The in vivo role of the mitochondrial p53 cell death program

描述(由申请人提供)：P53肿瘤抑制因子的复杂的凋亡功能是其体内抗肿瘤活性的中心。除了作为多个凋亡基因转录调控因子的经典作用外，P53还发挥着不依赖于转录的凋亡活性。在前一个赠款周期中，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基因Bcl2家族中抗和促凋亡成员的蛋白质-蛋白质相互作用直接作用于线粒体，从而执行已知最短的P53死亡信号通路：1)在P53依赖的死亡过程中，应激诱导的一小部分野生型P53迅速移位到线粒体。这是一种普遍的P53反应，发生在原代、永生和转化的培养细胞中，以及正常组织中，在所有P53诱导的应激反应中，如DNA损伤、低氧和癌基因去调控。2)大多数P53蛋白运输到外膜，而非肿瘤相关的P53突变体通过其DNA结合域与BclXL和Bcl2相互作用，并通过释放细胞色素C、Smac等凋亡激活剂诱导Bak寡聚和外膜通透性。3)故意将P53基因定位于线粒体足以诱导P53基因缺失的肿瘤细胞的凋亡和集落抑制。肿瘤来源的p53反式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而表现为“双重打击”。4)在受辐射的小鼠中，线粒体P53易位在对辐射敏感的组织中引发第一波快速的细胞死亡。在胸腺--原型反应组织--中，这一波后来被p53的转录程序加强。5)在易位机制方面，MDM2型E3连接酶的单核苷酸基化促进了线粒体P53的易位。细胞质不是细胞核，而是一个独立的、独特的P53池，该池变得压力稳定，是P53易位的主要来源。当P53到达线粒体时，通过应激诱导的P53-HAUSP复合体，P53经历线粒体Hausp的快速去泛素化，产生具有凋亡活性的非泛素化的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对P53缺失、ARF缺失和P53突变的肿瘤细胞具有有效的肿瘤杀伤作用。这项提案将重点放在这项前景光明的研究的下一个重要阶段。它将产生相关的动物模型，并确定P53在线粒体的参与和作用程度。AIMS 1和2将建立转基因和可切换的有丝分裂蛋白53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程序是否参与了缺血组织损伤的急性病理过程。目的4测试线粒体P53除了触发Bax/Bak脂孔外，是否还激活通透性转换孔(PTP)。

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).