Molecular dissection of Oncoprotein-induced apoptosis

描述(由申请人提供)：Myc癌蛋白诱导的细胞凋亡被认为是防止肿瘤出现的主要先天机制。然而，尽管许多不同的蛋白质都被认为是下游的关键凋亡效应因子，但将Myc与基础的凋亡机制联系起来的分子机制在很大程度上还不清楚。Myc使细胞对广泛的侮辱诱导的细胞凋亡敏感，这表明Myc起着启动一些决定细胞凋亡的中心决定因素的作用。与此一致，我们的工作直接表明线粒体是Myc促凋亡作用的快速和直接的目标。基于这一观察，我们使用有偏倚的蛋白质组学方法来鉴定在人和啮齿动物成纤维细胞中随着Myc的急性激活而迅速改变丰度或迁移率的Bclxl相互作用蛋白。已经确定了三个候选蛋白--已知的促凋亡BH3蛋白Bim，最近被发现由Myc靶基因编码的具有共同BH3结构域的新蛋白PDCD10，以及小G蛋白Rab7。我们建议使用一组独特的可切换的Myc小鼠转基因模型，在体外和体内的几个组织中，通过结合分子、细胞和分子生物学和遗传学策略来验证这些候选Myc凋亡效应器。最后，我们建议扩展我们最初的蛋白质组学研究，以建立一个伴随Myc激活而迅速发生的线粒体蛋白质组变化的图谱，特别是重点放在最有可能成为Myc凋亡效应靶标的线粒体外层隔室的限制性蛋白质上。这些研究将提供新的见解，将放松调控的增殖与其固有的肿瘤抑制凋亡失效安全机制联系起来，以及这种联系是如何在癌细胞中被切断的。

DESCRIPTION (provided by applicant): The induction of apoptosis by the Myc oncoprotein is considered to be a major innate mechanism preventing the emergence of neoplasia. However, although many, diverse proteins have each been proposed as key downstream apoptotic effectors, the molecular mechanisms linking Myc with the basal the apoptotic machinery are largely unknown. Myc sensitizes cells to apoptosis induced by a wide range of insults, suggesting that Myc acts to prime some central determinant of apoptotic decision-making. Consistent with this, our work has directly implicated the mitochondrion as rapid and immediate target of Myc pro-apoptotic action. Based upon this observation, we have used a biased proteomic approach to identify Bcl-xL-interacting proteins that rapidly change in abundance or mobility following acute Myc activation in human and rodent fibroblasts. Three candidates have been identified - the known pro-apoptotic BH3 protein Bim, a novel protein with a consensus BH3 domain called PDCD10 recently shown to be encoded by a Myc target gene, and the small G protein Rab7. We propose to validate each of these candidate Myc apoptotic effectors by a combination of molecular, cell and molecular biological and genetics strategies, both in vitro and in several tissues in vivo using a unique set of switchable Myc mouse transgenic models. Finally, we propose to extend our initial proteomic study to establish a map of the proteomic changes in mitochondria that rapidly accompany Myc activation, with particular emphasis on the restricted proteins in the outer mitochondrial compartment most likely targeted by Myc apoptotic effectors. These studies will provide new insights into the molecular machinery linking deregulated proliferation with its innate tumor suppressive apoptotic failsafe, and how that linkage is severed in cancer cells.