Molecular Mechanism of Mule in DNA Damage Response and Tumorigenesis

骡DNA损伤反应和肿瘤发生的分子机制

• 批准号: 8384853
• 负责人: Qing Zhong
• 金额: $ 4.66万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2013-06-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8384853
• 关键词: Adaptor Signaling Protein; Affinity; Affinity Chromatography; Apoptosis; Apoptotic; BH3 Domain; Binding; Binding Sites; Biochemical; Biological Assay; Biological Process; Cancer Etiology; Caspase; Cell Cycle Arrest; Cell Cycle Checkpoint; Cell Death; Cell Extracts; Cell division; Cell physiology; Cell-Free System; Cells; Cessation of life; Conflict (Psychology); DNA; DNA Damage; DNA damage checkpoint; DNA lesion; Dose; Embryo; Enzymes; Equilibrium; Event; Exposure to; Fibroblasts; Fractionation; Genetic; Genotoxic Stress; Human; In Vitro; Knockout Mice; Link; MCL1 protein; Malignant Neoplasms; Maps; Mediating; Mitochondria; Modification; Molecular; Oncogenic; Pathway interactions; Patients; Phenotype; Phosphorylation; Phosphotransferases; Physiological Processes; Play; Proteins; RNA Interference; Regulation; Role; Serine; Signal Transduction; Specificity; Structure; Structure-Activity Relationship; Surface; System; Testing; Therapeutic Intervention; Threonine; Time; Translations; Tumor Suppression; Tyrosine; Ubiquitin; Ubiquitination; anticancer research; base; cancer cell; cell injury; clinically significant; cytochrome c; cytotoxic; design; insight; knockout gene; multicatalytic endopeptidase complex; novel; overexpression; protein protein interaction; reconstitution; release factor; repaired; research study; response; tumorigenesis; ubiquitin ligase; ubiquitin-protein ligase

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.

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损伤，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。一旦这个细胞死亡 途径被触发，细胞毒因子从线粒体释放出来激活caspase，从而诱导 细胞凋亡。受损细胞的凋亡潜能受损会导致细胞不受限制地继续分裂 这通常会发展成癌症。事实上，细胞凋亡的错误调控与许多癌症有关。 了解调节DNA损伤信号的分子机制及其如何传递到 因此，线粒体启动细胞凋亡对癌症研究非常重要。生化方法是 用于重建DNA损伤诱导的细胞凋亡。关键的抗死亡蛋白Mcl-1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通过降解 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 底物破坏的特异性和时机。建立了一种生化分析方法来寻找这种酶。 从人细胞提取液中克隆了一个新的泛素连接酶Mule(Mcl-1泛素连接酶E3)。骡子 通过BH3结构域的直接相互作用促进Mcl-1的泛素修饰，Mule是 在Mcl-1介导的细胞凋亡途径中不可缺少。除了Mcl-1，Mule还泛化其他底物 包括P53，从而为细胞凋亡途径增加了另一个有趣的联系。在这项建议中，该机制由 靶向不同底物的Mule的激活及其对DNA的差异性调控 将对损坏信号进行调查。将研究Mule在DNA损伤反应中的生物学功能 目的1.我们新培养的Mule基因敲除小鼠胚胎成纤维细胞将被用于研究 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的结构与功能关系，特别是对Mule泛素连接酶活性的要求 因为它在DNA损伤反应中的作用。我们将描述泛素化和降解的特征 目的2.泛素链的形成，E3的活性(Mule与MDM2)，贡献 Mcl-1和P53在Mule依赖的DNA损伤反应中的表达及其调控机制 泛素化将以此为目标进行研究。在目标3中，我们将进一步描述Mule的相互作用 与其底物结合；特别是将P53的结合位点映射到Mule。我们将研究它们之间的相互作用 骡子和底物受到DNA损伤的调节。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，我们将更多地关注后 Mule的翻译修饰，特别是Mule在DNA损伤反应中的磷酸化及其讨论 其中涉及的潜在机制。这些实验应该揭示新的机制来调节DNA 损伤信号以及它们如何通过Mule传递，从而提供了对 控制细胞凋亡和DNA损伤反应的调控网络。对这一点的机械理解 途径将帮助我们破译癌症中细胞凋亡是如何被解除调控的，并有可能确定新的靶点 治疗性干预。