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

项目总结 X连锁智力残疾(XLID)影响大约1,000名男性。最近，我们发现 编码O-GlcNAc转移酶(OGT)的基因突变是XLID的原因。这些突变 在OGT的TPR结构域产生氨基酸取代的变体，被认为参与了 蛋白质之间的相互作用。核质和胞质蛋白Ser/Thr残基的修饰 通过OGT添加单个多糖(O-连接N-乙酰氨基葡萄糖，O-GlcNAc)会影响稳定性， 许多核蛋白和胞质蛋白的定位、活性和蛋白质-蛋白质相互作用。类似于 在哺乳动物中，数以千计的核蛋白和胞质蛋白被O-GlcNAc磷酸化修饰。不像 磷酸化，这是由过多的激酶和一组较小的磷酸酶，O- GlcN酰化是单一转移酶(OGT)活性的结果，可被单一水解酶去除 (O-GlcNAc水解酶，OGA)。有人认为O-GlcNAc的修饰是一种调节性的 修改，因为它已被证明是全局可诱导的，并且在 检测了蛋白质。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互动组。具体目标是利用我们在O-GlcNAc生物学和 OGT的酶学以及我们创新的标记、浓缩和基于质谱学的方法 对于应用于正常或Cas9神经谱系的位点定位和交互作用组鉴定- 改造的人类胚胎干细胞。在目标1中，我们发展了一种新的方法来研究 位点特异的O-GlcNAc修饰和修饰的蛋白质并将其与浓缩策略相结合 和串联质谱学方法来确定细胞类型和XLID基因的O-GlcNAc循环率 依赖的态度。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 当邻近标记以依赖于细胞类型和XLID基因的方式进行时。成功者 完成这些目标不仅将使O-GlcNAc生物界受益，更重要的是 确定受XLID影响的特定OGT目标和绑定合作伙伴，以获得未来详细的假设驱动 学习。

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.