Anticancer drug-induced BH3-only protein.Bak interactions

描述（由申请人提供）：由于细胞凋亡是一种生物化学上独特的细胞死亡形式，是化疗或激素控制在癌细胞中引发的主要反应之一，因此对细胞凋亡过程的调控有很大的兴趣。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起着关键作用。特别是，人们认为Bax和Bak引起线粒体外膜通透性（MOMP）；抗凋亡家族成员如Bcl-2抑制MOMP；仅bh3亚家族的成员通过结合Bax/Bak（“直接激活剂”）或结合抗凋亡药物（“致敏剂”）促进MOMP。Bax和Bak如何被激活以及它们如何引起MOMP尚不完全清楚。在初步研究中，我们检测了bh3蛋白介导的Bak活化。利用纯化蛋白，我们首次证明了Bak寡聚化是一种遗传机制

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.