Molecular Mechanisms of PTEN and USP7 Regulation

PTEN和USP7调控的分子机制

• 批准号: 10576979
• 负责人: Daniel R. Dempsey
• 金额: $ 24.9万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-17 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10576979
• 关键词: Acetylation; Active Sites; Automobile Driving; Binding; Biochemical; Biological; Biomolecular Nuclear Magnetic Resonance; C-terminal; Catalytic Domain; Cell Nucleus; Cell Proliferation; Cell membrane; Cells; Chemicals; Ciona intestinalis; Crystallography; Cytosol; Data; Deubiquitination; Disease; Double Minutes; Enzyme Inhibition; Enzymes; Evaluation; Excision; Genome Stability; Homologous Gene; Human; Hydrolase; Label; Link; Lipids; Location; Lysine; Malignant Neoplasms; Maps; Mediating; Membrane; Membrane Lipids; Methods; Modification; Molecular; Molecular Conformation; Monoubiquitination; Multiple Hamartoma Syndrome; Mus; Mutagenesis; Mutation; N-terminal; Nuclear; PI3K/AKT; Pathologic; Pathway interactions; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipids; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Positioning Attribute; Post-Translational Protein Processing; Post-Translational Regulation; Process; Property; Proteins; Proto-Oncogene Proteins c-akt; Regulation; Reporting; Research; Resolution; Role; Second Messenger Systems; Series; Set protein; Signal Transduction; Site; Structure; Surface; TP53 gene; Tail; Therapeutic Intervention; Titrations; Tumor Suppressor Proteins; Ubiquitin; Ubiquitination; alpha helix; autism spectrum disorder; career; cell growth; crosslink; enzyme structure; experience; human disease; improved; inorganic phosphate; insulin signaling; loss of function; mimicry; multicatalytic endopeptidase complex; novel therapeutics; prevent; protein protein interaction; skills; targeted treatment; tripolyphosphate; tumor; ubiquitin isopeptidase; ubiquitin-protein ligase; voltage

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.

项目摘要。此K99/R00建议涉及关键蛋白的结构，功能和调节 参与细胞信号，PTEN和USP7。 PTEN是一种肿瘤抑制脂质磷酸酶，可催化 从膜磷脂磷脂酰肌醇（3,4,5） - 三磷酸（PIP3）中去除3'-磷酸盐（PIP3） 生成pip2。由于PIP3是细胞生长和胰岛素信号传导的关键调节剂，因此必须使用PTEN 活动受到严格控制。在癌症中经常观察到功能PTEN突变的丧失。 Pten是 通过N末端泛素化和C末端磷酸化对翻译后的调节，但详细的 这些翻译后修饰（PTM）的结构和机械影响尚不清楚。 USP7是一种Cys水解酶，是去泛素酶（DUB），催化泛素/赖氨酸的裂解 异肽键。 USP7的泛素化蛋白底物包括PTEN和MDM2。 PTEN的去泛素化 据报道，抑制其从细胞质到核的易位。而且，已显示USP7 增强MDM2的细胞稳定性，这很重要，因为MDM2是主要的E3泛素连接酶 肿瘤抑制蛋白p53。目前尚不清楚哪些分子特征有选择地驱动USP7的底物，并且 如何在细胞中调节它。 USP7通过磷酸化对其N和C末端进行了修改， 乙酰化但这些PTM的调节作用尚不清楚。在这里，我们将解决PTEN和USP7 使用新的和新兴的半合成方法通过PTM调节。这些半合成方法 可以促进PTM的特定地点和化学计量的安装及其模拟物中的PTEN和USP7。目的 1试图使用结构来定义通过C末端尾磷酸化调节PTEN的分子基础 方法。 PTEN的构象闭合是由其C末端尾部的光谱驱动的 380、382、383和385导致抑制酶，质膜结合减少，增加 稳定。这个目标员工生物分子NMR，晶体学和诱变，以了解结构性 和构象闭合的机理基础。 AIM 2将采用一系列生化和细胞 定义lys13单泛素化功能的方法，增强了我们对这种PTM的理解 可能会促进PTEN从细胞质到核的穿梭。 AIM 3将解决哪些分子 功能驱动USP7的底物选择性以及USP7 PTM（Ser18，Tyr1091，Thr1092和Tyr1093） PhoterphoryLation; LYS1096乙酰化和泛素化）调节其功能。总体而言，这些提出的研究 可以大大增强我们对PTEN和USP7功能和调节的理解，这可以聚焦 治疗的可能目标。此外，该建议还可以提高PI的科学技能广度和 他试图为独立学术生涯的课程绘制一门课程。