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: 10268153
• 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/10268153
• 关键词: Address; Affect; Alzheimer' s Disease; Awareness; Axon; Benchmarking; Biological Markers; Biology; Biophysics; Blindness; Cells; Cellular biology; Cerebellar Diseases; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cytoskeleton; Data; Data Set; Development; Diagnostic; Disease; Enzymes; Etiology; Family; Foundations; Funding; Genes; Genetic; Goals; Guanosine Triphosphate; Health; Hippocampus (Brain); Homeostasis; Homology Modeling; Knowledge; Life Cycle Stages; Link; Maintenance; Manuscripts; Mass Spectrum Analysis; Mentors; Messenger RNA; Mission; Mitotic; Molecular; Mus; Mutate; Mutation; Nerve Degeneration; Nervous system structure; Neurons; Opsin; Patients; Phase; Phenocopy; Photoreceptors; Play; Point Mutation; Post-Translational Protein Processing; Proteins; Public Health; Pyramidal Cells; RPGR gene; Rattus; Research; Research Personnel; Retina; Retinal Photoreceptors; Retinitis Pigmentosa; Role; Sampling; Structural Models; Structure; Subcellular structure; Techniques; Testing; Therapeutic; Training; Tubulin; United States National Institutes of Health; Virus; base; biophysical techniques; brain tissue; career; cell type; cofactor; combinatorial; diagnostic biomarker; genetic manipulation; hippocampal cell loss; insight; neurodevelopment; next generation sequencing; novel; polarized cell; prevent; trafficking; transcriptome sequencing; 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.

项目摘要 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）。 尽管对谷氨酰化有了更多的认识，但我们对以下问题的理解存在差距：（1）谷氨酰化是如何发生的， （2）哪些特异性谷氨酰胺酶是海马神经元细胞凋亡所必需的？ 发育，以及（3）神经退行性状态如何影响谷氨酰化水平。在弥补这些差距时， 长期的目标是了解谷氨酰化如何注释细胞骨架的特殊作用。的 中心假设是谷氨酰化的稳态，最丰富的翻译后修饰 在神经系统中，对神经元和视网膜的健康是必不可少的， 作为一种生物标志物。中心假设将受到三个具体目标的挑战：（1）获得 对谷氨酰胺酶微管蛋白酪氨酸连接酶样5（TTLL 5）如何识别新的 非微管蛋白靶点（RPGR-ORF 15，视网膜色素变性（RP）中最常见的突变基因）， nal感光细胞，并评估由结构复合体影响的点突变如何影响oculo RPGR-ORF 15谷氨酰化和视蛋白运输。(2)确定9种谷氨酰化酶中的哪一种是必需的 海马锥体细胞骨架的发展，功能和维护，并确定哪些glu- 在神经变性状态如阿尔茨海默病（AD）中，他莫昔酶可能受到干扰，和（3）检查 谷氨酰化水平如何影响神经退行性病变样本。这种RP和AD的并列高- 揭示了谷氨酰化在维持细胞骨架结构和功能方面的基本作用， IED极化细胞类型，如光感受器和海马神经元。该研究具有重要意义- 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TTLL 5辅因子相互作用突变的RP患者。 通过下一代测序（NGS）鉴定的RPGR-ORF 15的主要结构域（CID）或RPGR-ORF 15的基本结构域（BD）。是-- 因为合理的治疗是结构依赖性的，特别是因为我们需要CID-BD的结构， 目前还没有CID的同源模型。更广泛地说，对谷氨酰- ase识别非微管蛋白视网膜靶点将为理解TTLL如何副催化再活化奠定基础。 配体用作底物靶的识别衔接子。这将成为一个统一的主题， TTLL超家族作为一个家族被NGS进一步探索并暗示为疾病的病因。同样地， 明确海马细胞中谷氨酰化的遗传要求将拓宽我们对 该谷氨酰化酶在海马细胞骨架的发育和维持中起作用。这 扩展的理解将奠定基础，以评估谷氨酰化作为诊断生物标志物，以检测 CSF和脑组织中的神经变性。