Molecular Mechanisms of PTEN and USP7 Regulation

项目摘要。这个K99/R00建议涉及关键蛋白质的结构、功能和调节 参与细胞信号转导、PTEN和USP7。PTEN是一种肿瘤抑制因子脂质磷酸酶，它催化 膜磷脂磷脂酰肌醇(3，4，5)-三磷酸(PIP3)脱除3‘-磷酸 以生成PIP2。由于PIP3是细胞生长和胰岛素信号的关键调节因子，因此PTEN势在必行 活动受到严格控制。功能丧失PTEN突变在癌症中经常被观察到。PTEN是 翻译后受N端泛素化和C端磷酸化调控，但详细的 这些翻译后修饰(PTM)的结构和机械影响还没有被很好地理解。 USP7是一种Cys水解酶，是一种脱泛素酶(DUB)，催化泛素/赖氨酸的切割 异肽键。USP7的S泛素化蛋白底物包括PTEN和MDM2。PTEN的去泛素化 据报道，它能抑制其从胞浆到细胞核的移位。此外，USP7已经被证明是 增强MDM2的细胞稳定性，这一点很重要，因为MDM2是一种主要的E3泛素连接酶 肿瘤抑制蛋白P53。目前尚不清楚是什么分子特征选择性地驱动了USP7的S底物和 它是如何在细胞中被调节的。USP7的N-端和C-端都是通过磷酸化和 乙酰化，但这些PTM的调节作用尚不清楚。在这里，我们将讨论PTEN和USP7如何 由PTMS使用新的和新兴的半合成方法进行管理。这些半合成方法 可促进PTMS及其模拟物在PTEN和USP7中的特定部位和化学计量比安装。目标 1试图通过C末端的尾部磷酸化来确定PTEN调节的分子基础 接近了。PTEN的构象关闭是由其C末端尾部的位置磷酸化驱动的 380、382、383和385导致抑制酶、减少质膜结合和增加 稳定性。这一目标利用生物分子核磁共振、结晶学和诱变来理解结构 构象闭合的力学基础。目标2将采用一系列生化和细胞 方法定义Lys13单泛素化的功能，增强我们对PTM是如何 可能促进PTEN从胞浆穿梭到胞核。目标3将解决哪些分子 特征驱动USP7的S底物选择性以及USP7 PTM(Ser18、Tyr1091、Thr1092和Tyr1093)如何 磷酸化；Lys1096乙酰化和泛素化)调节其功能。总的来说，这些拟议的研究 可以大大提高我们对PTEN和USP7的功能和调控的理解 可能的治疗目标。此外，这项建议还可以增加PI的科学技能和 当他寻求为独立的学术生涯规划一条道路时，他的经历。

Project Summary. This K99/R00 proposal concerns the structure, function, and regulation of key proteins involved in cell signaling, PTEN and USP7. PTEN is a tumor suppressor lipid phosphatase that catalyzes the removal of the 3'-phosphate from the membrane phospholipid phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to generate PIP2. Since PIP3 is a key regulator of cell growth and insulin signaling, it is imperative that PTEN activity be tightly controlled. Loss of function PTEN mutations are frequently observed in cancer. PTEN is post-translationally regulated by N-terminal ubiquitination and C-terminal phosphorylation but the detailed structural and mechanistic impacts of these post-translational modifications (PTMs) are not well understood. USP7 is a Cys hydrolase that is a deubiquitinase (DUB), catalyzing the cleavage of the ubiquitin/lysine isopeptide bond. USP7's ubiquitinated protein substrates include PTEN and MDM2. Deubiquitination of PTEN is reported to inhibit its translocation from the cytosol to the nucleus. Moreover, USP7 has been shown to enhance the cellular stability of MDM2, and this is important because MDM2 is an E3 ubiquitin ligase for major tumor suppressor protein p53. It is unclear what molecular features drive USP7's substrate selectively and how it is regulated in the cell. USP7 is modified on both its N- and C-termini by phosphorylation and acetylation but the regulatory roles of these PTMs are unclear. Here, we will address how PTEN and USP7 are regulated by PTMs using new and emerging semi-synthetic approaches. These semi-synthetic methods can facilitate site-specific and stoichiometric installation of PTMs and their mimics into PTEN and USP7. Aim 1 seeks to define the molecular basis for PTEN regulation by C-terminal tail phosphorylation using structural approaches. Conformational closure of PTEN is driven by phosphorylation of its C-terminal tail at positions 380, 382, 383, and 385 resulting in an inhibited enzyme, reduced plasma membrane binding, and increased stability. This aim employs biomolecular NMR, crystallography, and mutagenesis to understand the structural and mechanistic basis for conformational closure. Aim 2 will employ a series of biochemical and cellular methods to define the function of Lys13 monoubiquitination, enhancing our understanding of how this PTM may promote the shuttling of PTEN from the cytosol to the nucleus. Aim 3 will address what molecular features drive USP7’s substrate selectivity and how USP7 PTMs (Ser18, Tyr1091, Thr1092, and Tyr1093 phosphorylation; Lys1096 acetylation and ubiquitination) regulate its function. Overall, these proposed studies can greatly enhance our understanding of the function and regulation of PTEN and USP7 which can spotlight possible targets for therapy. In addition, this proposal can also increase the PI's breadth of scientific skills and experiences as he seeks to chart a course for an independent academic career.