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Utilizing Causal X-Linked Intellectual Disability Variants to Gain Insight into the O-GlcNAc Transferase Enzyme

利用因果 X 连锁智力障碍变异来深入了解 O-GlcNAc 转移酶

基本信息

批准号：
10607359
负责人：
Johnathan Martin Mayfield
金额：
$ 4.68万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-01-01 至 2025-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10607359
关键词：
Academia Adaptive Behaviors Address Affect Binding Biochemical Biology CRISPR/Cas technology Carbohydrates Catalytic Domain Cell physiology Cells Cellular Assay ChIP-seq Characteristics Clinical Complex Critical Thinking DNA Data Development Developmental Gene Disability phenotype Disease Engineering Environment Enzymatic Biochemistry Enzymes Excision Gene Expression Gene Expression Regulation Genes Genetic Transcription Global Change Glycobiology Goals Human Engineering Kinetics Laboratories Lead Learning Link Mass Spectrum Analysis Mentorship Metabolic Methylation Missense Mutation Modeling Mutation N-terminal Neurodevelopmental Disability Neuronal Differentiation Nuclear Nutrient O-GlcNAc transferase Outcome Patients Phenotype Positioning Attribute Post-Translational Protein Processing Proteins Recombinants Research Role Serine Syndrome Technical Expertise Testing Threonine Transcriptional Regulation Transferase United States Universities Variant Western Blotting Work X-linked intellectual disability base career casein kinase II causal variant chromatin remodeling design differential expression embryonic stem cell experience glycosyltransferase human embryonic stem cell human male in vitro Assay insight male nerve stem cell neurodevelopment novel peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase protein protein interaction relating to nervous system response sensor skills transcriptome sequencing transcriptomics

项目摘要

Summary: Approximately 1 in 500 males in the United States are affected by X-Linked Intellectual Disability (XLID). Our laboratory has previously characterized several mutations in the O-GlcNAc Transferase (OGT) gene that are causal for a syndromal form of XLID and have recently discovered three novel missense mutations in the catalytic domain with clinical collaborators. OGT is an essential glycosyltransferase that is solely responsible for the addition of the post-translational modification beta-N-acetylglucosamine (O-GlcNAc) onto serines and threonines of target nuclear and cytosolic proteins. OGT and O-GlcNAc have been implicated in a variety of cellular processes and diseases including neurodevelopment, transcriptional regulation, and XLID. Previous work by our lab biochemically characterized mutations in the Tetratricopeptide Repeat (TPR) domain of OGT, but a unifying downstream effect on transcription regulation responsible for the XLID phenotype has yet to be elucidated. Given both catalytic domain and TPR domain mutations are causal for XLID, our hypothesis is that both the novel catalytic domain variants and previously described TPR domain variants cause a dysregulation of gene expression by an inability to fully glycosylate key target proteins involved in transcriptional regulation due to a loss of OGT targeting (TPR domain) or reduction in OGT catalytic efficiency (catalytic domain). This hypothesis is supported by data demonstrating that TPR domain variants have altered transcription compared to wildtype and that catalytic domain variants can be causal for XLID. To test our hypothesis, we will biochemically characterize the novel catalytic domain variants, and we will determine changes to gene expression for both catalytic domain variants and previously characterized TPR domain variants. Our lab is uniquely poised to address this hypothesis due to our expertise in O-GlcNAc biology, previous work with XLID variants, and our possession of Cas9-engineered male human embryonic stem cells expressing TPR domain variants of OGT. In aim 1, we will use in vitro assays and whole cell assays to determine changes in the biochemical characteristics of the novel catalytic domain variants including thermal stability, kinetic parameters, and impact on global O- GlcNAc levels when expressed in cellulo. In aim 2, we will determine changes in gene expression between all characterized variants as we differentiate CRISPR/Cas9-engineered human embryonic stem cells to neural precursor cells. When combined with ChIP-Seq data, we can evaluate the impact of OGT variants on regulation of gene expression. Based on preliminary data, we will also investigate Tet2 and HCF1 as potential OGT interactors/substrates to explain the dysregulation of gene expression. These approaches will help elucidate how variants deficient in different functions result in the same XLID neurodevelopmental phenotype in the patient. Furthermore, this research will take place at the Complex Carbohydrate Research Center at the University of Georgia under the direction of Dr. Lance Wells, that will provide the trainee an excellent environment to learn general and specialized biochemical skills as well as critical thinking skills under exemplary mentorship.

概括：在美国，大约每 500 名男性中就有 1 名患有 X 连锁智力障碍 (XLID)。我们的实验室之前已经鉴定了 O-GlcNAc 转移酶 (OGT) 基因中的几个突变，这些突变是 XLID 综合征形式的病因，并且最近在催化中发现了三种新的错义突变与临床合作者的领域。 OGT 是一种必需的糖基转移酶，仅负责在丝氨酸和苏氨酸上添加翻译后修饰 β-N-乙酰氨基葡萄糖 (O-GlcNAc) 目标核蛋白和胞质蛋白。 OGT 和 O-GlcNAc 与多种细胞过程和疾病，包括神经发育、转录调节和 XLID。我们之前的工作 OGT 四肽重复序列 (TPR) 结构域的实验室生化特征突变，但统一下游对导致 XLID 表型的转录调控的影响尚未阐明。给定催化结构域和 TPR 结构域突变都是 XLID 的原因，我们的假设是，新的催化结构域变体和之前描述的 TPR 结构域变体导致基因失调由于无法完全糖基化参与转录调控的关键靶蛋白而表达 OGT 靶向性丧失（TPR 结构域）或 OGT 催化效率（催化结构域）降低。这个假设数据证明 TPR 结构域变体与野生型相比改变了转录催化结构域变异可能是 XLID 的原因。为了验证我们的假设，我们将通过生化方法表征新的催化结构域变体，我们将确定两者基因表达的变化催化结构域变体和先前表征的 TPR 结构域变体。我们的实验室具有独特的优势由于我们在 O-GlcNAc 生物学方面的专业知识、之前对 XLID 变体的研究以及我们的研究，解决了这一假设拥有表达 OGT TPR 结构域变体的 Cas9 工程男性胚胎干细胞。在目标1，我们将使用体外测定和全细胞测定来确定生化特征的变化新型催化域变体的研究，包括热稳定性、动力学参数以及对全局 O- 的影响在纤维素中表达时的 GlcNAc 水平。在目标 2 中，我们将确定所有基因之间基因表达的变化当我们将 CRISPR/Cas9 工程化的人类胚胎干细胞与神经细胞区分开来时，我们发现了特征变异前体细胞。结合 ChIP-Seq 数据，我们可以评估 OGT 变异对调控的影响的基因表达。根据初步数据，我们还将研究Tet2和HCF1作为潜在的OGT 相互作用/底物来解释基因表达的失调。这些方法将有助于阐明如何不同功能缺陷的变异会导致患者出现相同的 XLID 神经发育表型。此外，这项研究将在英国大学复杂碳水化合物研究中心进行佐治亚州在Lance Wells博士的指导下，将为学员提供良好的学习环境在模范指导下的一般和专业生化技能以及批判性思维技能。