Molecular Mechanism of Mule in DNA Damage Response and Tumorigenesis

描述（由申请人提供）：癌症仍然是每年影响大量新患者的主要杀手。癌症的一个主要原因是对DNA损伤的不适当反应。DNA损伤后，当DNA损伤无法修复时，细胞将通过线粒体装置启动凋亡。一旦这个细胞死亡途径被触发，细胞毒性因子从线粒体释放，激活半胱天冬酶，诱导细胞凋亡。受损细胞的凋亡潜能受损导致细胞不受限制地继续分裂，经常发展成癌症。事实上，细胞凋亡的错误调控与许多癌症有关。因此，了解调节DNA损伤信号的分子机制以及它们如何传递到线粒体以启动细胞凋亡对癌症研究非常重要。利用生物化学方法重建DNA损伤诱导的细胞凋亡。Mcl-1是一种关键的抗死亡蛋白，它的降解是在DNA损伤时触发线粒体释放细胞色素c所必需的。Mcl-1通过泛素-蛋白酶体途径降解。在泛素-蛋白酶体系统中，泛素连接酶决定底物破坏的特异性和时间。建立了从人细胞提取物中寻找该酶的生化检测方法，并克隆了一种新的泛素连接酶Mule （Mcl-1泛素连接酶e3）。Mule通过BH3结构域直接相互作用促进Mcl-1的泛素修饰，在Mcl-1介导的凋亡通路中是必不可少的。除了Mcl-1， Mule还泛素化包括p53在内的其他底物，从而为细胞凋亡途径增加了另一个有趣的联系。在本研究中，我们将研究Mule在DNA损伤信号下对不同底物的激活和差异调控机制。我们将在第一期研究中研究Mule在DNA损伤应答中的生物学功能。我们新生成的Mule敲除小鼠胚胎成纤维细胞将用于研究Mule在DNA损伤诱导的凋亡和细胞周期检查点中的功能。我们还将研究Mule的结构和功能关系，特别是Mule的泛素连接酶活性对其在DNA损伤应答中的功能的要求。我们将在Aim 2中描述Mule对Mcl-1和p53的泛素化和降解。泛素链的形成，E3活性（Mule与Mdm2）， Mcl-1和p53在Mule依赖性DNA损伤反应中的作用，以及Mcl-1和p53泛素化的调控机制将在此目的中进行研究。在Aim 3中，我们将进一步表征Mule与其底物的相互作用；特别是将p53的结合位点定位到Mule。我们将研究DNA损伤如何调节Mule和底物之间的相互作用。更重要的是，我们将通过串联亲和纯化寻找调节Mule活性的新蛋白因子。在Aim 4中，我们将更多地关注Mule的翻译后修饰，特别是在DNA损伤反应中的磷酸化，并讨论其可能参与的机制。这些实验将揭示介导DNA损伤信号的新机制以及它们如何通过Mule转导，从而为控制细胞凋亡和DNA损伤反应的调控网络提供基本见解。对这一途径的机制理解将有助于我们破译细胞凋亡在癌症中是如何解除调控的，并有可能确定治疗干预的新靶点。公共卫生相关性：细胞凋亡的错误调控被认为是肿瘤发生的主要机制之一，特别是在暴露于外源性DNA损伤剂和正常生理过程中。这一建议将揭示介导DNA损伤信号的新机制，从而为控制细胞凋亡的调控网络提供基本见解。对这一途径的机制理解将有助于我们破译细胞凋亡在癌症中是如何解除调控的，并有可能确定治疗干预的新靶点。

DESCRIPTION (provided by applicant): Cancer remains a major killer that impacts a large number of new patients every year. One leading reason for cancer is an inappropriate response to DNA damage. Upon DNA damage, the cell will initiate apoptosis through the mitochondria apparatus when DNA damage is beyond repair. Once this cell death pathway is triggered, cytotoxic factors are released from mitochondria to activate caspases that induce apoptosis. An impaired apoptotic potential of damaged cells leads to continued cell division without restriction that frequently develops into cancer. Indeed, mis-regulation of apoptosis is associated with many cancers. Understanding the molecular mechanisms that regulate DNA damage signals and how they are transmitted to mitochondria to initiate apoptosis is therefore important for cancer research. Biochemical approaches are utilized to reconstitute DNA damage-induced apoptosis. Degradation of Mcl-1, a key anti-death protein, is required to trigger cytochrome c release from mitochondria upon DNA damage. Mcl-1 is degraded through the ubiquitin-proteasome pathway. In the ubiquitin-proteasome system, the ubiquitin ligases determine the specificity and timing of substrates destruction. A biochemical assay was established to search for such enzyme from human cell extracts, and a novel ubiquitin ligase Mule (Mcl-1 ubiquitin ligase e3) was cloned. Mule promotes ubiquitin modification of Mcl-1 through direct interaction through a BH3 domain, and Mule is indispensable for Mcl-1 mediated apoptotic pathway. Besides Mcl-1, Mule also ubiquitinates other substrates including p53 thus adding another intriguing link to the apoptosis pathway. In this proposal, the mechanism by which Mule activation and the differential regulation of Mule target different substrates in response to DNA damage signals will be investigated. The biological function of Mule in DNA damage response will be studied in Aim 1. Our newly generated Mule knockout mouse embryonic fibroblast cells will be utilized to study the function of Mule in DNA damage induced apoptosis and cell cycle checkpoints. We will also investigate the structure and function relationship of Mule, especially the requirement of the ubiquitin ligase activity of Mule for its function in DNA damage response in this aim. We will characterize the ubiquitination and degradation of Mcl-1 and p53 by Mule in Aim 2. Ubiquitin chain formation, E3 activity (Mule versus Mdm2), the contribution of Mcl-1 and p53 in Mule-dependent DNA damage response, and regulatory mechanisms of Mcl-1 and p53 ubiquitination will be investigated in this aim. In Aim 3, we will further characterize the interaction of Mule with its substrates; especially map the binding site of p53 to Mule. We will study how the interaction between Mule and substrates are regulated by DNA damage. More importantly, we will search for novel protein factors modulating Mule activity through tandem affinity purification. In Aim 4, we will put more focus on post- translation modification of Mule, especially phosphorylation of Mule in DNA damage response and discuss potential mechanisms involved in it. These experiments should reveal novel mechanisms mediating DNA damage signals and how they are transduced through Mule, thereby providing fundamental insights into regulatory networks controlling apoptosis and DNA damage response. A mechanistic understanding of this pathway will help us decipher how apoptosis is deregulated in cancer and potentially identify new targets for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Misregulation of apoptosis is considered to be one of the major mechanisms for tumorigenesis, especially upon exposure to exogenous DNA damaging agents and during normal physiological processes. This proposal will disclose a novel mechanism mediating DNA damage signals, thereby providing fundamental insights into regulatory networks controlling apoptosis. A mechanistic understanding of this pathway will help us decipher how apoptosis is deregulated in cancer and potentially identify new targets for therapeutic intervention.