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: 10318174
• 负责人: Josephine Chu Ferreon
• 金额: $ 31.7万
• 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 2023-07-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10318174
• 关键词: Adopted; Affect; Amino Acids; Attenuated; Binding; Binding Sites; Biological Assay; C-terminal; 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 cell pluripotency; 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; promoter; recruit; scaffold; self assembly; self-renewal; seryl-proline; single molecule; stem cell biology; stem cell therapy; stem cells; stemness; synthetic peptide; transcription factor; translational applications; 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.

项目摘要 干细胞多能性的纳米结构和功能 这项拟议的研究将确定Nanog进行的相互作用的结构基础，这是一种表观遗传学的 调节细胞增殖和分化的重编程因子。自我更新需要Nanog 胚胎干细胞和重新编程，将体细胞转化为多能干细胞。 Nanog与其他> 100种蛋白质相互作用，但是内在无序的区域使Nanog难以研究。 调查人员将利用他们在本质上受干扰蛋白的丰富经验来剖析纳米 在两个阶段的功能：他们将决定磷酸化和自组装如何调节纳米稳定性和 活动（AIM 1），他们将确定Nanog与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 Hub功能的分子机制，也许 生产铅化合物，用于未来的药物发现工作，以靶向纳米依赖性细胞增殖和 多能性，这与使用干细胞或试图破坏癌症的治疗干预措施有关 干细胞。 第一个目的将使用溶液NMR光谱和其他方法来揭示如何在 Nanog害虫降解信号改变了Ser-Pro二肽处的局部骨干构象并影响Nanog 细胞中通过磷酸化依赖性脯氨酸异构酶PIN1的水平。重新编程测定将使用 测试扰动纳米/PIN1相互作用的影响。单分子荧光方法将用于 表征分离的纳米C末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纳米寡聚。这些实验将提供基础 - 在组装状态中，人们对纳米的可用性的心理见解是其作为分子枢纽功能的基础。 第二个目标将确定纳米纳克充当“通往多能的门户”的机制。这 研究人员将使用不自然的氨基酸光链接，化学交联和质谱法 - 基于蛋白质组学以鉴定干细胞中的纳米结合伴侣，以发现 多个结合伙伴，并绘制支持结合的纳米序列。他们将确定 这些相互作用的功能重要性是通过扰动伴侣和/或在体细胞中的Nanog上的绑定位点的功能重要性 细胞重编程测定。这些实验将证明关键合作伙伴对Nanog的重要性 功能，并将揭示哪些Nanog区域招募这些伴侣来推动多能。两个合作伙伴 它们结合的Nanog区域可能是调节纳米活性的有用靶标。他们会的 确定纳米如何影响转录因子Oct4和Sox2的结合以及彼此之间的结合 纳米启动子。这些实验将揭示纳米支架如何控制的大分子组件 干细胞多能。