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Deubiquitination regulation of c-Myc

c-Myc 的去泛素化调控

基本信息

批准号：
9012778
负责人：
Mu-Shui Dai
金额：
$ 38.33万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-01 至 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9012778
关键词：
Affect Binding Biochemical Biogenesis Biology Boxing Burkitt Lymphoma Cell Culture Techniques Cell Nucleolus Cell Proliferation Cells Complex Cultured Cells DNA Polymerase II DNA Polymerase III Deubiquitinating Enzyme Deubiquitination Event Family member Gene Targeting Genes Genetic Transcription Goals Growth Health Homeostasis Hot Spot Human In Vitro Investigation Lead Link MCF10A cells Malignant Neoplasms Mammary Tumorigenesis Mediating Messenger RNA Molecular Mus Mutation Normal Cell Nucleoplasm Oncogenes Oncogenic Phosphorylation Play Post-Translational Protein Processing Proteins Proteolysis Proto-Oncogene Proteins c-myc Recombinant DNA Recruitment Activity Regulation Reporting Research Ribosomes Role Serine Serum Signal Transduction Stress System Testing Threonine Translations Ubiquitin Ubiquitination base c-myc Genes cancer therapy cell growth feeding high throughput screening in vivo insight killings knock-down malignant breast neoplasm mammary epithelium mouse model multicatalytic endopeptidase complex mutant novel novel therapeutics overexpression promoter protein degradation response small molecule inhibitor therapeutic development therapeutic target tumor xenograft tumorigenesis ubiquitin ligase ubiquitin-protein ligase ubiquitin-specific protease

项目摘要

DESCRIPTION (provided by applicant): The c-Myc oncoprotein is essential for normal cell growth and proliferation. However, overexpression of c-Myc occurs in most human cancers. Thus, its level and activity must be tightly regulated during normal cell homeostasis. The ubiquitination-proteasome system plays a key role in controlling c-Myc levels and activity. c-Myc normally undergoes rapid ubiquitin-dependent proteolysis, but it is transiently stabilized by key phosphorylation events in response to growth signals. Phosphorylation of Serine 62 (S62) stabilizes c-Myc, whereas phosphorylation of Threonine 58 (T58) promotes c-Myc ubiquitination by the SCFFbw7 ubiquitin ligase and proteasomal degradation, mainly in the nucleolus. Like other post-translational modifications, ubiquitination can be reversed by the action of deubiquitinating enzymes (DUBs). While several ubiquitin ligases have been identified for c-Myc, only one DUB, USP28, has been reported to target c-Myc. We have recently discovered that the nucleolar deubiquitinating enzyme USP36 is a novel c-Myc regulator. USP36 binds to c-Myc and deubiquitinates c-Myc in cells and in vitro. Overexpression of wild-type USP36, but not its catalytic-inactive C131A mutant, stabilizes c-Myc and enhances c-Myc-driven transcription. Knockdown of USP36 reduces c-Myc levels and drastically suppresses cell proliferation. Importantly, USP36 interacts with the nucleolar Fbw7γ and abolishes Fbw7γ-mediated c-Myc degradation. In contrast, USP28 antagonizes Fbw7-mediated c-Myc degradation. Since the bulk of c-Myc is degraded in the nucleolus, our discovery leads to the novel hypothesis that USP36 functions as a crucial regulator of c-Myc by deubiquitinating c-Myc in the nucleolus. Interestingly, we found that USP36 itself is a c-Myc target gene, suggesting that USP36 and c-Myc form a positive feed-forward regulatory loop. To gain further insight into the role of USP36 in the regulation of c-Myc protein stability, activity and oncogenicity, we will investigate the molecular and biochemical mechanisms underlying the regulation of c-Myc by USP36 in Aim 1, including how USP36 interplays with Fbw7γ to regulate c-Myc in the nucleolus, whether it interplays with USP28 in the dynamic control of c-Myc ubiquitination, and the importance of c-Myc-USP36 feed-forward regulation. We will elucidate the functional consequences of USP36 regulation of c-Myc in cells in Aim 2 by analyzing whether USP36 regulates c-Myc binding and turnover at target gene promoters, whether it promotes c-Myc-dependent ribosome biogenesis, and whether it promotes c-Myc's oncogenic potential in cells and in vivo. Finally, we will elucidate whether USP36 is a therapeutic target using cell based and mouse models as proposed in Aim 3, including the investigation of USP36 deregulation in human breast cancers, whether deletion of USP36 inhibits c-Myc-driven mammary tumorigenesis in mice, and high-throughput screening of small molecule inhibitors for USP36. Achieving these goals will provide critical insight into how c-Myc is properly regulated by dynamic ubiquitination and deubiquitination, how deregulation of this dynamic contributes to tumorigenesis, and how USP36 can be targeted in human cancers.

