The Role of the O-GlcNAc Modification in X-linked Intellectual Disability

O-GlcNAc 修饰在 X 连锁智力障碍中的作用

• 批准号: 10607367
• 负责人: Lance Wells
• 金额: $ 37.07万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607367
• 关键词: Affect; Amino Acid Substitution; Automobile Driving; Binding; Biochemical; Biology; Catalysis; Catalytic Domain; Cells; Characteristics; Chromatin; Clinical; Complex; Data; Defect; Disability phenotype; Disease; Engineering; Enzymes; Epigenetic Process; Evolution; Exhibits; Gene Duplication; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic Screening; Hydrolase; In Vitro; Intellectual functioning disability; Isotopes; Knockout Mice; Lead; Ligation; Link; Mammals; Mass Spectrum Analysis; Mediating; Microelectrodes; Missense Mutation; Modeling; Modification; Molecular; Mutation; Nuclear; Nutrient; O-GlcNAc transferase; Outcome; Pathway interactions; Patients; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Polysaccharides; Post-Translational Protein Processing; Proteins; Publishing; Regulation; Research; Role; Sampling; Scaffolding Protein; Site; Structure; Substrate Specificity; Synapses; Synaptosomes; Testing; Transferase; Variant; Work; X-linked intellectual disability; causal variant; clinical phenotype; disability; embryonic stem cell; experimental study; genome wide screen; glucose transport; human embryonic stem cell; male; molecular modeling; nerve stem cell; neural; novel; overexpression; protein protein interaction; sample collection; screening; sensor; stem cell differentiation; stem cells; sugar nucleotide; synaptic function; synaptogenesis; tandem mass spectrometry; tool; transcriptome

PROJECT SUMMARY X-linked intellectual disability (XLID) affects approximately 1 in 500 males. Recently, we, and others, have discovered multiple unique missense mutations in the X-linked gene encoding O-GlcNAc transferase (OGT) that are causal for XLID. These mutations generate variants with amino acid substitutions in either the TPR domain or catalytic domain of OGT. The modification of Ser/Thr residues of thousands of nuclear and cytosolic proteins by the addition of a single glycan (O-linked N-acetylglucosamine, O-GlcNAc) by OGT can impact the stability, localization, activity, and protein-protein interactions of the modified proteins. Similar to phosphorylation, the O- GlcNAc modification is inducible and dynamic. However, unlike phosphorylation, which is mediated by hundreds of kinases and a smaller set of phosphatases, O-GlcNAc modification results from the activity of a single transferase (OGT) that can be removed by a single hydrolase (O-GlcNAc hydrolase, OGA). We have generated considerable published and preliminary data including biochemical characterization of several OGT-TPR variants, engineering of an isotope-based approach to quantitatively compare site-specific O-GlcNAc between samples and establish turnover rates, generation of Cas9-engineered RUES-1 stem cells harboring the XLID missense mutations, and defining the OGT TPR interactome including defining interactors, such as the epigenetic regulator TET2 and the synapse scaffolding protein PCLO, that show reduced interactions with XLID TPR variants. Based on our preliminary data, we will test the hypothesis that OGT-TPR variants have an altered interactome and define the impact of loss of interaction (Aim 1A). In Aim 1B, we will characterize novel XLID variants in the catalytic domain of OGT that, based on modeling and in cellulo expression studies, we hypothesize will be Km variants for the sugar nucleotide donor and perform genome wide screens to identify regulators of OGT expression that might be able to ameliorate the deficits of a catalytic variant. Finally, given the nearly identical and overlapping patient phenotypes for both TPR and catalytic domain XLID OGT variants, we will test the hypothesis that all variants share common downstream alterations, likely in gene regulation that may be modulated by TET2 and associated proteins (Aim 2) and/or synaptic activity modulated by PCLO and associated proteins (Aim 3) based on preliminary and published data. The successful completion of these aims, geared towards elucidating a mechanistic understanding of the impact of XLID OGT variants, will advance both the intellectual disability and O-GlcNAc fields.

项目摘要 X连锁的智力残疾（XLID）影响了大约500名男性中的1个。最近，我们和其他人 在编码O-GLCNAC转移酶（OGT）的X连锁基因中发现了多个独特的错义突变。 是Xlid的因果。这些突变在TPR结构域中产生具有氨基酸取代的变体 或OGT的催化域。数千核和胞质蛋白的Ser/THR残基的修饰 通过添加单个聚糖（O连接的N-乙酰葡萄糖，O-GLCNAC），OGT会影响稳定性， 修饰蛋白的定位，活性和蛋白质 - 蛋白质相互作用。与磷酸化相似，O- GlcNAC修饰是可诱导且动态的。但是，与磷酸化不同，该磷酸化由数百个介导 激酶和一组较小的磷酸酶，O-GLCNAC的修饰是由单个的活性产生的 可以通过单个水解酶（O-GLCNAC水解酶，OGA）去除的转移酶（OGT）。我们已经生成了 大量发布和初步数据，包括几种OGT-TPR的生化表征 变体，基于同位素的工程方法，用于定量比较特定于位点的O-GLCNAC 样品并建立周转率，生成CAS9工程的Rues-1干细胞具有Xlid 错义突变，并定义OGT TPR相互作用，包括定义相互作用者，例如 表观遗传调节剂TET2和突触脚手架蛋白PCLO，显示出与Xlid的相互作用降低 TPR变体。根据我们的初步数据，我们将测试OGT-TPR变体有变化的假设 互动组并定义了相互作用丧失的影响（AIM 1A）。在AIM 1B中，我们将描述新颖的Xlid OGT催化结构域中的变体，基于建模和纤维素表达研究，我们 假设将是糖核苷酸供体的​​KM变体，并执行基因组宽筛网以识别 OGT表达的调节剂可能能够改善催化变体的缺陷。最后，给定 TPR和催化域Xlid OGT变体几乎相同和重叠的患者表型，我们 将检验以下假设：所有变体都有共同的下游变化，可能在基因调节中可能 由TET2和相关蛋白（AIM 2）和/或由PCLO和/或突触活动调节 基于初步和已发布数据的相关蛋白质（AIM 3）。这些目标的成功完成， 旨在阐明对Xlid OGT变体影响的机械理解，将促进两者 智力残疾和O-GLCNAC领域。