T69E模拟磷酸化修饰对Bcl-2与Nur77相互作用的影响

doi:10.11938/cjmr20212901

摘要/Abstract

摘要：

Bcl-2蛋白是Bcl-2家族中主要行使抗凋亡功能的蛋白质.核孤儿受体蛋白Nur77可出核定位至线粒体与其发生相互作用，促使Bcl-2的功能发生逆转，由细胞保护者转变为细胞杀手，从而促发细胞凋亡.Bcl-2的功能受翻译后修饰调控，如位于其BH3与BH4基序间无序loop区上T69位点的磷酸化.由于缺乏全长Bcl-2蛋白的结构信息，目前loop区T69的磷酸化修饰对两种蛋白相互作用的影响尚不清楚，缺乏原子水平的相关研究.因此，我们构建了含有loop区的胞内全长Bcl-2，并用T69E突变体模拟稳定的磷酸化修饰，综合运用圆二色谱（CD）、免疫印迹（Western Blot）、核磁共振（NMR）等技术手段对其相互作用进行研究.我们发现相比只含结构区的Bcl-2/xl嵌合体，胞内全长的Bcl-2和Nur77具有更强的相互作用，并且T69E模拟磷酸化修饰减弱了Bcl-2与Nur77的相互作用.该项研究对基于Bcl-2和Nur77信号转导通路和癌症细胞靶向凋亡的研究具有重要的参考意义.

关键词: Bcl-2, 核磁共振（NMR）, 磷酸化, 蛋白质相互作用, 细胞凋亡因子

Abstract:

Bcl-2 mainly executes anti-apoptotic function in the Bcl-2 family. Nuclear orphan receptor Nur77 can be translocated from nucleus to mitochondria, then interact with Bcl-2 to reverse the function of Bcl-2 from a cell protector to a cell killer, thereby inducing cell apoptosis. The function of Bcl-2 is regulated by post-translational modification, such as the phosphorylation of T69 in the intrinsically disordered loop between BH3 and BH4 motifs. However, due to the lack of structural information of the full-length protein, the effect of phosphorylation modification of T69 in the loop region on the interaction of the two proteins is still unclear, and there is a lack of related research at the atomic level. Therefore, we constructed a full-length intracellular Bcl-2 containing loop region and T69E mutant to mimic stable phosphorylation modification, and investigated the interactions by combining circular dichroism (CD), Western blot and nuclear magnetic resonance (NMR) methods. It was observed that the full-length Bcl-2 had a stronger interaction with Nur77 compared to the Bcl-2/xl chimera, and the phosphorylation modification mimicked by T69E weakened the interaction between Bcl-2 and Nur77. The results will promote future research based on Bcl-2 and Nur77 signal transduction pathways and the apoptosis targeting cancer cells.

Key words: Bcl-2, nuclear magnetic resonance (NMR), phosphorylation, protein-protein interaction, apoptosis factor

中图分类号:

O482.53

徐倩,陈朗,胡翔颖,李从刚,刘乙祥,姜凌. T69E模拟磷酸化修饰对Bcl-2与Nur77相互作用的影响[J]. 波谱学杂志, 2022, 39(1): 87-95.

Qian XU,Lang CHEN,Xiang-ying HU,Cong-gang LI,Yi-xiang LIU,Ling JIANG. The Effect of T69E-mimicked Phosphorylation on the Interaction Between Bcl-2 and Nur77[J]. Chinese Journal of Magnetic Resonance, 2022, 39(1): 87-95.

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