Molecular Mechanisms of PTEN and USP7 Regulation

PTEN和USP7调控的分子机制

• 批准号: 10019574
• 负责人: Daniel R. Dempsey
• 金额: $ 10万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 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 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10019574
• 关键词: Acetylation; Active Sites; Address; Automobile Driving; Binding; Biochemical; Biological; Biomolecular Nuclear Magnetic Resonance; C-terminal; Catalytic Domain; Cell Nucleus; Cell Proliferation; Cell membrane; Cells; Chemicals; Ciona intestinalis; Crystallization; Crystallography; Cytosol; Data; Deubiquitination; Disease; Double Minutes; 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; Protein p53; Proteins; Proto-Oncogene Proteins c-akt; Regulation; Reporting; Research; Resolution; Role; Second Messenger Systems; Series; Set protein; Signal Transduction; Site; Structure; Surface; Tail; Therapeutic Intervention; Tumor Suppressor Proteins; Ubiquitin; Ubiquitination; alpha helix; autism spectrum disorder; base; 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-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。该 K99/R00 提案涉及关键蛋白质的结构、功能和调节 参与细胞信号传导、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 生成 PIP2。由于 PIP3 是细胞生长和胰岛素信号传导的关键调节因子，因此 PTEN 至关重要 活动受到严格控制。 PTEN 功能丧失突变在癌症中经常观察到。 PTEN 是 翻译后受 N 端泛素化和 C 端磷酸化调节，但详细信息 这些翻译后修饰 (PTM) 的结构和机制影响尚不清楚。 USP7 是一种半胱氨酸水解酶，是一种去泛素酶 (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 去泛素化 据报道可抑制其从细胞质到细胞核的易位。此外，USP7 已被证明 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选择性地驱动 USP7 的底物并 它如何在细胞中受到调节。 USP7 在其 N 端和 C 端均通过磷酸化进行修饰， 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 受到 PTM 使用新兴半合成方法的监管。这些半合成方法 可以促进 PTM 及其模拟物在 PTEN 和 USP7 中进行位点特异性和化学计量安装。目的 1 试图利用结构性的 C 末端尾部磷酸化来定义 PTEN 调节的分子基础 接近。 PTEN 的构象闭合是由其 C 末端尾部位置的磷酸化驱动的 380、382、383 和 385 导致酶受抑制、质膜结合减少并增加 稳定。该目标采用生物分子核磁共振、晶体学和诱变来了解结构 和构象闭合的机制基础。目标2将采用一系列生化和细胞技术 定义 Lys13 单泛素化功能的方法，增强我们对该 PTM 的理解 可能促进 PTEN 从细胞质到细胞核的穿梭。目标 3 将解决什么分子 特性驱动 USP7 的底物选择性以及 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。总体而言，这些拟议的研究 可以极大地增强我们对PTEN和USP7的功能和调控的理解，这可以突出 可能的治疗目标。此外，该提案还可以增加 PI 的科学技能和知识广度 他在寻求为独立学术生涯规划路线时的经历。