DEFINING STRUCTURAL AND GENETIC REQUISITES OF GLUTAMYLATION IN POLARIZED CELLS

定义极化细胞中谷氨酰化的结构和遗传要求

• 批准号: 9526727
• 负责人: James H Park
• 金额: $ 5.4万
• 依托单位: VETERANS BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9526727
• 关键词: Address; Address; Affect; Affect; Alzheimer' s Disease; Alzheimer' s Disease; Awareness; Awareness; Axon; Benchmarking; Benchmarking; Biological Markers; Biological Markers; Biology; Biology; Biophysics; Biophysics; Blindness; Blindness; Cells; Cells; Cellular biology; Cellular biology; Cerebellar Diseases; Cerebellar Diseases; Clustered Regularly Interspaced Short Palindromic Repeats; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Complex; Cytoskeleton; Cytoskeleton; Data; Data; Data Set; Data Set; Development; Development; Diagnostic; Diagnostic; Disease; Disease; Enzymes; Enzymes; Etiology; Etiology; Family; Family; Foundations; Foundations; Funding; Funding; Genes; Genes; Genetic; Genetic; Goals; Goals; Guanosine Triphosphate; Guanosine Triphosphate; Health; Health; Hippocampus (Brain); Hippocampus (Brain); Homeostasis; Homeostasis; Homology Modeling; Homology Modeling; Knowledge; Knowledge; Life Cycle Stages; Life Cycle Stages; Link; Link; Maintenance; Maintenance; Manuscripts; Manuscripts; Mass Spectrum Analysis; Mass Spectrum Analysis; Mentors; Mentors; Messenger RNA; Messenger RNA; Mission; Mission; Mitotic; Mitotic; Molecular; Molecular; Mus; Mus; Mutate; Mutate; Mutation; Mutation; Nerve Degeneration; Nerve Degeneration; Nervous system structure; Nervous system structure; Neurons; Neurons; Opsin; Opsin; Patients; Patients; Phase; Phase; Phenocopy; Phenocopy; Photoreceptors; Photoreceptors; Play; Play; Point Mutation; Point Mutation; Post-Translational Protein Processing; Post-Translational Protein Processing; Proteins; Proteins; Public Health; Public Health; Pyramidal Cells; Pyramidal Cells; RPGR gene; RPGR gene; Rattus; Rattus; Research; Research; Research Personnel; Research Personnel; Retina; Retina; Retinal; Retinal Photoreceptors; Retinal Photoreceptors; Retinitis Pigmentosa; Retinitis Pigmentosa; Role; Role; Sampling; Sampling; Structural Models; Structural Models; Structure; Structure; Subcellular structure; Subcellular structure; Techniques; Techniques; Testing; Testing; Therapeutic; Therapeutic; Training; Training; Tubulin; Tubulin; United States National Institutes of Health; United States National Institutes of Health; Virus; Virus; base; base; biophysical techniques; biophysical techniques; brain tissue; brain tissue; career; career; cell type; cell type; cofactor; cofactor; combinatorial; combinatorial; diagnostic biomarker; diagnostic biomarker; genetic manipulation; genetic manipulation; hippocampal cell loss; hippocampal cell loss; insight; insight; neurodevelopment; neurodevelopment; next generation sequencing; next generation sequencing; novel; novel; polarized cell; polarized cell; prevent; prevent; trafficking; trafficking; transcriptome sequencing; transcriptome sequencing; tyrosyltubulin ligase; tyrosyltubulin ligase

