune signaling.
  • The findings provide new insights into the intricate regulation of NOD-dependent inflammatory and autophagic pathways.

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