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

项目总结 X连锁智力残疾(XLID)影响大约500名男性中的1名。最近，我们和其他人已经 在编码O-GlcNAc转移酶(OGT)的X连锁基因上发现了多个独特的错义突变 是XLID的原因。这些突变产生了在TPR结构域中的氨基酸替换的变体 或OGT的催化结构域。数千种核质和胞质蛋白Ser/Thr残基的修饰 通过OGT添加单一的多糖(O-连接的N-乙酰氨基葡萄糖，O-GlcNAc)可以影响稳定性， 修饰蛋白质的定位、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，磷酸化是由数百人介导的 在激酶和较小的一组磷酸酶中，O-GlcNAc修饰是由单个 可被单一水解酶(O-GlcNAc水解酶，OGA)去除的转移酶(OGT)。我们已经产生了 大量已发表和初步的数据，包括几种OGT-TPR的生化特征 变种，基于同位素的方法来定量比较特定部位的O-GlcNAc和 采样并建立周转率，生成携带XLID的Cas9工程RUES-1干细胞 错义突变，以及定义OGT TPR交互作用组，包括定义交互作用子，如 与XLID相互作用减弱的表观遗传调节因子TET2和突触支架蛋白PCLO TPR变体。基于我们的初步数据，我们将检验OGT-TPR变体具有改变的假设 互动，并界定失去互动的影响(目标1A)。在目标1B中，我们将描述新的XLID OGT催化领域的变体，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识别 OGT表达的调节剂可能能够改善催化变异体的缺陷。最后，考虑到 TPR和催化域XLID OGT变体的患者表型几乎相同和重叠 将检验这一假设，即所有变异都有共同的下游改变，很可能是在基因调控方面，可能 受TET2及其相关蛋白(AIM 2)的调节和/或PCLO和 基于初步和已发表的数据的相关蛋白(目标3)。这些目标的成功实现， 旨在阐明对XLID OGT变体影响的机械论理解，将推动这两个 智力障碍和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.