DeSUMOylation regulation of c-Myc

项目摘要 虽然适当调节的c-Myc水平对于正常的细胞生长和增殖是必不可少的，但异常 C-Myc的过度表达和激活与大多数人类癌症有关。因此，c-Myc的水平和活性必须 在正常的细胞动态平衡期间受到严格的调控。C-Myc的快速周转受泛素- 依赖蛋白水解酶。C-Myc可被苏氨酸58磷酸化依赖的泛素泛素化 连接酶(E3)复合体SCFFbw7以及其他各种泛素E3。相反，c-Myc泛素化可以是 被去泛素酶(DUB)逆转，包括USP28、USP36和USP37。有趣的是， C-Myc也可以被类似泛素的小修饰物(SUMO)修饰。然而，c-Myc SUMO化的功能 目前仍不清楚，c-Myc是如何受去SUMO影响的还不清楚。我们最近确定了相扑 蛋白水解酶SENP1是一种新的c-Myc脱硫酶。SENP1直接结合和脱除c- MYC在细胞内和体外。过表达野生型(Wt)SENP1，但不表达其催化失活突变体(C603S)， 稳定c-Myc，增强c-Myc反式激活活性。一直以来，SENP1基因的敲除都会降低c- MYC水平并抑制细胞增殖。我们进一步证明c-Myc可以被泛素和 SUMO和SENP1介导的去SUMO减少c-Myc泛素化，提示SUMO 通过泛素-蛋白酶体系统促进c-Myc的降解。此外，SENP1脱SUMOylate USP28稳定SENP1，Fbw7降低SENP1水平。因此，c-Myc的水平和活性 可能受复杂的泛素化-糖基化串扰的动态控制。SENP1经常 人乳腺组织中c-Myc高表达与患者生存不良相关 癌症。综上所述，这些结果导致了一个新的假设，即SENP1作为c-C-1的关键调节因子发挥作用。 MYC通过c-Myc去SUMO。进一步了解SENP1在调节c-Myc蛋白中的作用 稳定性、活性和致瘤性，我们将研究其分子和生化机制。 目标1中SENP1对c-Myc的调节，包括SENP1如何促进c-Myc的稳定，c-Myc如何 被相扑和泛素共同修饰，以及它如何与Fbw7和USP28相互作用以动态控制c-Myc 营业额。我们将通过分析阐明SENP1在c-Myc介导的AIM 2基因调控中的作用 SENP1是否调节c-Myc在靶基因启动子上的结合和转换，是否调节特定的c-Myc- MYC靶基因对生长信号的响应，以及SENP1是否调节空间定位 C-Myc在细胞核内的分布。在目标3中，我们将测试SENP1是否增强c-Myc驱动的转化和 乳腺肿瘤发生，抑制SENP1是否抑制体内c-Myc驱动的肿瘤发生 抑制SENP1对乳腺癌是否有效。实现这些目标将提供关键的洞察力 C-Myc如何通过动态相扑修改进行适当调节，这一放松管制如何有助于 肿瘤的发生，以及SENP1是否是人类癌症的有前途的治疗靶点。

Project Summary While properly regulated levels of c-Myc are essential for normal cell growth and proliferation, aberrant overexpression and activation of c-Myc contribute to most human cancers. Thus, c-Myc level and activity must be tightly regulated during normal cell homeostasis. The rapid turnover of c-Myc is controlled by ubiquitin- dependent proteolysis. c-Myc can be ubiquitinated by the Threonine 58 phosphorylation-dependent ubiquitin ligase (E3) complex SCFFbw7 as well as various other ubiquitin E3s. Conversely, c-Myc ubiquitination can be reversed by the action of deubiquitinating enzymes (DUBs), including USP28, USP36 and USP37. Interestingly, c-Myc can also be modified by small ubiquitin-like modifiers (SUMOs). Yet the function of c-Myc SUMOylation is still unclear and how c-Myc is affected by deSUMOylation is unknown. We recently identified the SUMO protease SENP1 as a novel c-Myc deSUMOylating enzyme. SENP1 directly binds to and deSUMOylates c- Myc in cells and in vitro. Overexpression of wild-type (wt) SENP1, but not its catalytic-inactive mutant (C603S), stabilizes c-Myc and enhances c-Myc transactivation activity. Consistently, knockdown of SENP1 reduces c- Myc levels and suppresses cell proliferation. We further show that c-Myc can be co-modified by ubiquitin and SUMO and SENP1-mediated deSUMOylation reduces c-Myc ubiquitination, suggesting that SUMOylation promotes c-Myc degradation through the ubiquitin-proteasome system. In addition, SENP1 deSUMOylates USP28 whereas USP28 stabilizes SENP1 and Fbw7 reduces SENP1 levels. Thus, c-Myc levels and activity may be dynamically controlled by complex ubiquitination-SUMOylation crosstalk. SENP1 is frequently overexpressed, correlating with the high expression of c-Myc and poor patient survival, in human breast cancers. Together, these results lead to a novel hypothesis that SENP1 functions as a crucial regulator of c- Myc by deSUMOylating c-Myc. To gain further insight into the role of SENP1 in regulating c-Myc protein stability, activity and oncogenicity, we will investigate the molecular and biochemical mechanisms of the regulation of c-Myc by SENP1 in Aim 1, including how SENP1 contributes to c-Myc stabilization, how c-Myc is co-modified by SUMO and ubiquitin, and how it interplays with Fbw7 and USP28 to dynamically control c-Myc turnover. We will elucidate the role of SENP1 in c-Myc-mediated gene regulation in Aim 2 by analyzing whether SENP1 regulates c-Myc binding and turnover at target gene promoters, whether it regulates specific c- Myc target gene programs in response to growth signals, and whether SENP1 regulates the spatial localization of c-Myc in the nucleus. In Aim 3, we will test whether SENP1 potentiates c-Myc-driven transformation and mammary tumorigenesis, whether inhibiting SENP1 suppresses c-Myc-driven tumorigenesis in vivo, and whether SENP1 inhibition is efficacious in breast cancer. Achieving these goals will provide critical insight into how c-Myc is properly regulated by dynamic SUMO modifications, how deregulation of this contributes to tumorigenesis, and whether SENP1 is a promising therapeutic target in human cancers.