BACKGROUND BAX activation is a crucial step for commitment to apoptosis. Several activators, such as BimBH3-based therapeutic peptides and cleaved Bid (cBid) protein, can trigger BAX-mediated apoptosis, but it is unclear whether they proceed through the same pathway. METHODS Here we utilize PEGylation-based approach, which is shown to efficiently shield individual binding grooves in BAX from activators, to investigate and reveal that the activators take different routes to induce BAX-mediated apoptosis. Various spectroscopic/biochemical tools, including electron spin resonance, circular dichroism, fluorescence recovery after photobleaching, and label-transfer assay, were employed to reveal details in the processes. RESULTS We observe a key mutant BAX 164-PEG that acts differently in response to cBid and BimBH3 stimuli. While BimBH3 directly interacts with the trigger groove (TG) to induce the conformational changes in BAX that includes the release of α9 from the canonical groove (CG) and oligomerization, cBid engages with CG and works with mitochondrial lipids to fully activate BAX. CONCLUSION PEGylation-based approach is proven useful to shield individual binding grooves of BAX from apoptotic stimuli. Groove engagement in CG of BAX is required for a full cBid-induced BAX activation. This study has identified differences in the pathways involved during the initiation of BAX activation by full-length cBid protein versus synthetic BimBH3-based peptides. GENERAL SIGNIFICANCE Our finding is potentially valuable for therapeutic application as the pore-forming activity of 164-PEG is independent from the cBid-mediated apoptotic pathways, but can be administrated by the synthetic short peptides.

中文翻译：

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基于PEG化的策略，以鉴定参与凋亡BAX蛋白活化的途径。

背景技术BAX激活是致力于凋亡的关键步骤。几种激活剂，例如基于BimBH3的治疗性肽和裂解的Bid（cBid）蛋白，可以触发BAX介导的细胞凋亡，但尚不清楚它们是否沿着相同的途径进行。方法在这里，我们利用了基于PEG化的方法，该方法可有效屏蔽BAX与激活剂的结合槽，以研究并揭示激活剂采取不同的途径诱导BAX介导的细胞凋亡。各种光谱/生化工具，包括电子自旋共振，圆二色性，光漂白后的荧光恢复和标记转移分析，被用来揭示过程中的细节。结果我们观察到一个关键突变体BAX 164-PEG，其对cBid和BimBH3刺激的反应不同。虽然BimBH3直接与触发槽（TG）相互作用以诱导BAX的构象变化，包括从规范槽（CG）释放α9和低聚，但cBid与CG结合并与线粒体脂质协同工作以完全激活BAX。结论事实证明，基于聚乙二醇化的方法可有效保护BAX的单个结合槽免受凋亡刺激。要完全cBid诱导BAX激活，需要在BAX CG中使用凹槽。这项研究已经确定了全长cBid蛋白与合成的基于BimBH3的肽在BAX激活起始过程中涉及的途径的差异。一般意义我们的发现对于治疗应用具有潜在的价值，因为164-PEG的成孔活性独立于cBid介导的凋亡途径，