O-GlcNAc dynamics and the OGT interactome in variants causal for X-linked intellectual disability

导致 X 连锁智力障碍的变异中的 O-GlcNAc 动力学和 OGT 相互作用组

• 批准号: 10011894
• 负责人: Lance Wells
• 金额: $ 22.65万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-10 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10011894
• 关键词: Adopted; Affect; Amino Acid Substitution; Amino Acids; Amino Sugars; Automobile Driving; Binding; Biochemical; Biology; Cell Survival; Cells; Co-Immunoprecipitations; Code; Communities; DNA Polymerase II; Data; Detection; Development; Disease; Down-Regulation; Embryo Loss; Engineering; Enzymatic Biochemistry; Enzymes; Equilibrium; Evolution; Financial compensation; Future; Gene Duplication; Genes; Genetic Transcription; Genotype; Glutamine; Half-Life; Human Engineering; Hydrolase; Isotopes; Knockout Mice; Label; Ligation; Link; Mammals; Maps; Mass Spectrum Analysis; Mediating; Methods; Missense Mutation; Modification; Molecular; Mutation; Nature; Nuclear; Nutrient; O-GlcNAc transferase; Outcome; Patients; Pharmacology; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Polysaccharides; Post-Translational Protein Processing; Property; Proteins; Publishing; RNA; Research; Resolution; Role; Site; Technology; Transferase; Variant; Western Blotting; Work; X-linked intellectual disability; base; causal variant; cell type; embryonic stem cell; engineered stem cells; human embryonic stem cell; innovation; lead candidate; male; nerve stem cell; novel; overexpression; protein protein interaction; relating to nervous system; sample collection; sensor; tandem mass spectrometry; tool

PROJECT SUMMARY X-linked intellectual disability (XLID) affects approximately 1 in 1,000 males. Recently, we have discovered mutations in the gene encoding O-GlcNAc transferase (OGT) that are causal for XLID. These mutations generate variants with amino acid substitutions in the TPR domains of OGT that are thought to be involved in protein-protein interactions. The modification of Ser/Thr residues of nuclear and cytosolic proteins by the addition of a single glycan (O-linked N-acetylglucosamine, O-GlcNAc) by OGT impacts the stability, localization, activity, and protein-protein interactions of many nuclear and cytosolic proteins. Similar to phosphorylation, thousands of nuclear and cytosolic proteins in mammals are modified by O-GlcNAc. Unlike phosphorylation, which is mediated by a plethora of kinases and a smaller set of phosphatases, O- GlcNAcylation results from the activity of a single transferase (OGT) and can be removed by a single hydrolase (O-GlcNAc hydrolase, OGA). It has been suggested that the O-GlcNAc modification is a regulatory modification in that it has been demonstrated to be globally inducible and dynamic on a small subset of proteins examined. We hypothesize that the TPR variants observed in XLID are altering O-GlcNAc dynamics and/or the OGT interactome. The specific aims leverage our expertise in O-GlcNAc biology and the enzymology of OGT along with our innovative labeling, enrichment, and mass spectrometry-based approaches for site-mapping and interactome identification applied to neural lineages derived from normal or Cas9- engineered human embryonic stem cells. In Aim 1, we develop a novel method for examining the dynamics of both site-specific O-GlcNAc modification and the modified protein and couple this with enrichment strategies and tandem mass spectrometry approaches to define O-GlcNAc cycling rates in a cell type and XLID genotype dependent manner. In Aim 2, we define the OGT interactome using classical co-immunoprecipitation as well as proximity labeling approaches in a cell type and XLID genotype dependent manner. The successful completion of these aims will not only benefit the O-GlcNAc biology community but more importantly will identify specific OGT targets and binding partners impacted by XLID for future detailed hypothesis-driven studies.

项目摘要 X连锁智力残疾（XLID）影响约1千分之一的男性。最近，我们发现 编码O-GlcNAc转移酶（OGT）的基因中的突变是XLID的病因。这些突变 产生在OGT的TPR结构域中具有氨基酸取代的变体，所述变体被认为涉及 蛋白质相互作用核蛋白和胞浆蛋白的Ser/Thr残基的修饰 通过OGT添加单个聚糖（O-连接的N-乙酰葡糖胺，O-GlcNAc）影响稳定性， 定位，活性和蛋白质-蛋白质相互作用的许多核和胞质蛋白质。类似于 由于O-GlcNAc的磷酸化，哺乳动物中数以千计的核蛋白和胞质蛋白被O-GlcNAc修饰。不像 磷酸化，这是由过多的激酶和磷酸酶，O- GlcNAc酰化由单个转移酶（OGT）的活性引起，并且可以通过单个水解酶去除 （O-GlcNAc水解酶，OGA）。已经表明，O-GlcNAc修饰是调节性的。 修改，因为它已被证明是全球诱导和动态的一小部分， 蛋白质检测我们假设在XLID中观察到的TPR变体改变了O-GlcNAc动力学 和/或OGT相互作用组。具体目标利用我们在O-GlcNAc生物学和生物学领域的专业知识， OGT的酶学沿着我们创新的标记，富集和质谱为基础的方法 用于应用于源自正常或Cas9- 人类胚胎干细胞在目标1中，我们开发了一种新的方法来检查 位点特异性O-GlcNAc修饰和修饰的蛋白质，并将其与富集策略结合 和串联质谱法来确定细胞类型和XLID基因型中的O-GlcNAc循环速率 依赖的方式。在目标2中，我们也使用经典的免疫共沉淀来定义OGT相互作用组 作为以细胞类型和XLID基因型依赖性方式的邻近标记方法。成功 这些目标的完成不仅有利于O-GlcNAc生物界，而且更重要的是， 确定受XLID影响的特定OGT目标和结合伙伴，以供未来详细假设驱动 问题研究