The in vivo role of the mitochondrial p53 cell death program

描述（由申请人提供）：p53肿瘤抑制因子复杂的凋亡功能是其体内抗肿瘤活性的核心。除了作为多种凋亡基因的转录调节因子的经典作用外，p53还具有转录不依赖的凋亡活性。在上一个拨款周期中，我们阐明了后者的机制。我们发现野生型p53蛋白通过与线粒体通透性调节因子Bcl2家族的抗凋亡和促凋亡成员进行蛋白-蛋白相互作用，在线粒体中发挥直接作用，从而执行已知最短的p53死亡信号通路：1)在p53依赖性死亡过程中，一小部分应激诱导的野生型p53迅速易位到线粒体。这是一种普遍的p53反应，发生在原代、永生和转化培养细胞中，以及在p53诱导的整个应激范围（如DNA损伤、缺氧和癌基因解除管制）下的正常组织中。2)大多数p53转运到外膜，wtp53 -而不是肿瘤相关的p53突变体-通过其dna结合域与BclXL和Bcl2相互作用，诱导Bak寡聚化和外膜渗透，释放细胞色素C， Smac等凋亡激活因子。3)故意靶向p53到线粒体足以诱导p53缺陷肿瘤细胞凋亡和集落抑制。肿瘤衍生的p53转激活缺陷错义突变体同时失去了与BclXL相互作用的能力，这表明p53突变通过同时取消p53的转录和线粒体凋亡活性代表了“双重打击”。4)在受辐射小鼠中，线粒体p53易位触发了辐射敏感组织中快速的第一波细胞死亡。在胸腺——反应的原型组织——这种波后来被p53的转录程序加强。5)关于易位的机制，mdm2型E3连接酶的单泛素化促进了线粒体p53易位。与细胞核不同，细胞质中含有一个独立的、独特的p53库，它变得应力稳定，并作为p53易位的主要来源。到达线粒体后，p53通过应激诱导的p53-HAUSP复合体被线粒体HAUSP快速去泛素化，产生具有凋亡活性的非泛素化p53。6)在cmyc驱动的Burkitt淋巴瘤小鼠模型中，线粒体靶向野生型p53的逆转录病毒基因转移在体内可有效杀伤p53-null、ARF-null和p53-突变的肿瘤细胞。本建议侧重于这项有前途的研究的下一个重要阶段。将生成相关的动物模型，明确p53在线粒体的参与和作用程度。目的1和2将建立转基因和可切换的mitop53敲入小鼠模型，以评估线粒体p53程序对p53的急性遗传毒性反应和长期肿瘤抑制的贡献。目的3探讨线粒体p53程序是否有助于缺血性组织损伤的急性病理。Aim 4测试线粒体p53除了触发Bax/Bak脂质孔外，是否也激活通透性过渡孔（PTP）。公共卫生相关性：p53在人类中是一种重要的肿瘤抑制因子，因为它在细胞遭受DNA损伤后控制着强大的细胞死亡反应。除了在细胞核中开启其他死亡效应基因外，p53还通过蛋白质相互作用在线粒体中直接运行细胞死亡程序。重要的是，使用线粒体靶向p53融合蛋白，它的力量可以被利用作为p53介导的癌细胞细胞死亡的最短回路。基于这些研究，这项有前景的研究的下一个重要阶段是明确的：需要生成相关的动物模型，以充分定义线粒体p53程序在临床重要组织损伤的生理和病理生理反应中的程度。此外，需要评估线粒体p53程序对动物长期抑制的贡献。这是这项建议的要旨。此外，该建议旨在获得更多的机制洞察p53如何在线粒体中工作以渗透这些细胞器。

DESCRIPTION (provided by applicant): The complex apoptotic functions of the p53 tumor suppressor are central to its antineoplastic activity in vivo. Besides its well-understood classic action as a transcriptional regulator of multiple apoptotic genes, p53 also exerts a transcription-independent apoptotic activity. In the previous grant cycle we elucidated a mechanism for the latter. We showed that wild type p53 protein has a direct role at the mitochondria by engaging in protein-protein interactions with anti- and pro-apoptotic members of the Bcl2 family of mitochondrial permeability regulators, thereby executing the shortest known circuitry of p53 death signaling: 1) A fraction of stress-induced wild type p53 rapidly translocates to mitochondria during p53-dependent death. This is a universal p53 response and occurs in primary, immortal and transformed cultured cells, and in normal tissues upon the entire gamut of p53-inducing stresses such as DNA damage, hypoxia and oncogene deregulation. 2) The majority of p53 traffics to the outer membrane where wtp53 - but not tumor-associated p53 mutants - interacts with BclXL and Bcl2 via its DNA-binding domain and induces Bak oligomerization and outer membrane permeabilization with release of apoptotic activators like Cytochrome C, Smac etc. 3) Deliberate targeting of p53 to mitochondria is sufficient to induce apoptosis and colony suppression of p53-deficient tumor cells. Tumor-derived transactivation-deficient missense mutants of p53 concomitantly loose the ability to interact with BclXL, suggesting that p53 mutations represent `double-hits' by simultaneously abrogating the transcriptional and mitochondrial apoptotic activity of p53. 4) In irradiated mice, mitochondrial p53 translocation triggers a rapid first wave of cell death in radiosensitive tissues. In thymus - the prototype response tissue - this wave is later fortified by the transcriptional program of p53. 5) As to the mechanism of translocation, monoubiquitylation by Mdm2-type E3 ligases promotes mitochondrial p53 translocation. Rather than the nucleus, the cytoplasm contains a separate and distinct p53 pool that becomes stress-stabilized and serves as the major source for p53 translocation. Upon arrival at mitochondria, p53 undergoes rapid deubiquitylation by mitochondrial HAUSP via a stress-induced p53-HAUSP complex that generates the apoptotically active non-ubiquitylated p53. 6) Retroviral gene transfer of mitochondrial targeted wild-type p53 in a cMyc-driven mouse model of Burkitt's lymphoma shows effective tumor killing of p53-null, ARF-null and p53-mutant tumor cells in vivo. This proposal focuses on the next important phase of this promising research. It will generate relevant animal models and define the participation and extent of the p53 action at mitochondria. Aims 1 and 2 will establish transgenic and switchable mitop53 knock-in mouse models to assess the contribution of the mitochondrial p53 program to p53`s acute genotoxic response and long-term tumor suppression. Aim 3 explores whether the mitochondrial p53 program contributes to the acute pathology of ischemic tissue injury. Aim 4 tests whether mitochondrial p53 - beyond triggering the Bax/Bak- lipid pore - also activates the permeability transition pore (PTP). PUBLIC HEALTH RELEVANCE: p53 is a critical tumor suppressor in humans because it controls a powerful cell death response after cells sustain DNA damage. In addition to switching on other death effector genes in the nucleus, p53 also runs a direct cell death program at the mitochondria via protein interactions. Importantly, using mitochondrial targeted p53 fusion proteins, its power can be harnessed to act as the shortest circuit of p53-mediated cell death in cancer cells. Based on these studies, the next important phase of this promising research is clear: relevant animal models need to be generated to fully define the extent of this mitochondrial p53 program in physiologic and pathophysiologic responses to clinically important tissue insults. Also, the contribution of this mitochondrial p53 program to long-term suppression in animals needs to be assessed. This is the thrust of this proposal. Furthermore, this proposal aims at gaining more mechanistic insight into how p53 works at the mitochondria to permeabilize these organelles.