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Cell Death Dis. 2014 Jan 23;5:e1015. doi: 10.1038/cddis.2013.529.

The fine-tuning of TRAF2-GSTP1-1 interaction: effect of ligand binding and in situ detection of the complex.

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  • 1The NAST Centre for Nanoscience & Nanotechnology & Innovative Instrumentation, University of Tor Vergata, Rome, Italy.
  • 2Department of Experimental Medicine and Surgery, University of Tor Vergata, Rome, Italy.
  • 3Department of Biomedical Sciences, University of Chieti G D'Annunzio, CeSI Center of Excellence on Aging, Chieti, Italy.
  • 4Department of Clinical Sciences and Translational Medicine, University of Tor Vergata, Rome, Italy.
  • 5Laboratory of Metabolomics and Proteomics, Children's Hospital IRCCS Bambino Gesù, Rome, Italy.
  • 6Laboratory of Experimental Oncology, Istituto Ortopedico Rizzoli IRCCS, Bologna, Italy.
  • 71] The NAST Centre for Nanoscience & Nanotechnology & Innovative Instrumentation, University of Tor Vergata, Rome, Italy [2] Department of Experimental Medicine and Surgery, University of Tor Vergata, Rome, Italy.

Abstract

We provide the first biochemical evidence of a direct interaction between the glutathione transferase P1-1 (GSTP1-1) and the TRAF domain of TNF receptor-associated factor 2 (TRAF2), and describe how ligand binding modulates such an equilibrium. The dissociation constant of the heterocomplex is K(d)=0.3 μM; however the binding affinity strongly decreases when the active site of GSTP1-1 is occupied by the substrate GSH (K(d)≥2.6 μM) or is inactivated by oxidation (Kd=1.7 μM). This indicates that GSTP1-1's TRAF2-binding region involves the GSH-binding site. The GSTP1-1 inhibitor NBDHEX further decreases the complex's binding affinity, as compared with when GSH is the only ligand; this suggests that the hydrophobic portion of the GSTP1-1 active site also contributes to the interaction. We therefore hypothesize that TRAF2 binding inactivates GSTP1-1; however, analysis of the data, using a model taking into account the dimeric nature of GSTP1-1, suggests that GSTP1-1 engages only one subunit in the complex, whereas the second subunit maintains the catalytic activity or binds to other proteins. We also analyzed GSTP1-1's association with TRAF2 at the cellular level. The TRAF2-GSTP1-1 complex was constitutively present in U-2OS cells, but strongly decreased in S, G2 and M phases. Thus the interaction appears regulated in a cell cycle-dependent manner. The variations in the levels of individual proteins seem too limited to explain the complex's drastic decline observed in cells progressing from the G0/G1 to the S-G2-M phases. Moreover, GSH's intracellular content was so high that it always saturated GSTP1-1. Interestingly, the addition of NBDHEX maintains the TRAF2-GSTP1-1 complex at low levels, thus causing a prolonged cell cycle arrest in the G2/M phase. Overall, these findings suggest that a reversible sequestration of TRAF2 into the complex may be crucial for cell cycle progression and that multiple factors are involved in the fine-tuning of this interaction.

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