DeSUMOylation regulation of c-Myc

项目摘要 虽然适当调节的c-Myc水平对于正常细胞生长和增殖是必不可少的，但异常的c-Myc表达对于正常细胞生长和增殖是必需的。 c-Myc的过表达和活化导致大多数人类癌症。因此，c-Myc水平和活性必须 在正常的细胞内稳态中受到严格的调节。c-Myc的快速更新是由泛素控制的， 依赖性蛋白水解c-Myc可被Three 58磷酸化依赖性泛素泛素化 连接酶（E3）复合物SCFFbw 7以及各种其它泛素E3。相反，c-Myc泛素化可以是 通过去泛素化酶（DUB）的作用逆转，包括USP 28、USP 36和USP 37。有趣的是， c-Myc也可以被小泛素样修饰物（SUMO）修饰。然而，c-Myc SUMO化的功能 目前还不清楚，c-Myc如何受到去SUMO化的影响也是未知的。我们最近发现相扑 蛋白酶SENP 1作为一种新的c-Myc去SUMO化酶。SENP 1直接结合并使c-去SUMO化， Myc在细胞和体外的表达。野生型（wt）SENP 1的过表达，但不表达其催化失活突变体（C603 S）， 稳定c-Myc并增强c-Myc反式激活活性。一致地，SENP 1的敲低降低了c- Myc水平和抑制细胞增殖。我们进一步表明，c-Myc可以被泛素共修饰， SUMO和SENP 1介导的去SUMO化减少c-Myc泛素化，表明SUMO化 通过泛素-蛋白酶体系统促进c-Myc降解。此外，SENP 1去SUMO化 USP 28稳定SENP 1，而Fbw 7降低SENP 1水平。因此，c-Myc水平和活性 可以通过复杂的泛素化-SUMO化串扰动态控制。SENP 1经常 在人乳腺癌组织中过表达，与c-Myc的高表达和患者生存率低相关， 癌的总之，这些结果导致了一个新的假设，即SENP 1作为c- 通过使c-Myc去SUMO化。为了进一步了解SENP 1在调节c-Myc蛋白中的作用， 稳定性，活性和致癌性，我们将研究的分子和生化机制， 目的1中SENP 1对c-Myc的调节，包括SENP 1如何促进c-Myc的稳定，c-Myc是如何被 由SUMO和泛素共同修饰，以及它如何与Fbw 7和USP 28相互作用以动态控制c-Myc 周转我们将阐明SENP 1在c-Myc介导的Aim 2基因调控中的作用， SENP 1是否调节靶基因启动子处的c-Myc结合和周转，是否调节特异性c-Myc结合和周转， Myc靶基因对生长信号的反应程序，以及SENP 1是否调节空间定位 c-Myc在细胞核中的表达。在目标3中，我们将测试SENP 1是否增强c-Myc驱动的转化， 乳腺肿瘤发生，是否抑制SENP 1抑制体内c-Myc驱动的肿瘤发生，以及 SENP 1抑制是否在乳腺癌中有效。实现这些目标将提供关键的洞察力， c-Myc如何被动态SUMO修饰正确调节，这种调节的解除如何有助于 肿瘤发生，以及SENP 1是否是人类癌症中有希望的治疗靶点。

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.