Conformational Rearrangements in the Pro-apoptotic Protein, Bax, as It Inserts into Mitochondria A CELLULAR DEATH SWITCH
Conformational Rearrangements in the Pro-apoptotic Protein, Bax, as It Inserts into Mitochondria A CELLULAR DEATH SWITCH
复制标题
DOI：
10.1074/jbc.m114.593897
复制
发表时间：
2014-11-21
影响因子：
4.8
通讯作者：
Tjandra, Nico
中科院分区：
文献类型：
--
作者：
Gahl, Robert F.;He, Yi;Tjandra, Nico
B细胞淋巴瘤2(Bcl2)家族蛋白通过线粒体途径调节细胞凋亡的激活。这个家族中支持和反对细胞凋亡的成员相互保持控制，直到正确的时间致力于细胞凋亡。这一承诺的不归路是线粒体膜的通透性。促凋亡成员Bax从胞浆到线粒体的移位是这一事件的分子特征。我们使用了一种新的方法来可靠地检测一对荧光团之间的Forster共振能量转移(FRET)，以确定Bax在活细胞中发生这种移位时分子内的构象变化和分子间的接触。在胞浆中，我们的FRET测量表明，C-末端螺旋暴露在蛋白质的核心中，而不是隐藏在核心中。此外，荧光相关光谱(FCS)显示，胞内Bax的扩散速度比预期慢得多，这表明可能形成了络合物或瞬时的膜相互作用。将C-末端螺旋(α9)与α4螺旋进行交联降低了发生这些相互作用的可能性。易位后，我们的FRET测量表明，Bax分子通过涉及BH3结构域(螺旋2)和C末端螺旋的两个不同界面在线粒体中形成同源低聚物。这些发现对可能与其他调控细胞凋亡所必需的Bcl-2蛋白的接触有一定的意义。
The B-cell lymphoma 2 (Bcl-2) family of proteins regulates the activation of apoptosis through the mitochondria pathway. Pro-and anti-apoptotic members of this family keep each other in check until the correct time to commit to apoptosis. The point of no return for this commitment is the permeabilization of the outer mitochondrial membrane. Translocation of the pro-apoptotic member, Bax, from the cytosol to the mitochondria is the molecular signature of this event. We employed a novel method to reliably detect Forster resonance energy transfer (FRET) between pairs of fluorophores to identify intra-molecular conformational changes and inter-molecular contacts in Bax as this translocation occurs in live cells. In the cytosol, our FRET measurement indicated that the C-terminal helix is exposed instead of tucked away in the core of the protein. In addition fluorescence correlation spectroscopy (FCS) showed that cytosolic Bax diffuses much slower than expected, suggesting possible complex formation or transient membrane interaction. Cross-linking the C-terminal helix (alpha 9) to helix alpha 4 reduced the potential of those interactions to occur. After translocation, our FRET measurements showed that Bax molecules form homo-oligomers in the mitochondria through two distinct interfaces involving the BH3 domain (helix alpha 2) and the C-terminal helix. These findings have implications for possible contacts with other Bcl-2 proteins necessary for the regulation of apoptosis.