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

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

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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，大约有500名男性中有1个受X连锁智力残疾（XLID）的影响。我们的实验室先前已经表征了O-GLCNAC转移酶（OGT）基因的几个突变 Xlid综合征形式的因果关系，最近在催化中发现了三个新的错义突变与临床合作者的领域。 OGT是一个必不可少的糖基转移酶，完全负责将翻译后修饰β-N-乙酰葡萄糖（O-GLCNAC）添加到丝氨酸和苏氨酸靶核和胞质蛋白。 OGT和O-GLCNAC已与各种细胞有关过程和疾病，包括神经发育，转录调节和XLID。我们的先前工作 OGT的四肽重复（TPR）结构域中的实验室生化表征突变，但统一对负责XLID表型的转录调控的下游影响尚未阐明。给出催化结构域和TPR结构域突变都是Xlid的因果关系，我们的假设是两者都是新颖的催化结构域变体和先前描述的TPR结构域变体引起基因失调通过无法全面糖基化的关键靶靶蛋白来表达参与转录调节的表达 OGT靶向（TPR结构域）的丧失或OGT催化效率（催化域）的降低。这个假设数据表明，与WildType相比，TPR域变体改变了转录该催化域变体可能是Xlid的因果关系。为了检验我们的假设，我们将在生化上表征新型的催化域变体，我们将确定两者的基因表达变化催化域变体和先前表征的TPR结构域变体。我们的实验室独特地准备由于我们在O-GLCNAC生物学方面的专业知识，Xlid变体的先前工作以及我们的拥有表达OGT的TPR结构域变体的Cas9工程男性人类胚胎干细胞。在 AIM 1，我们将使用体外测定和全细胞测定法来确定生化特征的变化新型催化结构域的变体，包括热稳定性，动力学参数以及对全局O-的影响 GlcNAC水平在纤维素中表达。在AIM 2中，我们将确定所有所有人之间基因表达的变化当我们将CRISPR/CAS9工程的人类胚胎干细胞与神经区分开时，变体的特征是前体细胞。与芯片序列数据结合使用时，我们可以评估OGT变体对调节的影响基因表达。基于初步数据，我们还将研究TET2和HCF1作为潜在的OGT 相互作用/底物解释基因表达的失调。这些方法将有助于阐明缺乏不同功能的变异会导致患者的相同XLID神经发育表型。此外，这项研究将在大学的复杂碳水化合物研究中心进行佐治亚州在兰斯·威尔斯（Lance Wells）博士的指导下，这将为实习生提供一个学习的绝佳环境一般和专业的生化技能以及范式指导下的批判性思维技能。