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 X连锁的智力残疾（XLID）在美国影响了500名男性中约1个。我们有 确定了Xlid因果的O-GLCNAC转移酶基因（OGT）中的几个突变，但是 表型基础的机制尚不清楚。 OGT是必不可少的核眼质糖基转移酶 这可以通过单个β-N-乙酰葡萄糖（O-GLCNAC）修饰核和胞质蛋白。 OGT有 成千上万的底物和O-GLCNAC提供了一组潜水功能，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 与激酶不同，OGT是唯一负责哺乳动物细胞内O-GLCNAC修饰的酶。 因此，OGT底物选择性的机制是关注的主要领域。认为N末端 OGT的四肽重复序列（TPR）负责OGT底物选择部分通过招募 将OGT靶向特定底物和细胞结构域的伴侣蛋白。所有OGT Xlid变体都是 这里研究的局部与TPR进行了，导致了我们关于OGT突变的机制的假设 导致Xlid：OGT Xlid变体而不是中断OGT的稳定性或催化活性 蛋白质 - 蛋白质相互作用受损，这些异常蛋白质相互作用会导致细胞中断 导致Xlid表型的功能。数据证明所有OGT的数据都支持了这一假设 Xlid变体具有热稳定，催化功能性，并且具有与野生型（WT）OGT相当的动力学。 为了检验我们的假设，我们将使用公正的接近蛋白质组学方法来定义WT和Xlid OGT 相互作用，并确定异常相互作用的细胞影响。我们很毒，解决这个问题 假设由于我们在O-GLCNAC生物学，质谱法和拥有Cas9-的专业知识而引起的假设 设计的男性人类胚胎干细胞表达每个OGT XLID变体。在AIM 1中，我们将使用 近端蛋白质组学方法，生物形式，以识别OGT TPR相互作用。 WT OGT和Xlid变体相互作用体 将进行比较以确定验证的异常相互作用和单个相互作用。在AIM 2中，我们将评估 XLID表型中异常相互作用的分子贡献，使用各种评估来评估 交互者的特征（本地化，表达，翻译后修改）和功能 与OGT相互作用丧失的后果（酶促，转录组，信号通路分析）。这些 方法不仅可以定义OGT TPR突变如何引起Xlid的模型，还可以确定一个wt ogt TPR Interactome，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 佐治亚大学，将实习生置于一个很好的环境中，以学习一般和专业 杰出心态下的生化技巧。培训计划虽然专注于将实习生开发到 独立科学家，还整合了DVM临床培训和研究开发，以确保受训者 在作为兽医临床科学家职业生涯中取得成功所需的技能。