In Vitro Inhibitory Mechanism Effect of TRAIP on the Function of TRAF2 Revealed by Characterization of Interaction Domains.

Eijaz Bhat,Chang Kim,Hyun Park,Sunghwan Kim

doi:10.3390/ijms19082457

Abstract

TRAF-interacting protein (TRAIP), a negative regulator of TNF-induced-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, inhibits adaptor protein TRAF2 by direct interaction and is critical in apoptosis, cell proliferation, antiviral response, and embryonic development. Although the critical function of TRAIP in NF-κB signaling is well-known, the molecular inhibitory mechanism of TRAIP remains unclear. We found that the TRAIP coiled-coil domain altered its stoichiometry between dimer and trimer in a concentration-dependent manner. Additionally, the TRAIP RING domain induced even higher-ordered assembly, which was necessary for interacting with the TRAF-N domain of TRAF2 but not TRAF1. Characterization of the TRAF-N domains of TRAF1 and TRAF2, the tentative TRAIP-binding region of TRAFs, suggested the molecular basis of the inhibitory effect of TRAIP on TRAF2 in NF-κB signaling.

Highlights

Tumor necrosis factor (TNF)-receptor associated factor (TRAF) proteins, TRAF1–TRAF7, are major signaling molecules that transduce signals for the TNF receptor (TNFR) and interleukin-1 receptor/Toll like receptor family signaling pathways in mammals and play critical roles in the regulation of the immune system and apoptosis [1,2,3]
It has been suggested that the coiled-coil domain of TRAF-interacting protein (TRAIP) interacts with the TRAF2 coiled-coil domain (TRAF-N) to inhibit TRAF2 function [12], we found that only the coiled-coil domain of TRAIP failed to form a complex with the TRAF2 TRAF-N domain in vitro
We found that the TRAF-N domain of TRAF proteins is important for trimer formation and stabilization of the TRAF domain, which is the functional unit of TRAF

Summary

Introduction

Tumor necrosis factor (TNF)-receptor associated factor (TRAF) proteins, TRAF1–TRAF7, are major signaling molecules that transduce signals for the TNF receptor (TNFR) and interleukin-1 receptor/Toll like receptor family signaling pathways in mammals and play critical roles in the regulation of the immune system and apoptosis [1,2,3]. In addition to the main function, several novel functions of TRAIP, including proliferation, chromosome alignment at early mitotic progression, and DNA damage response, have been reported [13,14,15]. Through these multi-functional effects, TRAIP is involved in many important cellular signaling pathways. The main inhibitory function of TRAIP in TNF-induced NF-κB activation was introduced a few decades ago, the molecular mechanism of TRAIP-TRAF2 interaction-mediated inhibition of TRAF2 function is not fully understood. Our biochemical analysis of TRAIP and TRAF1/2 provides insight into the molecular basis of TRAIP-mediated inhibition of TRAF function via a direct interaction in TNF-induced NF-κB activation

Results and Discussion

Purification and Characterization of TRAF-N Domains of TRAF1 and TRAF2

Native-PAGE Shift Assay

Solubility Assay

Thermostability Assay

Complex Association Assay by Size-Exclusion Chromatography