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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要 X 连锁智力障碍 (XLID) 影响大约千分之一的男性。最近，我们发现编码 O-GlcNAc 转移酶 (OGT) 的基因突变是导致 XLID 的原因。这些突变产生 OGT 的 TPR 结构域中氨基酸取代的变体，这些变体被认为参与蛋白质-蛋白质相互作用。核蛋白和胞质蛋白的 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）会影响稳定性，许多核蛋白和胞浆蛋白的定位、活性和蛋白-蛋白相互作用。类似于 O-GlcNAc 磷酸化后，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，由大量激酶和一小部分磷酸酶介导，O- GlcNAc 酰化是由单一转移酶 (OGT) 的活性引起的，并且可以通过单一水解酶去除（O-GlcNAc 水解酶，OGA）。有人建议 O-GlcNAc 修饰是一种调节修改，因为它已被证明在一小部分上是全局可诱导的和动态的检查蛋白质。我们假设 XLID 中观察到的 TPR 变体正在改变 O-GlcNAc 动力学和/或 OGT 相互作用组。具体目标利用了我们在 O-GlcNAc 生物学方面的专业知识和 OGT 酶学以及我们创新的标记、富集和基于质谱的方法用于位点作图和相互作用组识别，应用于源自正常或 Cas9- 的神经谱系工程化人类胚胎干细胞。在目标 1 中，我们开发了一种新方法来检查动态位点特异性 O-GlcNAc 修饰和修饰的蛋白质，并将其与富集策略结合起来和串联质谱方法来定义细胞类型和 XLID 基因型中的 O-GlcNAc 循环速率依赖方式。在目标 2 中，我们也使用经典的免疫共沉淀定义了 OGT 相互作用组作为以细胞类型和 XLID 基因型依赖方式进行邻近标记的方法。成功者这些目标的完成不仅有利于 O-GlcNAc 生物学界，更重要的是确定受 XLID 影响的特定 OGT 靶点和结合伙伴，以供未来详细的假设驱动研究。