NANOG STRUCTURE AND FUNCTION IN STEM CELL PLURIPOTENCY

干细胞多能性中的纳米结构和功能

• 批准号: 9814562
• 负责人: Josephine Chu Ferreon
• 金额: $ 5.61万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9814562
• 关键词: Adopted; Affect; Amino Acids; Attenuated; Binding; Binding Sites; Biological Assay; C-terminal; Cell Culture Techniques; Cell Nucleus; Cell Proliferation; Cells; Chemicals; DNA; Data; Dipeptides; Disease; Distant; Epigenetic Process; Fluorescence; Future; Genetic Transcription; Growth; In Vitro; Lead; Length; Maps; Mass Spectrum Analysis; Measures; Mediating; Methods; Modeling; Modification; Molecular; Molecular Conformation; Multiple Partners; Mutation; Mutation Analysis; N-terminal; NMR Spectroscopy; Peptides; Peptidylprolyl Isomerase; Phosphorylation; Pluripotent Stem Cells; Positioning Attribute; Post-Translational Protein Processing; Proline; Proteins; Proteomics; Research; Research Personnel; Signal Transduction; Site; Somatic Cell; Spectrum Analysis; Stem cells; Structure; Techniques; Testing; Therapeutic Intervention; Time; Tryptophan; Ubiquitination; Vertebral column; anti-cancer; base; cancer stem cell; cell type; conformer; crosslink; deletion analysis; drug discovery; embryonic stem cell; experience; experimental study; in vivo; induced pluripotent stem cell; insight; interdisciplinary approach; macromolecular assembly; pluripotency; prevent; prolyl-serine; promoter; recruit; scaffold; self assembly; self-renewal; seryl-proline; single molecule; stem cell biology; stem cell therapy; stemness; synthetic peptide; transcription factor; unnatural amino acids

Project Summary Nanog Structure and Function in Stem Cell Pluripotency This proposed research will determine the structural basis for interactions made by Nanog, an epigenetic reprogramming factor that regulates cellular proliferation and differentiation. Nanog is required for self-renewal of embryonic stem cells and for re-programming, the transformation of somatic cells to pluripotent stem cells. Nanog interacts with >100 other proteins, but intrinsically disordered regions make Nanog difficult to study. The investigators will use their extensive experience with intrinsically disordered proteins to dissect Nanog function in two stages: they will determine how phosphorylation and self-assembly regulate Nanog stability and activity (Aim 1), and they will determine how interactions between Nanog and partners give rise to pluripotency (Aim 2). These experiments will establish the molecular mechanisms of Nanog hub function, and perhaps produce lead compounds for future drug discovery efforts to target Nanog-dependent cell proliferation and pluripotency, which is relevant to therapeutic interventions that utilize stem cells or that seek to destroy cancer stem cells. The first Aim will use solution NMR spectroscopy and other methods to reveal how phosphorylation in the Nanog PEST degradation signal alters local backbone conformation at Ser-Pro dipeptides and affects Nanog levels in cells via the phosphorylation-dependent proline isomerase Pin1. Reprogramming assays will be used to test the effects of perturbing Nanog/Pin1 interaction. Single molecule fluorescence methods will be used to characterize the structure and dynamics of the isolated Nanog C-terminal domain and to determine how the N- terminal domain modulates Nanog oligomerization in vitro and in vivo. These experiments will provide funda- mental insight into Nanog availability in the assembled state thought to underlie its function as a molecular hub. The second Aim will determine the mechanisms by which Nanog acts as the ‘gateway to pluripotency’. The investigators will use unnatural amino acid photocrosslinking, chemical crosslinking and mass spectrometry- based proteomics to identify Nanog binding partners in stem cells, to uncover any correlations between multiple binding partners, and to map the Nanog sequences that support binding. They will determine the functional importance of these interactions by perturbing the partner and/or its binding site on Nanog in somatic cell reprogramming assays. These experiments will demonstrate the importance of key partners to Nanog function and will reveal which regions of Nanog recruit these partners to drive pluripotency. Both the partners and the regions of Nanog that they bind may be useful targets for modulating Nanog activity. They will determine how Nanog influences binding of the transcription factors Oct4 and Sox2 to each other and to the Nanog promoter. These experiments will reveal how Nanog scaffolds macromolecular assemblies that control stem cell pluripotency.

项目概要 Nanog 干细胞多能性的结构和功能 这项拟议的研究将确定 Nanog（一种表观遗传物质）相互作用的结构基础。 调节细胞增殖和分化的重编程因子是自我更新所必需的。 胚胎干细胞和重新编程，将体细胞转化为多能干细胞。 Nanog 与超过 100 种其他蛋白质相互作用，但本质上无序的区域使 Nanog 难以研究。 研究人员将利用他们在本质无序蛋白质方面的丰富经验来剖析 Nanog 分两个阶段发挥作用：它们将决定磷酸化和自组装如何调节 Nanog 稳定性和 活动（目标 1），他们将确定 Nanog 和合作伙伴之间的相互作用如何产生多能性 （目标 2）。这些实验将建立 Nanog 中枢功能的分子机制，或许还有可能​​。 生产用于未来药物发现工作的先导化合物，以靶向 Nanog 依赖性细胞增殖和 多能性，与利用干细胞或试图消灭癌症的治疗干预相关 干细胞。 第一个目标将使用溶液核磁共振波谱和其他方法来揭示磷酸化过程 Nanog PEST 降解信号改变 Ser-Pro 二肽的局部主链构象并影响 Nanog 将使用通过磷酸化依赖性脯氨酸异构酶 Pin1 检测细胞中的水平。 将使用单分子荧光方法来测试扰动 Nanog/Pin1 相互作用的影响。 表征分离的 Nanog C 端结构域的结构和动力学，并确定 N- 末端结构域在体外和体内调节 Nanog 寡聚化，这些实验将提供基础。 对 Nanog 在组装状态下可用性的心理洞察被认为是其作为分子中心功能的基础。 第二个目标将确定 Nanog 作为“多能性之门”的机制。 研究人员将使用非天然氨基酸光交联、化学交联和质谱法- 基于蛋白质组学来识别干细胞中的 Nanog 结合伙伴，以揭示之间的任何相关性 多个结合伙伴，并绘制支持结合的 Nanog 序列。 通过扰乱体细胞中 Nanog 上的伴侣和/或其结合位点，这些相互作用的功能重要性 这些实验将证明 Nanog 关键合作伙伴的重要性。 功能并将揭示 Nanog 的哪些区域招募这些合作伙伴来驱动多能性。 它们结合的 Nanog 区域可能是调节 Nanog 活性的有用靶标。 确定 Nanog 如何影响转录因子 Oct4 和 Sox2 彼此之间以及与 这些实验将揭示 Nanog 支架如何控制大分子组装体。 干细胞多能性。