描述（由申请人提供）：c-Myc 癌蛋白对于正常细胞生长和增殖至关重要。然而，c-Myc 的过度表达发生在大多数人类癌症中。因此，在正常细胞稳态过程中必须严格调节其水平和活性。泛素化蛋白酶体系统在控制 c-Myc 水平和活性方面发挥着关键作用。 c-Myc 通常会经历快速的泛素依赖性蛋白水解，但它会通过响应生长信号的关键磷酸化事件而短暂稳定。丝氨酸 62 (S62) 的磷酸化可稳定 c-Myc，而苏氨酸 58 (T58) 的磷酸化可通过 SCFFbw7 泛素连接酶促进 c-Myc 泛素化和蛋白酶体降解（主要在核仁中）。与其他翻译后修饰一样，泛素化可以通过去泛素化酶 (DUB) 的作用来逆转。虽然已鉴定出多种针对 c-Myc 的泛素连接酶，但据报道，只有一种 DUB（USP28）能够靶向 c-Myc。我们最近发现核仁去泛素化酶 USP36 是一种新型的 c-Myc 调节因子。 USP36 在细胞内和体外与 c-Myc 结合并使 c-Myc 去泛素化。野生型 USP36 的过表达（而非其催化失活的 C131A 突变体）可稳定 c-Myc 并增强 c-Myc 驱动的转录。 USP36 的敲低会降低 c-Myc 水平并显着抑制细胞增殖。重要的是，USP36 与核仁 Fbw7γ 相互作用并消除 Fbw7γ 介导的 c-Myc 降解。相反，USP28 拮抗 Fbw7α 介导的 c-Myc 降解。由于大部分 c-Myc 在核仁中被降解，我们的发现引出了一个新的假设：USP36 通过使核仁中的 c-Myc 去泛素化而发挥 c-Myc 的关键调节作用。有趣的是，我们发现USP36本身是c-Myc靶基因，表明USP36和c-Myc形成正前馈调节环。为了进一步了解USP36在c-Myc蛋白稳定性、活性和致癌性调节中的作用，我们将在目标1中研究USP36调节c-Myc的分子和生化机制，包括USP36如何与Fbw7γ相互作用来调节核仁中的c-Myc，是否与USP28相互作用动态控制c-Myc 泛素化以及 c-Myc-USP36 前馈调节的重要性。我们将通过分析 USP36 是否调节靶基因启动子处的 c-Myc 结合和周转、是否促进 c-Myc 依赖性核糖体生物发生以及是否促进 c-Myc 在细胞和体内的致癌潜力，阐明 USP36 在 Aim 2 中对细胞中 c-Myc 的调节功能后果。最后，我们将利用目标 3 中提出的基于细胞和小鼠模型阐明 USP36 是否是一个治疗靶点，包括研究人类乳腺癌中 USP36 失调、USP36 缺失是否抑制小鼠 c-Myc 驱动的乳腺肿瘤发生，以及 USP36 小分子抑制剂的高通量筛选。实现这些目标将为了解 c-Myc 如何通过动态泛素化和去泛素化进行正确调节、这种动态的放松管制如何促进肿瘤发生以及 USP36 如何靶向人类癌症提供重要见解。