Defining the OGT Interactive and its Role in X-Linked Intellectual Disability - Corrected Resubmission - Stephen Pre Doc Fellowship

定义 OGT Interactive 及其在 X 连锁智力障碍中的作用 - 更正重新提交 - Stephen Pre Doc Fellowship

• 批准号: 9911290
• 负责人: Hannah Michelle Stephen
• 金额: $ 4.21万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-02 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9911290
• 关键词: Address; Affect; Area; Binding; Biochemical; Biological Assay; Biology; C-terminal; Catalytic Domain; Cell Survival; Cell physiology; Cells; Characteristics; Chromosome abnormality; Clinical; Data; Development; Disability phenotype; Disease; ES Cell Line; Engineering; Ensure; Environment; Enzymes; Exhibits; Fellowship; Future; Gene Mutation; Genetic Transcription; Glucose; Impairment; Individual; Interruption; Kinetics; Label; Lead; Learning; Link; Mammalian Cell; Mass Spectrum Analysis; Mediating; Mentorship; Methods; Missense Mutation; Modeling; Modification; Molecular; Mutation; N-terminal; Neurons; Neurophysiology - biologic function; Nuclear; Nutrient; O-GlcNAc transferase; Outcome; Pathway Analysis; Patients; Phenotype; Phosphorylation; Phosphotransferases; Play; Post-Translational Protein Processing; Proteins; Proteomics; Regulation; Research; Resolution; Resources; Role; Scientist; Signal Pathway; Signal Transduction; Substrate Interaction; Synapses; Testing; Therapeutic Intervention; Training; Transcriptional Activation; Transferase; United States; Universities; Variant; Western Blotting; X-linked intellectual disability; base; career; causal variant; detection of nutrient; embryonic stem cell; gene function; glycosyltransferase; human embryonic stem cell; human male; interest; lymphoblast; male; nerve stem cell; neurodevelopment; pre-doctoral; protein protein interaction; recruit; relating to nervous system; research and development; sensor; skills; stem cells; synaptic function; transcription factor; transcriptomics

Project Summary X-Linked Intellectual Disability (XLID) affects approximately 1 in 500 males in the United States. We have identified several mutations in the O-GlcNAc Transferase gene (OGT) that are causal for XLID, but the mechanism underlying the phenotype is unknown. OGT is an essential nucleocytoplasmic glycosyltransferase that modifies nuclear and cytosolic proteins with a single β-N-Acetyl-Glucosamine (O-GlcNAc). OGT has thousands of substrates and O-GlcNAc serves a diverse set of functions, including modulating nutrient sensing, transcription, and synaptic function. The O-GlcNAc modification is considered analogous to phosphorylation, but unlike kinases, OGT is the only enzyme responsible for the O-GlcNAc modification within the mammalian cell. Therefore, the mechanism of OGT substrate selectivity is a major area of interest. It is thought that the N-terminal tetratricopeptide repeats (TPRs) of OGT are responsible for OGT substrate selection in part by recruitment of partner proteins that target OGT to specific substrates and cellular domains. All of the OGT XLID variants being studied here are localized to the TPRs, leading to our hypothesis on the mechanism by which OGT mutations lead to XLID: that rather than interrupting the stability or catalytic activity of OGT, the OGT XLID variants exhibit impaired protein-protein interactions and that these anomalous protein interactions cause disruptions in cellular function that lead to the XLID phenotype. This hypothesis is supported by data demonstrating that all of the OGT XLID variants are thermally stable, catalytically functional, and kinetically comparable to the wild-type (WT) OGT. To test our hypothesis, we will use an unbiased proximity proteomic approach to define the WT and XLID OGT interactomes, and identify the cellular impact of aberrant interactions. We are uniquely poised to address this hypothesis due to our expertise in O-GlcNAc biology, mass spectrometry, and our possession of Cas9- engineered male human embryonic stem cells expressing each OGT XLID variant. In Aim 1, we will use a proximity proteomic method, BioID, to identify OGT TPR interactors. WT OGT and XLID variant interactomes will be compared to identify aberrant interactions and individual interactions validated. In Aim 2, we will assess the molecular contribution of aberrant interactions in the XLID phenotype, using a variety of assays to assess the interactor’s characteristics (localization, expression, post-translational modifications) and functional consequences of its loss of interaction with OGT (enzymatic, transcriptomic, signaling pathway analyses). These approaches will not only define a model for how OGT TPR mutations cause XLID, but also identify a WT OGT TPR interactome, an essential resource for the field. This research will take place in Dr. Lance Wells’ lab at the University of Georgia, placing the trainee in an excellent environment to learn general and specialized biochemical skills under outstanding mentorship. The training plan, while focused on developing the trainee into an independent scientist, also integrates DVM clinical training and research development to ensure the trainee gains the skills necessary to be successful in a career as a veterinary clinician scientist.

