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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.

总结：在美国，大约每500名男性中就有1人受到X连锁智力残疾（XLID）的影响。我们一个实验室先前已经表征了O-GlcNAc转移酶（OGT）基因中的几种突变，导致XLID综合征，最近发现了三个新的错义突变的催化与临床合作者。OGT是一种必需的糖基转移酶，其单独负责在丝氨酸和苏氨酸上添加翻译后修饰β-N-乙酰葡糖胺（O-GlcNAc）靶向核蛋白和胞质蛋白。OGT和O-GlcNAc与多种细胞凋亡有关。过程和疾病，包括神经发育，转录调控和XLID。我们以前的工作实验室生化特征的突变在四三肽重复（TPR）结构域的OGT，但一个统一的对负责XLID表型的转录调节的下游作用尚未阐明。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，催化结构域变体和先前描述的TPR结构域变体引起基因表达的失调。通过不能完全糖基化参与转录调控的关键靶蛋白， OGT靶向丧失（TPR结构域）或OGT催化效率降低（催化结构域）。这一假设 TPR结构域变体与野生型相比转录改变的数据支持了这一结论并且催化结构域变体可以是XLID的病因。为了验证我们的假设，我们将从生物化学的角度表征新的催化结构域变体，我们将确定两者的基因表达变化，催化结构域变体和先前表征的TPR结构域变体。我们的实验室是独一无二的由于我们在O-GlcNAc生物学方面的专业知识，先前对XLID变体的研究，以及我们的研究，拥有表达OGT的TPR结构域变体的Cas9工程化的男性人胚胎干细胞。在目的1，我们将使用体外试验和全细胞试验来确定生物化学特性的变化的新的催化结构域变体，包括热稳定性，动力学参数，并对全球的O- 当在细胞中表达时的GlcNAc水平。在目标2中，我们将确定所有基因表达之间的变化，当我们将CRISPR/Cas9工程化的人胚胎干细胞分化为神经干细胞时，前体细胞当与ChIP-Seq数据相结合时，我们可以评估OGT变体对调控的影响的基因表达。基于初步数据，我们还将研究Tet 2和HCF 1作为潜在的OGT interactors/substrates来解释基因表达的失调。这些方法将有助于阐明不同功能缺陷的变体在患者中导致相同的XLID神经发育表型。此外，这项研究将在密歇根大学的复合碳水化合物研究中心进行。在兰斯威尔斯博士的指导下，格鲁吉亚，这将为受训者提供一个良好的学习环境一般和专业的生化技能，以及批判性思维能力的示范指导下。