PROJECT SUMMARY Through mass spectrometry, glutamylation is becoming recognized as an important post-translational modifi- cation (PTM) in polarized neuronal cells such as the retinal photoreceptor and hippocampal pyramidal cells. Despite this greater awareness of glutamylation, there exist gaps in our understanding of (1) how glutamyla- tion specifically occurs in the photoreceptor, (2) which specific glutamylase(s) are necessary for hippocampal development, and (3) how a neurodegenerative state affects glutamylation levels. In addressing those gaps, the long-term goal is to understand how glutamylation annotates the cytoskeleton for specialized roles. The central hypothesis is that the homeostasis of glutamylation, the most abundant post-translational modification in the nervous system, is essential to neuronal and retinal health and that aberrant glutamylation levels may serve as an adjunctive biomarker. The central hypothesis will be challenged by three specific aims: (1) gain a structural understanding of how a glutamylase, Tubulin Tyrosine Ligase-Like 5 (TTLL5) recognizes a novel non-tubulin target (RPGR-ORF15, the most commonly mutated gene in retinitis pigmentosa (RP)) in the reti- nal photoreceptor and assess how point mutations informed by the structural complex impacts in oculo RPGR-ORF15 glutamylation and opsin trafficking. (2) Identify which of the nine glutamylases are necessary for hippocampal pyramidal cytoskeleton development, function, and maintenance and to determine which glu- tamylase(s) could be perturbed in a neurodegenerative state like Alzheimer's disease (AD), and (3) examine how glutamylation levels are affected in neurodegenerative samples. This juxtaposition of RP and AD high- lights the fundamental role glutamylation plays in maintaining cytoskeletal structure and functions across var- ied polarized cell types such as photoreceptors and hippocampal neurons. The proposed research is signifi- cant because defining the structural requirements of glutamylation in the photoreceptor will have relevance to basic photoreceptor biology and help RP patients who carry mutations in TTLL5's Cofactor Interaction Do- main (CID) or RPGR-ORF15's Basic Domain (BD) identified by Next Generation Sequencing (NGS). Be- cause rational therapeutics is structure dependent, a structure of the CID-BD is needed especially since we currently possess no homology models for CID. More broadly, a structural understanding of how a glutamyl- ase recognizes a non-tubulin retinal target will lay the groundwork to understand how TTLL paracatalytic re- gions functions as recognition adaptors for substrate targets. This will emerge as a unifying theme for the TTLL superfamily as the family is further explored and implicated as an etiology of disease by NGS. Likewise, defining the genetic requirements of glutamylation in the hippocampal cells will broaden our understanding of which glutamylases play a role in the development and maintenance of the hippocampal cytoskeleton. This expanded understanding will lay the foundation to assess glutamylation as a diagnostic biomarker to detect neurodegeneration in CSF and brain tissue.

项目概要 通过质谱分析，谷氨酰化被认为是一种重要的翻译后修饰。 极化神经元细胞（如视网膜感光细胞和海马锥体细胞）中的阳离子（PTM）。 尽管人们对谷氨酰化作用有了更高的认识，但我们对（1）谷氨酰化如何- 化特别发生在光感受器中，（2）哪些特定的谷氨酰胺酶是海马所必需的 发育，以及（3）神经退行性状态如何影响谷氨酰化水平。在解决这些差距时， 长期目标是了解谷氨酰化如何注释细胞骨架的特殊作用。这 中心假设是谷氨酰化（最丰富的翻译后修饰）的稳态 在神经系统中，对于神经元和视网膜健康至关重要，异常的谷氨酰化水平可能会 作为辅助生物标志物。中心假设将受到三个具体目标的挑战：（1）获得 谷氨酰胺酶、微管蛋白酪氨酸连接酶样 5 (TTLL5) 如何识别小说的结构理解 视网膜中的非微管蛋白靶点（RPGR-ORF15，视网膜色素变性（RP）中最常见的突变基因） 最终的光感受器并评估结构复杂性所告知的点突变如何影响眼 RPGR-ORF15 谷氨酰化和视蛋白运输。 (2) 确定九种谷氨酰胺酶中哪一种是必需的 用于海马锥体细胞骨架的发育、功能和维护，并确定哪些葡萄糖 淀粉酶可能会在阿尔茨海默病 (AD) 等神经退行性疾病中受到干扰，并且 (3) 检查 神经退行性样本中谷氨酰化水平如何受到影响。 RP 和 AD 的并置高 阐明了谷氨酰化在维持各种细胞骨架结构和功能中所起的基本作用 ied 极化细胞类型，例如光感受器和海马神经元。拟议的研究具有重要意义 不能，因为定义光感受器中谷氨酰化的结构要求将与 基本的光感受器生物学并帮助携带 TTLL5 辅因子相互作用突变的 RP 患者 通过下一代测序 (NGS) 鉴定出主 (CID) 或 RPGR-ORF15 的基本结构域 (BD)。是- 因为合理的治疗是结构依赖的，所以需要 CID-BD 的结构，特别是因为我们 目前没有 CID 的同源模型。更广泛地说，对谷氨酰如何 ase 识别非微管蛋白视网膜靶点将为了解 TTLL ​​副催化再催化奠定基础 gions 充当底物目标的识别适配器。这将作为一个统一的主题出现 NGS 进一步探索了 TTLL ​​超家族并将其作为疾病的病因。同样地， 定义海马细胞谷氨酰化的遗传要求将拓宽我们的理解 其中谷氨酰胺酶在海马细胞骨架的发育和维持中发挥作用。这 扩大了解将为评估谷氨酰化作为诊断生物标志物来检测奠定基础 脑脊液和脑组织的神经变性。