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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：
9245658
负责人：
Mu-Shui Dai
金额：
$ 37.65万
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/9245658
关键词：
Affect Binding Biochemical Biogenesis Biology 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 MYC Box Malignant Neoplasms Mammary Tumorigenesis Mediating Messenger RNA Molecular Mus Mutation Normal Cell Nucleoplasm Oncogenic Oncoproteins 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 public health relevance 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（由申请方提供）：c-Myc癌蛋白是正常细胞生长和增殖所必需的。然而，c-Myc的过度表达发生在大多数人类癌症中。因此，其水平和活性必须在正常细胞稳态期间严格调节。泛素化-蛋白酶体系统在控制c-Myc水平和活性中起关键作用。c-Myc通常经历快速的泛素依赖性蛋白水解，但它通过响应生长信号的关键磷酸化事件而瞬时稳定。丝氨酸62（S62）的磷酸化稳定c-Myc，而苏氨酸58（T58）的磷酸化通过SCFFbw 7泛素连接酶和蛋白酶体降解促进c-Myc泛素化，主要在核仁中。像其他翻译后修饰一样，泛素化可以通过去泛素化酶（DUBs）的作用逆转。虽然已经鉴定了c-Myc的几种泛素连接酶，但仅报道了一种DUB，USP 28，靶向c-Myc。我们最近发现核仁去泛素化酶USP 36是一种新的c-Myc调节剂。USP 36在细胞和体外与c-Myc结合并使c-Myc去泛素化。野生型USP 36的过表达，而不是其催化失活的C131 A突变体，稳定了c-Myc并增强了c-Myc驱动的转录。USP 36的敲低降低了c-Myc水平并显著抑制了细胞增殖。重要的是，USP 36与核仁Fbw 7 γ相互作用并消除Fbw 7 γ介导的c-Myc降解。相反，USP 28拮抗Fbw 7 β介导的c-Myc降解。由于大部分c-Myc在核仁中被降解，我们的发现导致了新的假设，即USP 36通过使核仁中的c-Myc去泛素化而作为c-Myc的关键调节剂发挥作用。有趣的是，我们发现USP 36本身是c-Myc靶基因，这表明USP 36和c-Myc形成了一个正前馈调控环。为了进一步了解USP 36在调节c-Myc蛋白稳定性、活性和致癌性中的作用，我们将在Aim 1中研究USP 36调节c-Myc的分子和生化机制，包括USP 36如何与Fbw 7 γ相互作用以调节核仁中的c-Myc，它是否与USP 28相互作用以动态控制c-Myc泛素化，以及c-Myc-USP 36前馈调节的重要性。我们将通过分析USP 36是否调节c-Myc在靶基因启动子的结合和周转，是否促进c-Myc依赖性核糖体生物合成，以及是否促进c-Myc在细胞和体内的致癌潜力，阐明USP 36调节c-Myc在细胞中的功能后果。最后，我们将使用目标3中提出的基于细胞的模型和小鼠模型阐明USP 36是否是治疗靶点，包括研究USP 36在人类乳腺癌中的失调，USP 36的缺失是否抑制小鼠中c-Myc驱动的乳腺肿瘤发生，以及高通量筛选USP 36的小分子抑制剂。实现这些目标将为c-Myc如何通过动态泛素化和去泛素化进行适当调节，这种动态的失调如何有助于肿瘤发生以及USP 36如何在人类癌症中靶向提供关键的见解。