Molecular dissection of Oncoprotein-induced apoptosis

描述（由申请人提供）：Myc癌蛋白诱导细胞凋亡被认为是防止肿瘤发生的主要先天机制。然而，尽管许多不同的蛋白质都被认为是关键的下游凋亡效应因子，但将Myc与基本凋亡机制联系起来的分子机制在很大程度上是未知的。Myc使细胞对多种损伤诱导的凋亡敏感，这表明Myc在凋亡决策中起着重要的决定作用。与此一致，我们的工作直接暗示线粒体是Myc促凋亡作用的快速和直接目标。基于这一观察，我们使用了一种有偏向的蛋白质组学方法来鉴定人类和啮齿动物成纤维细胞中Myc急性激活后，bcl - xl相互作用蛋白的丰度或流动性迅速变化。已经确定了三种候选蛋白——已知的促凋亡BH3蛋白Bim，一种具有共识BH3结构域的新蛋白PDCD10，最近被证明由Myc靶基因编码，以及小G蛋白Rab7。我们建议通过结合分子、细胞和分子生物学和遗传学策略，在体外和体内的几种组织中使用一组独特的可切换的Myc小鼠转基因模型来验证这些候选Myc凋亡效应物。最后，我们建议扩展我们最初的蛋白质组学研究，以建立线粒体中伴随Myc激活的蛋白质组学变化图，特别强调线粒体外室中最有可能被Myc凋亡效应物靶向的限制性蛋白质。这些研究将提供新的见解，了解将不受管制的增殖与其固有的肿瘤抑制凋亡故障安全联系起来的分子机制，以及这种联系如何在癌细胞中被切断。

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.