Molecular Mechanism of Mule in DNA Damage Response and Tumorigenesis

项目摘要 癌症仍然是每年影响大量新患者的主要杀手。一 癌症的主要原因是对DNA损伤的不适当反应。一旦DNA受损，细胞就会 当DNA损伤无法修复时，通过线粒体装置启动细胞凋亡。一旦细胞死亡 当细胞凋亡通路被触发时，细胞毒性因子从线粒体释放以激活半胱天冬酶， 凋亡受损细胞的凋亡潜能受损导致细胞继续分裂而不受限制 经常发展成癌症。事实上，细胞凋亡的错误调节与许多癌症有关。 了解调节DNA损伤信号的分子机制以及它们如何被传递到 因此，线粒体启动细胞凋亡对于癌症研究是重要的。生物化学方法是 用于重建DNA损伤诱导的细胞凋亡。Mcl-1是一种关键的抗死亡蛋白， 所需的触发细胞色素c释放线粒体DNA损伤。Mcl-1是通过 泛素-蛋白酶体途径在泛素-蛋白酶体系统中，泛素连接酶决定了蛋白质的结构。 底物破坏的特异性和时机。建立了一种生物化学方法来寻找这种酶 从人细胞提取物中克隆了一种新的泛素连接酶Mule（Mcl-1 ubiquitin ligase e3）。骡子 通过BH 3结构域的直接相互作用促进Mcl-1的泛素修饰，Mule是 在Mcl-1介导的凋亡途径中不可或缺。除了Mcl-1，Mule还泛素化其他底物 包括p53，从而增加了另一个有趣的连接到细胞凋亡途径。在该提案中， 其中Mule激活和Mule的差异调节响应于DNA而靶向不同的底物， 将研究损坏信号。Mule在DNA损伤反应中的生物学功能 目标1。我们新产生的Mule基因敲除小鼠胚胎成纤维细胞将用于研究 Mule在DNA损伤诱导的细胞凋亡和细胞周期检查点中的作用。我们还将调查 Mule的结构和功能关系，特别是对Mule的泛素连接酶活性的要求 在DNA损伤反应中的作用。我们将描述的泛素化和降解的 Mule在Aim 2中的Mcl-1和p53。泛素链形成，E3活性（Mule与Mdm 2）， Mcl-1和p53在骡依赖性DNA损伤反应中的作用及其调控机制 泛素化将在这一目标进行研究。在目标3中，我们将进一步描述Mule的相互作用 尤其是p53与Mule的结合位点。我们将研究 Mule和底物受DNA损伤调控。更重要的是，我们将寻找新的蛋白质因子 通过串联亲和纯化调节Mule活性。在目标4中，我们将更加注重后 Mule的翻译修饰，特别是Mule的磷酸化在DNA损伤反应中的作用，并讨论了 这些实验应该揭示新的机制，介导DNA 损伤信号以及它们是如何通过Mule传导的，从而为以下方面提供了基本的见解： 调控网络控制细胞凋亡和DNA损伤反应。机械地理解这个 细胞凋亡通路将帮助我们破译细胞凋亡在癌症中是如何失调的，并可能确定新的靶点。 治疗干预

Project Summary 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 acitivity 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.