A Novel Motif in the Crohn's Disease Susceptibility Protein, NOD2, Allows TRAF4 to Down-regulate Innate Immune Responses

Craig R. Homer,Derek W. Abbott,Christine McDonald,Jill M. Marinis

doi:10.1074/jbc.m110.189308

Abstract

The Crohn's disease and early onset sarcoidosis susceptibility protein, NOD2, coordinates innate immune signaling pathways. Because dysregulation of this coordination can lead to inflammatory disease, maintaining appropriate activation of the NOD2 signaling pathway is paramount in immunologic homeostasis. In this work, we identify the atypical tumor necrosis factor-associated factor (TRAF) family member, TRAF4, as a key negative regulator of NOD2 signaling. TRAF4 inhibits NOD2-induced NF-κB activation and directly binds to NOD2 to inhibit NOD2-induced bacterial killing. We find that two consecutive glutamate residues in NOD2 are required for interaction with TRAF4 and inhibition of NOD2 signaling because mutation of these residues abrogated both TRAF4 binding and inhibition of NOD2. This work identifies a novel negative regulator of NOD2 signaling. Additionally, it defines a TRAF4 binding motif within NOD2 involved in termination of innate immune signaling responses.

Highlights

Upon intracellular exposure to a breakdown product of bacterial peptidoglycan, muramyl dipeptide (MDP), NOD2 binds to the scaffolding protein kinase, RIP2, via caspase recruitment domain interactions [1, 7]
The reciprocal experiment was performed in which MEKK4 was immunoprecipitated from lysates and TRAF4 binding was assayed by Western blotting
To determine if TRAF6 or other tumor necrosis receptor-associated factor (TRAF) family members bind to MEKK4, FLAG-TRAF2, FLAG-TRAF3, FLAGTRAF4, or Omni-TRAF6 was cotransfected with HA-MEKK4 into HEK293T cells

Summary

Introduction

Upon intracellular exposure to a breakdown product of bacterial peptidoglycan, muramyl dipeptide (MDP), NOD2 binds to the scaffolding protein kinase, RIP2 (receptor-interacting protein 2), via caspase recruitment domain interactions [1, 7]. TRAF4 Binds to and Inhibits NOD2 Signaling with MEKK4 and find that, like MEKK4, TRAF4 negatively regulates MDP-induced NF-␬B activation and killing of intracellular bacteria. NOD2 Binds to TRAF4 and Bridges TRAF4 to RIP2—In the absence of MDP, MEKK4 is thought to maintain low levels of NOD2-induced NF-␬B activation by sequestering RIP2, 1940 JOURNAL OF BIOLOGICAL CHEMISTRY

Results

Conclusion