The fine-tuning of TRAF2-GSTP1-1 interaction: effect of ligand binding and in situ detection of the complex.
The fine-tuning of TRAF2-GSTP1-1 interaction: effect of ligand binding and in situ detection of the complex.
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DOI：
10.1038/cddis.2013.529
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发表时间：
2014-01-23
影响因子：
9
通讯作者：
中科院分区：
文献类型：
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我们提供了谷胱甘肽转移酶P1-1 （GSTP1-1）与TNF受体相关因子2 （TRAF2）的TRAF结构域之间直接相互作用的第一个生化证据，并描述了配体结合如何调节这种平衡。杂络合物的解离常数Kd=0.3 μM；而当GSTP1-1的活性位点被底物GSH占据（Kd≥2.6 μM）或被氧化失活（Kd=1.7 μM）时，GSTP1-1的结合亲和力明显降低。这表明GSTP1-1的traf2结合区涉及gsh结合位点。与GSH为唯一配体时相比，GSTP1-1抑制剂NBDHEX进一步降低了复合物的结合亲和力；这表明GSTP1-1活性位点的疏水部分也有助于相互作用。因此，我们假设TRAF2结合使GSTP1-1失活；然而，使用考虑到GSTP1-1二聚体性质的模型分析数据表明，GSTP1-1在复合物中只参与一个亚基，而第二个亚基保持催化活性或与其他蛋白质结合。我们还在细胞水平上分析了GSTP1-1与TRAF2的关联。TRAF2-GSTP1-1复合物在u - 2s细胞中组成性存在，但在S、G2和M期强烈减少。因此，这种相互作用似乎以细胞周期依赖的方式受到调节。单个蛋白质水平的变化似乎太有限，无法解释在细胞从G0/G1到S-G2-M阶段观察到的复合物的急剧下降。而且，GSH在细胞内的含量非常高，总是使GSTP1-1饱和。有趣的是，NBDHEX的加入将TRAF2-GSTP1-1复合物维持在低水平，从而导致细胞周期阻滞在G2/M期延长。总的来说，这些发现表明，TRAF2在复合物中的可逆隔离可能对细胞周期进程至关重要，并且多种因素参与了这种相互作用的微调。
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 Kd=0.3 μM; however the binding affinity strongly decreases when the active site of GSTP1-1 is occupied by the substrate GSH (Kd≥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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影响因子：
2.9
作者：
De Luca, Anastasia;Federici, Luca;Caccuri, Anna Maria
通讯作者：
Caccuri, Anna Maria
影响因子：
11.2
作者：
Federici, Luca;Lo Sterzo, Carlo;Caccuri, Anna Maria
通讯作者：
Caccuri, Anna Maria
影响因子：
4.8
作者：
Ricci, G;De Maria, F;Caccuri, AM
通讯作者：
Caccuri, AM
影响因子：
32.4
作者：
Kelliher, MA;Grimm, S;Leder, P
通讯作者：
Leder, P
DOI：
10.1165/rcmb.2011-0195oc
发表时间：
2012-04-01
影响因子：
6.4
作者：
Battisti, Sonia;Valente, Davide;Palumbo, Camilla
通讯作者：
Palumbo, Camilla