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

项目摘要 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 发现了O-GlcNAc转移酶基因(OGT)中的几个突变，这些突变是XLID的原因，但 这种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 它用单一的β-N-乙酰-氨基葡萄糖(O-GlcNAc)修饰核蛋白和细胞质蛋白。OGT有 数以千计的底物和O-GlcNAc具有不同的功能，包括调节营养传感， 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底物选择性的机理是一个重要的研究领域。据认为，N-末端 OGT的四肽重复序列(TPR)部分通过招募 针对特定底物和细胞结构域的OGT的伙伴蛋白。所有的OGT XLID变体都是 这里的研究局限于TPR，导致了我们关于OGT突变机制的假设 导致XLID：OGT XLID变体不仅没有中断OGT的稳定性或催化活性，反而表现出 受损的蛋白质-蛋白质相互作用以及这些异常的蛋白质相互作用导致细胞内的破坏 导致XLID表型的功能。这一假设得到了数据的支持，这些数据表明，所有的OGT XLID变异体具有热稳定性、催化功能和与野生型(WT)OGT类似的动力学特征。 为了验证我们的假设，我们将使用一种无偏见的邻近蛋白质组方法来定义WT和XLID OGT 相互作用，并确定异常相互作用的细胞影响。我们正独一无二地准备解决这个问题 假设是由于我们在O-GlcNAc生物学、质谱学方面的专业知识，以及我们拥有Cas9- 表达每个OGT XLID变体的工程化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，用于鉴定OGT TPR相互作用子。WT OGT和XLID变异互作 将进行比较，以识别异常交互和验证的个体交互。在目标2中，我们将评估 使用多种检测方法评估XLID表型中异常相互作用的分子贡献 互动者的特点(本地化、表达、翻译后修饰)和功能 失去与OGT相互作用的后果(酶、转录、信号通路分析)。这些 方法不仅将定义OGT TPR突变如何导致XLID的模型，还将识别WT OGT TPR互动组，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 佐治亚大学，将学员安排在一个良好的环境中学习普通和专业 在出色的导师指导下的生化技能。培训计划，虽然重点是将受训人员发展为 一位独立的科学家，还整合了DVM临床培训和研究开发，以确保学员 获得在兽医临床科学家的职业生涯中取得成功所需的技能。

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.