Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan.
Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan.
Biochim Biophys Acta Gen Subj. 2020 Apr;1864(4):129541. doi: 10.1016/j.bbagen.2020.129541. Epub 2020 Jan 24.
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.
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.
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.
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.
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.
BAX 的激活是细胞凋亡的关键步骤。几种激活剂，如基于 BimBH3 的治疗性肽和裂解 Bid（cBid）蛋白，可触发 BAX 介导的细胞凋亡，但尚不清楚它们是否通过相同的途径进行。
我们采用聚乙二醇化方法，该方法可有效地将 BAX 中的各个结合沟从激活剂中屏蔽，以研究和揭示激活剂采用不同的途径诱导 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、光漂白后荧光恢复和标记转移测定，用于揭示过程中的细节。
我们观察到一种关键的突变体 BAX 164-PEG，它对 cBid 和 BimBH3 刺激的反应不同。虽然 BimBH3 直接与触发沟（TG）相互作用，诱导 BAX 的构象变化，包括α9 从经典沟（CG）的释放和寡聚化，但 cBid 与 CG 结合，并与线粒体脂质一起作用以完全激活 BAX。
聚乙二醇化方法被证明可有效屏蔽 BAX 的各个结合沟免受凋亡刺激。BAX 的 CG 结合是 cBid 诱导的 BAX 完全激活所必需的。这项研究确定了全长 cBid 蛋白与合成的基于 BimBH3 的肽诱导 BAX 激活起始过程中涉及的途径的差异。
我们的发现对于治疗应用具有潜在价值，因为 164-PEG 的孔形成活性与 cBid 介导的凋亡途径无关，但可由合成的短肽进行给药。
