Petit Patrice X, Dupaigne Pauline, Pariselli Fabrizio, Gonzalvez François, Etienne François, Rameau Carole, Bernard Sophie
Institut Cochin, INSERM U567/CNRS UMR8104, Université Paris Descartes, France.
FEBS J. 2009 Nov;276(21):6338-54. doi: 10.1111/j.1742-4658.2009.07345.x. Epub 2009 Oct 1.
BH3 interacting domain death agonist (Bid), a pro-apoptotic member of the Bcl-2 family of proteins, is activated through cleavage by caspase-8. The active C-terminal fragment of Bid (tBid) translocates to the mitochondria where it interacts with cardiolipins at contact sites and induces the release of cytochrome c by a mechanism that is not yet fully understood. It has been shown that the alpha-helices alphaH6 and alphaH7 (which create the hairpin-forming domain of tBid) mediate the insertion of Bid into mitochondrial membranes and are essential for the cytochrome c-releasing activity. In the present study, we focused on the interaction between the alphaH6 and the mitochondrial membrane. By the use of single-cell electropermeabilization associated with flow cytometric analysis of intact cells, we demonstrated that H6 is able to induce cell death when used in the micromolar range. We also studied the interactions of the alphaH6 with artificial monolayers. We showed that the presence of negatively charged cardiolipins greatly enhances the insertion of alphaH6 into the phospholipid monolayer. The modification of two charged amino acid residues in alphaH6 abolished its insertion and also its in vivo effects. Furthermore, the negative values of the excess areas of mixing indicate that attractive interactions between cardiolipins and alphaH6 occur in the mixed monolayers. Fluorescence microscopy observations revealed that alphaH6 significantly disrupts cardiolipin packing and stabilizes the fluid lipid phase. These results suggest that cardiolipins at the contact sites between the two mitochondrial membranes could mediate the binding of tBid via alphaH6.
BH3相互作用结构域死亡激动剂（Bid）是Bcl-2蛋白家族的促凋亡成员，通过caspase-8的切割而被激活。Bid的活性C末端片段（tBid）易位至线粒体，在接触位点与心磷脂相互作用，并通过一种尚未完全了解的机制诱导细胞色素c的释放。研究表明，α螺旋αH6和αH7（形成tBid的发夹结构域）介导Bid插入线粒体膜，并且对于细胞色素c释放活性至关重要。在本研究中，我们聚焦于αH6与线粒体膜之间的相互作用。通过将单细胞电穿孔与完整细胞的流式细胞术分析相结合，我们证明当在微摩尔范围内使用时，H6能够诱导细胞死亡。我们还研究了αH6与人工单层膜的相互作用。我们发现带负电荷的心磷脂的存在极大地增强了αH6插入磷脂单层膜的能力。αH6中两个带电荷氨基酸残基的修饰消除了其插入能力及其体内效应。此外，混合过量面积的负值表明在混合单层膜中心磷脂与αH6之间存在吸引相互作用。荧光显微镜观察显示，αH6显著破坏心磷脂的堆积并稳定脂质流体相。这些结果表明，两个线粒体膜接触位点的心磷脂可通过αH6介导tBid的结合。
