Anticancer drug-induced BH3-only protein.Bak interactions

描述(申请人提供)：由于细胞凋亡是一种生化上不同的细胞死亡形式，是由化疗或激素操纵在癌细胞中引发的主要反应之一，因此人们对了解细胞凋亡过程的调控一直很感兴趣。过去二十年的研究表明，Bcl-2家族成员在细胞的生死决定中发挥着关键作用。特别是，Tat Bax和Bak被认为引起线粒体外膜通透性(MOMP)；抗凋亡家族成员如Bc l-2抑制MOMP；仅BH3亚家族成员通过与Bax/Bak结合(“直接激活剂”)或结合抗凋亡剂(“敏化剂”)促进MOMP。Bax和Bak是如何被激活的，以及它们是如何引起MOMP的，目前还不完全清楚。在初步研究中，我们检测了BH3蛋白介导的Bak激活。使用纯化的蛋白质，我们第一次证明了Bak齐聚是一种 两步过程，包括初始形成BH3蛋白*Bak异源二聚体，然后在形成Bak同源低聚物期间释放仅BH3蛋白。我们还绘制了BH3-Only蛋白和Bak上的相互作用结构域，并证实它们参与了Bak在细胞环境中的激活。重要的是，这一分析的结果表明，Bak齐聚作用与目前BAX激活的每一种模型都有很大的不同。此外，我们的结果表明，Noxa与Bim和TBID一样，是一种仅激活BH3的蛋白质。为了在我们的结果的基础上，我们现在建议1)检查仅BH3-肽与Bak单体结合的影响，从而确认假定的结合位置并洞察使Bak为齐聚做准备的结构变化；2)阐明Bak的结构 通过形态、生化和计算方法的组合来提供关于Bak的作用和MOMP过程的新信息；以及3)研究Noxa的翻译后调控，我们已经确定Noxa是 BAK在硼替佐米诱导淋巴细胞凋亡过程中的激活。如果成功，拟议的实验不仅将为Bak激活过程提供新的见解，而且还将获得关于一种日益广泛使用的抗淋巴瘤药物的作用的重要新信息。

DESCRIPTION (provided by applicant): Because apoptosis, a biochemically distinct form of cell death, is one of the major responses triggered in cancer cells by either chemotherapy or hormonal manipulation, there has been substantial interest in understanding regulation of the apoptotic process. Studies performed over the past two decades have demonstrated that Bcl-2 family members play critical roles in cellular life/death decisions. In particular, it is thought tat Bax and Bak cause mitochondrial outer membrane permeabilization (MOMP); antiapoptotic family members such as Bcl-2 inhibit MOMP; and members of the BH3-only subfamily facilitate MOMP by either binding to Bax/Bak ("direct activators") or binding the antiapoptotics ("sensitizers"). How Bax and Bak are activated and how they cause MOMP is incompletely understood. In preliminary studies, we have examined BH3-only protein-mediated Bak activation. Using purified proteins, we have shown for the first time that Bak oligomerization is a two-step process involving initial formation of a BH3 protein*Bak heterodimer followed by release of the BH3-only protein during formation of Bak homo-oligomers. We have also mapped the interacting domains on the BH3-only protein and Bak and have confirmed their involvement during Bak activation in a cellular context. Importantly, the results of this analysis suggest substantial differences between Bak oligomerization and each of the current models of Bax activation. Moreover, our results implicate Noxa, like Bim and tBid, as an activator BH3-only protein. To build on our results, we now propose to 1) examine the effect of BH3-only peptide binding on the Bak monomer, thereby confirming the putative binding site and providing insight into the structural changes that prepare Bak for oligomerization; 2) elucidate the structure of Bak oligomers through a combination of morphological, biochemical and computational approaches designed to provide new information about the action of Bak and the process of MOMP; and 3) examine posttranslational regulation of Noxa, which we have identified as a critical participant in Bak activation during bortezomib-induced apoptosis in lymphoid cells. If successful, the proposed experiments will not only provide new insight into the process of Bak activation, but also yield important new information about action of an increasingly widely used anti-lymphoma agent.