项目概要 在美国，大约每 500 名男性中就有 1 人患有 X 连锁智力障碍 (XLID)。我们有 确定了 O-GlcNAc 转移酶基因 (OGT) 中的几个突变是导致 XLID 的原因，但 表型背后的机制尚不清楚。 OGT 是一种必需的核细胞质糖基转移酶 用单个 β-N-乙酰葡萄糖胺 (O-GlcNAc) 修饰核蛋白和胞浆蛋白。奥格特有 数以千计的底物和 O-GlcNAc 具有多种功能，包括调节营养感应、 转录和突触功能。 O-GlcNAc 修饰被认为类似于磷酸化，但是 与激酶不同，OGT 是哺乳动物细胞内唯一负责 O-GlcNAc 修饰的酶。 因此，OGT底物选择性的机制是人们感兴趣的主要领域。据认为，N 端 OGT 的四肽重复序列 (TPR) 部分通过招募 OGT 底物来选择 将 OGT 靶向特定底物和细胞结构域的伙伴蛋白。所有 OGT XLID 变体均是 这里的研究局限于 TPR，导致我们对 OGT 突变机制的假设 导致 XLID：OGT XLID 变体不会破坏 OGT 的稳定性或催化活性，而是表现出 蛋白质-蛋白质相互作用受损，这些异常的蛋白质相互作用会导致细胞的破坏 导致 XLID 表型的功能。这一假设得到了数据的支持，表明所有 OGT XLID 变体具有热稳定性、催化功能，并且在动力学上与野生型 (WT) OGT 相当。 为了检验我们的假设，我们将使用无偏近蛋白质组学方法来定义 WT 和 XLID OGT 相互作用组，并确定异常相互作用对细胞的影响。我们有独特的准备来解决这个问题 由于我们在 O-GlcNAc 生物学、质谱分析方面的专业知识以及我们拥有 Cas9- 表达每个 OGT XLID 变体的工程男性人类胚胎干细胞。在目标 1 中，我们将使用 邻近蛋白质组学方法 BioID 用于识别 OGT TPR 相互作用因子。 WT OGT 和 XLID 变异相互作用组 将进行比较以识别异常的相互作用并验证个体相互作用。在目标 2 中，我们将评估 XLID 表型中异常相互作用的分子贡献，使用多种测定法来评估 交互者的特征（定位、表达、翻译后修饰）和功能 失去与 OGT 相互作用的后果（酶学、转录组学、信号通路分析）。这些 方法不仅可以定义 OGT TPR 突变如何导致 XLID 的模型，还可以识别 WT OGT TPR 相互作用组，该领域的重要资源。这项研究将在 Lance Wells 博士的实验室进行 佐治亚大学，为学员提供良好的环境来学习普通知识和专业知识 在出色的指导下获得生化技能。培训计划，同时注重将受训者培养成 一名独立科学家，还整合了 DVM 临床培训和研究开发，以确保学员 获得作为兽医临床科学家在职业生涯中取得成功所需的技能。