Deubiquitination regulation of c-Myc

 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。

 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.