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: 10320946
• 负责人: Hannah Michelle Stephen
• 金额: $ 4.34万
• 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/10320946
• 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.

项目摘要 在美国，X连锁智力残疾（XLID）影响大约1/500的男性。我们有 在O-GlcNAc转移酶基因（OGT）中发现了几种导致XLID的突变，但 表型的潜在机制尚不清楚。OGT是一种必需的核质糖基转移酶 用单个β-N-乙酰氨基葡萄糖（O-GlcNAc）修饰细胞核和胞质蛋白。OGT拥有 成千上万的底物和O-GlcNAc提供多种功能，包括调节营养感测， 转录和突触功能。O-GlcNAc修饰被认为类似于磷酸化，但 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感兴趣的主要领域。据认为，N-末端 OGT的四肽重复序列（TPR）部分通过募集 将OGT靶向特定底物和细胞结构域的伴侣蛋白。所有的OGT XLID变体 我们研究的OGT基因定位于TPRs，这导致了我们关于OGT突变的机制的假设。 导致XLID：OGT XLID变体没有中断OGT的稳定性或催化活性， 蛋白质-蛋白质相互作用受损，这些异常的蛋白质相互作用导致细胞分裂， 导致XLID表型的功能。这一假设得到了数据的支持，表明所有的OGT XLID变体是热稳定的、催化功能的，并且在动力学上与野生型（WT）OGT相当。 为了验证我们的假设，我们将使用无偏倚的邻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的细胞影响。我们有能力解决这个问题 假设由于我们在O-GlcNAc生物学，质谱学方面的专业知识，以及我们拥有Cas9- 表达每种OGT XLID变体的工程化男性人胚胎干细胞。在目标1中，我们将使用 邻近蛋白质组学方法BioID鉴定OGT TPR相互作用物。WT OGT和XLID变体相互作用体 将进行比较，以识别异常相互作用和经验证的个体相互作用。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，使用各种测定来评估 相互作用物的特征（定位、表达、翻译后修饰）和功能 其与OGT相互作用丧失的后果（酶、转录组学、信号通路分析）。这些 这种方法不仅可以定义OGT TPR突变如何导致XLID的模型，而且还可以识别WT OGT TPR相互作用组，该领域的重要资源。这项研究将在兰斯威尔斯博士的实验室进行， 格鲁吉亚大学，将受训者置于良好的环境中学习一般和专业知识 在出色的指导下掌握生化技能。培训计划虽然侧重于将受训者培养成 一个独立的科学家，还整合了DVM临床培训和研究开发，以确保学员 获得必要的技能是成功的职业生涯作为一个兽医临床科学家。