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凋亡功能对其体内凋亡活性至关重要。除了其作为多个凋亡基因的转录调节因子的经典作用外，p53还发挥不依赖于转录的凋亡活性。在上一个赠款周期中，我们阐明了后者的机制。我们发现野生型p53蛋白通过与线粒体渗透性调节剂的Bcl 2家族的抗凋亡和促凋亡成员进行蛋白质-蛋白质相互作用而在线粒体中具有直接作用，从而执行已知最短的p53死亡信号传导回路：1）在p53依赖性死亡期间，一部分应激诱导的野生型p53快速易位到线粒体。这是一种普遍的p53反应，发生在原代、永生和转化的培养细胞中，以及在正常组织中，在整个范围的p53诱导应激如DNA损伤、缺氧和癌基因失调时。2)大多数p53运输到外膜，其中wtp 53-而不是肿瘤相关的p53突变体-通过其DNA结合结构域与BclXL和Bcl 2相互作用，并诱导巴克寡聚化和外膜透化，同时释放凋亡激活剂如细胞色素C，3）有意地将p53靶向线粒体足以诱导p53缺陷型肿瘤细胞的凋亡和集落抑制。肿瘤衍生的反式激活缺陷的错义突变体的p53伴随松散的能力，与BclXL相互作用，这表明p53突变代表“双重打击”，同时废除转录和线粒体凋亡活性的p53。4)在受辐射的小鼠中，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-原型反应组织-这种波后来被p53的转录程序强化。5)至于易位的机制，Mdm 2型E3连接酶的单泛素化促进线粒体p53易位。而不是细胞核，细胞质包含一个单独的和独特的p53池，成为应力稳定，并作为p53易位的主要来源。在到达线粒体后，p53通过应激诱导的p53-HAUSP复合物被线粒体HAUSP快速去泛素化，该复合物产生具有抗肿瘤活性的非泛素化p53。6)在伯基特淋巴瘤的cMyc驱动的小鼠模型中，线粒体靶向野生型p53的逆转录病毒基因转移显示出对p53-null、ARF-null和p53-突变体肿瘤细胞的体内有效肿瘤杀伤。这项建议的重点是这项有前途的研究的下一个重要阶段。它将产生相关的动物模型，并确定线粒体中p53作用的参与和程度。目的1和2将建立转基因和可转换的mitop 53基因敲入小鼠模型，以评估线粒体p53程序对p53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学。目的4测试线粒体p53 -除了触发Bax/巴克-脂质孔-是否也激活渗透性转换孔（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。除了启动细胞核中的其他死亡效应基因外，p53还通过蛋白质相互作用在线粒体上运行直接的细胞死亡程序。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来作为癌细胞中p53介导的细胞死亡的最短回路。基于这些研究，这一有前途的研究的下一个重要阶段是明确的：需要产生相关的动物模型，以充分确定这种线粒体p53程序在生理和病理生理反应的程度，临床上重要的组织损伤。此外，需要评估这种线粒体p53程序对动物长期抑制的贡献。这就是这项建议的主旨。此外，该提议旨在获得更多的机制洞察力，了解p53如何在线粒体中发挥作用，使这些细胞器透化。

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.