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 Kd¼0.3lM; however the binding affinity strongly decreases when the active site of GSTP1-1 is occupied by the substrate GSH (KdZ2.6lM) or is inactivated by oxidation (Kd¼1.7lM). 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

De Luca, A., Mei, G., Rosato, N., Nicolai, E., Federici, L., Palumbo, C., et al. (2014). The fine-tuning of TRAF2-GSTP1-1 interaction: effect of ligand binding and in situ detection of the complex. CELL DEATH & DISEASE, 5(1) [10.1038/cddis.2013.529.].

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

De Luca, A;MEI, GIAMPIERO;ROSATO, NICOLA;NICOLAI, ELEONORA;PALUMBO, CAMILLA;CACCURI, ANNA MARIA
2014-01-01

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 Kd¼0.3lM; however the binding affinity strongly decreases when the active site of GSTP1-1 is occupied by the substrate GSH (KdZ2.6lM) or is inactivated by oxidation (Kd¼1.7lM). 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
2014
Pubblicato
Rilevanza internazionale
Articolo
Esperti anonimi
Settore BIO/10 - BIOCHIMICA
Settore BIO/11 - BIOLOGIA MOLECOLARE
English
Con Impact Factor ISI
TRAF; GST; MAPK; NBDHEX; osteosarcoma
De Luca, A., Mei, G., Rosato, N., Nicolai, E., Federici, L., Palumbo, C., et al. (2014). The fine-tuning of TRAF2-GSTP1-1 interaction: effect of ligand binding and in situ detection of the complex. CELL DEATH & DISEASE, 5(1) [10.1038/cddis.2013.529.].
De Luca, A; Mei, G; Rosato, N; Nicolai, E; Federici, L; Palumbo, C; Pastore, A; Serra, M; Caccuri, Am
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/92807
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