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

项目摘要 X连锁智力残疾（XLID）影响大约1/500的男性。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， 与XLID有因果关系。这些突变产生在TPR结构域中具有氨基酸取代的变体， 或OGT的催化结构域。数千种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）可影响稳定性， 修饰蛋白的定位、活性和蛋白质-蛋白质相互作用。与磷酸化类似，O- GlcNAc修饰是可诱导的和动态的。然而，与磷酸化不同的是，磷酸化是由数百个 激酶和磷酸酶的一个较小的集合，O-GlcNAc修饰的结果从一个单一的活性， 在一个实施方案中，所述酶是可通过单一水解酶（O-GlcNAc水解酶，OGA）去除的葡萄糖转移酶（OGT）。我们已经生成 相当多的已发表和初步数据，包括几种OGT-TPR的生化表征 变体，工程化基于同位素的方法以定量比较 样品并建立周转率，产生携带XLID的Cas9工程化的RUES-1干细胞 错义突变，以及定义OGT TPR相互作用物组，包括定义相互作用物，如 表观遗传调节因子TET 2和突触支架蛋白PCLO，显示与XLID的相互作用减少 TPR变体。基于我们的初步数据，我们将检验OGT-TPR变体改变了 相互作用组，并定义相互作用丧失的影响（目标1A）。在目标1B中，我们将表征新型XLID OGT催化结构域中的变体，基于建模和细胞表达研究，我们 假设将是糖核苷酸供体的Km变体，并进行全基因组筛选以鉴定 OGT表达的调节剂，其可能能够改善催化变体的缺陷。最后，鉴于 TPR和催化结构域XLID OGT变体几乎相同和重叠的患者表型，我们 将测试所有变体共享共同下游改变的假设，可能在基因调控中， 由TET 2和相关蛋白（Aim 2）调节和/或由PCLO调节的突触活性， 相关蛋白（目标3）的基础上初步和公布的数据。这些目标的圆满实现， 旨在阐明XLID OGT变体影响的机械理解，将推进 智力残疾和O-GlcNAc领域。

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.