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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：
8911127
负责人：
Mu-Shui Dai
金额：
$ 39.56万
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/8911127
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 Homeostasis Hot Spot Human In Vitro Investigation Lead Link MCF10A cells Malignant Neoplasms Mammary Tumorigenesis Mediating Messenger RNA Molecular Mus Mutation Normal Cell Nucleoplasm Oncogene Proteins 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 inhibitor/antagonist insight killings malignant breast neoplasm mammary epithelium mouse model multicatalytic endopeptidase complex mutant novel novel therapeutics overexpression promoter protein degradation public health relevance response small molecule 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癌蛋白是正常细胞生长和增殖所必需的。然而，c-Myc的过度表达在大多数人类癌症中都存在。因此，在正常的细胞动态平衡过程中，其水平和活性必须受到严格的调控。泛素化-蛋白酶体系统在控制c-Myc水平和活性方面起着关键作用。C-Myc通常经历快速的泛素依赖的蛋白分解，但它在生长信号的响应下被关键的磷酸化事件瞬时稳定。丝氨酸62(S62)的磷酸化稳定了c-Myc，而苏氨酸58(T58)的磷酸化促进了SCFFbw7泛素连接酶的泛素化和蛋白酶体的降解，主要是在核仁中。像其他翻译后修饰一样，泛素化可以被去泛素化酶(DUBS)的作用逆转。虽然c-Myc有几种泛素连接酶被鉴定，但只有一种DUB，USP28，被报道以c-Myc为靶点。我们最近发现核仁去泛素化酶USP36是一种新的c-Myc调节因子。USP36与c-Myc结合并在细胞内和体外去泛素化c-Myc。野生型USP36的过表达，而不是其催化失活的C131a突变体，稳定了c-Myc并增强了c-Myc驱动的转录。USP36基因的敲除降低了c-Myc水平，并显著抑制了细胞的增殖。重要的是，USP36与核仁Fbw7γ相互作用，并取消Fbw7γ介导的c-Myc降解。相反，USP28拮抗Fbw7介导的c-Myc降解。由于c-Myc的大部分在核仁中被降解，我们的发现导致了一个新的假设，即USP36通过去泛素化核仁中的c-Myc而作为c-Myc的关键调节因子发挥作用。有趣的是，我们发现USP36本身是一个c-Myc靶基因，这表明USP36和c-Myc形成了一个正前馈调控环。为了进一步了解USP36在c-Myc蛋白稳定性、活性和致瘤性调控中的作用，我们将在目标1中研究USP36调控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，它是否与USP28在c-Myc泛素化的动态调控中相互作用，以及c-Myc-USP36前馈调控的重要性。我们将通过分析USP36是否调控c-Myc在靶基因启动子上的结合和周转，是否促进c-Myc依赖的核糖体生物发生，以及是否促进c-Myc在细胞和体内的致癌潜力，来阐明USP36调控c-Myc在AIM 2细胞中的功能后果。最后，我们将像目标3中建议的那样，使用基于细胞的模型和小鼠模型来阐明USP36是否是一个治疗靶点，包括USP36在人类乳腺癌中的去调控研究，USP36的缺失是否抑制c-Myc驱动的小鼠乳腺肿瘤的发生，以及USP36小分子抑制剂的高通量筛选。实现这些目标将为c-Myc如何受到动态泛素化和去泛素化的适当调控，这种动态调控的解除如何促进肿瘤发生，以及USP36如何在人类癌症中被靶向提供关键的见解。