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。这个K99/R00提案涉及关键蛋白质的结构、功能和调控 参与细胞信号传导的蛋白质，PTEN和USP7。PTEN是一种肿瘤抑制性脂质磷酸酶，其催化肿瘤细胞凋亡。 从膜磷脂磷脂酰肌醇（3，4，5）-三磷酸（PIP3）中除去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，因此必须使PTEN 活动要严格控制。在癌症中经常观察到功能丧失的PTEN突变。pten是 在蛋白质表达后，蛋白质的N端泛素化和C端磷酸化都受到调节，但在蛋白质表达后， 这些翻译后修饰（PTM）的结构和机制影响还没有被很好地理解。 USP7是一种Cys水解酶，其是一种去泛素酶（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MDM 2。PTEN的去泛素化 据报道抑制其从胞质溶胶到细胞核的易位。此外，USP7已被证明 增强MDM2的细胞稳定性，这很重要，因为MDM2是一种E3泛素连接酶，用于主要的 肿瘤抑制蛋白p53。目前还不清楚是什么分子特征选择性地驱动USP7的底物， 它在细胞中是如何被调节的。USP 7在其N-和C-末端均通过磷酸化修饰， 乙酰化，但这些PTM的调节作用尚不清楚。在这里，我们将讨论如何PTEN和USP7 使用新的和新兴的半合成方法由PTM调节。这些半合成方法 可以促进PTM及其模拟物位点特异性和化学计量安装到PTEN和USP7中。目的 1试图使用结构分析来确定通过C-末端尾部磷酸化进行PTEN调节的分子基础。 接近。PTEN的构象关闭是由其C-末端尾的磷酸化驱动的， 380、382、383和385，导致酶受抑制，质膜结合减少， 稳定这一目标利用生物分子NMR、晶体学和诱变来理解结构 和构象闭合的机制基础。Aim 2将采用一系列生物化学和细胞 方法来定义Lys13单泛素化的功能，增强我们对这种PTM如何 可能促进PTEN从胞质到细胞核的穿梭。目标3将解决分子 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。总的来说，这些研究 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，这一建议还可以增加PI的科学技能的广度， 他试图为独立的学术生涯绘制一个课程。