Institut Cochin, CNRS UMR8104 (Centre National de la Recherche Scientifique), INSERM U 567, Université Paris-Descartes, Paris, France.
PLoS One. 2010 Feb 22;5(2):e9342. doi: 10.1371/journal.pone.0009342.
The pro-apoptotic effector Bid induces mitochondrial apoptosis in synergy with Bax and Bak. In response to death receptors activation, Bid is cleaved by caspase-8 into its active form, tBid (truncated Bid), which then translocates to the mitochondria to trigger cytochrome c release and subsequent apoptosis. Accumulating evidence now indicate that the binding of tBid initiates an ordered sequences of events that prime mitochondria from the action of Bax and Bak: (1) tBid interacts with mitochondria via a specific binding to cardiolipin (CL) and immediately disturbs mitochondrial structure and function idependently of its BH3 domain; (2) Then, tBid activates through its BH3 domain Bax and/or Bak and induces their subsequent oligomerization in mitochondrial membranes. To date, the underlying mechanism responsible for targeting tBid to mitochondria and disrupting mitochondrial bioenergetics has yet be elucidated.
The present study investigates the mechanism by which tBid interacts with mitochondria issued from mouse hepatocytes and perturbs mitochondrial function. We show here that the helix alphaH6 is responsible for targeting tBid to mitochondrial CL and disrupting mitochondrial bioenergetics. In particular, alphaH6 interacts with mitochondria through electrostatic interactions involving the lysines 157 and 158 and induces an inhibition of state-3 respiration and an uncoupling of state-4 respiration. These changes may represent a key event that primes mitochondria for the action of Bax and Bak. In addition, we also demonstrate that tBid required its helix alphaH6 to efficiently induce cytochrome c release and apoptosis.
Our findings provide new insights into the mechanism of action of tBid, and particularly emphasize the importance of the interaction of the helix alphaH6 with CL for both mitochondrial targeting and pro-apoptotic activity of tBid. These support the notion that tBid acts as a bifunctional molecule: first, it binds to mitochondrial CL via its helix alphaH6 and destabilizes mitochondrial structure and function, and then it promotes through its BH3 domain the activation and oligomerization of Bax and/or Bak, leading to cytochrome c release and execution of apoptosis. Our findings also imply an active role of the membrane in modulating the interactions between Bcl-2 proteins that has so far been underestimated.
促凋亡效应因子 Bid 与 Bax 和 Bak 协同诱导线粒体凋亡。在死亡受体激活后，Bid 被 caspase-8 切割成其活性形式 tBid（截断 Bid），然后易位到线粒体，引发细胞色素 c 释放和随后的细胞凋亡。越来越多的证据表明，tBid 的结合引发了一系列有序的事件，使 Bax 和 Bak 能够作用于线粒体：（1）tBid 通过与心磷脂（CL）的特定结合与线粒体相互作用，并立即独立于其 BH3 结构域扰乱线粒体结构和功能；（2）然后，tBid 通过其 BH3 结构域激活 Bax 和/或 Bak，并诱导它们随后在线粒体膜中寡聚化。迄今为止，尚不清楚将 tBid 靶向线粒体并破坏线粒体生物能的潜在机制。
本研究调查了 tBid 与来自小鼠肝细胞的线粒体相互作用并扰乱线粒体功能的机制。我们在这里表明，螺旋 αH6 负责将 tBid 靶向线粒体 CL 并破坏线粒体生物能。特别是，αH6 通过涉及赖氨酸 157 和 158 的静电相互作用与线粒体相互作用，并诱导状态 3 呼吸抑制和状态 4 呼吸解偶联。这些变化可能代表一个关键事件，使线粒体为 Bax 和 Bak 的作用做好准备。此外，我们还证明 tBid 有效地诱导细胞色素 c 释放和细胞凋亡需要其螺旋 αH6。
我们的研究结果为 tBid 的作用机制提供了新的见解，特别强调了螺旋 αH6 与 CL 的相互作用对 tBid 的线粒体靶向和促凋亡活性的重要性。这些支持了 tBid 作为双功能分子的观点：首先，它通过其螺旋 αH6 与线粒体 CL 结合并破坏线粒体结构和功能，然后通过其 BH3 结构域促进 Bax 和/或 Bak 的激活和寡聚化，导致细胞色素 c 释放和凋亡执行。我们的研究结果还暗示了膜在调节 Bcl-2 蛋白之间相互作用方面的积极作用，而这一点迄今为止一直被低估。
