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Functional Analysis of O-GlcNAc using Synthetic Protein Chemistry

使用合成蛋白质化学对 O-GlcNAc 进行功能分析

基本信息

批准号：
10298804
负责人：
Matthew Robert Pratt
金额：
$ 40.17万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10298804
关键词：
Acetylation Affect Amyloid Animals Biochemical Biochemistry Biological Biology Biophysics Carbohydrates Cellular biology Charcot-Marie-Tooth Disease Chemicals Collaborations Cryoelectron Microscopy Data Development Diabetes Mellitus Diffusion Disease Embryonic Development Excision Functional disorder Funding Goals Grant HSPB1 gene Heat shock proteins Kinetics Knowledge Ligation Malignant Neoplasms Mammals Measures Methods Michigan Modification Molecular Molecular Chaperones Monosaccharides Mus Mutagenesis Mutation Nerve Degeneration Neurodegenerative Disorders Neurons Neuropathy O-GlcNAc transferase Parkinson Disease Pathogenicity Pathway interactions Patients Peripheral Nervous System Diseases Phosphorylation Play Point Mutation Polymorph Post-Translational Protein Processing Protein Chemistry Proteins Proteomics Publishing Recombinant Proteins Research Role Serine Serine/Threonine Phosphorylation Silicon Dioxide Site Structure Testing Therapeutic Threonine Time Toxic effect Western Blotting Work alpha synuclein amyloid formation amyloid structure base biophysical properties dark matter experimental study glycosylation human disease in vivo monomer mutant prevent programs protein aggregation protein function public health relevance stereochemistry sugar synthetic protein unnatural amino acids

项目摘要

Modified Project Summary/Abstract (The abstract contained no specific references to the in vivo mouse experiments and therefore is unchanged): O-GlcNAc modification is a dynamic protein-modification that is absolutely required for embryonic development in mammals, and is misregulated in diseases, including diabetes, neurodegeneration and cancer. Although approximately 1,000 proteins are modified by O-GlcNAc, the effects of the vast majority of these modifications on protein function are completely unknown. The long-term goal of our research program is to fill in these missing gaps by determining the biochemical consequences of O-GlcNAc on proteins that are key to human disease. To accomplish this goal, we use a combination of carbohydrate and synthetic protein chemistries to build O-GlcNAc modified proteins for subsequent biological experiments. This chemical approach is uniquely enabling, as it is currently the only way to generate homogeneous and site-specifically O-GlcNAc modified proteins. We have been very successful and have used synthetic proteins to determine that O-GlcNAc has a multifaceted role in preventing the amyloid aggregation of proteins in neurodegenerative diseases. Specifically, we have found that O-GlcNAc both directly inhibits the aggregation of amyloid forming proteins and activates the activity of certain small chaperones. In this proposal we will continue to build on these discoveries. In Aim 1, we will determine how O-GlcNAc inhibits the early stages of α-synuclein amyloid formation. In Aim 2, we will test whether O-GlcNAc alters the structure/toxicity relationships of α-synuclein amyloids. In Aim 3, we examine how O-GlcNAc alters the small heat shock protein interactome. Finally, in Aim 4, we will determine if O-GlcNAc can rescue the activity of mutant chaperones that cause Charcot-Marie-Tooth disease. At the conclusion of these independent aims, we will have further unravelled the mechanisms by which O-GlcNAc inhibits protein aggregation and provided critical data to support the ongoing efforts to target O-GlcNAc therapeutically.

修改后的项目摘要/摘要（摘要中没有具体提及体内小鼠实验，因此未更改）：O-GlcNAc 修饰是一种动态蛋白质修饰，是哺乳动物胚胎发育所必需的，并且在糖尿病、神经退行性疾病和癌症等疾病中受到错误调节。尽管 O-GlcNAc 修饰了大约 1,000 种蛋白质，但其中绝大多数修饰对蛋白质功能的影响完全未知。我们研究计划的长期目标是通过确定 O-GlcNAc 对人类疾病关键蛋白质的生化影响来填补这些缺失的空白。为了实现这一目标，我们结合碳水化合物和合成蛋白质化学来构建 O-GlcNAc 修饰蛋白质，用于后续生物实验。这种化学方法具有独特的功效，因为它是目前生成同质且位点特异性 O-GlcNAc 修饰蛋白的唯一方法。我们非常成功，并使用合成蛋白质来确定 O-GlcNAc 在预防神经退行性疾病中蛋白质淀粉样蛋白聚集方面具有多方面的作用。具体来说，我们发现O-GlcNAc既可以直接抑制淀粉样蛋白形成蛋白的聚集，又可以激活某些小分子伴侣的活性。在本提案中，我们将继续以这些发现为基础。在目标 1 中，我们将确定 O-GlcNAc 如何抑制 α-突触核蛋白淀粉样蛋白形成的早期阶段。在目标 2 中，我们将测试 O-GlcNAc 是否改变 α-突触核蛋白淀粉样蛋白的结构/毒性关系。在目标 3 中，我们研究了 O-GlcNAc 如何改变小型热休克蛋白相互作用组。最后，在目标 4 中，我们将确定 O-GlcNAc 是否可以挽救引起腓骨肌萎缩症的突变伴侣的活性。在完成这些独立目标后，我们将进一步揭示 O-GlcNAc 抑制蛋白质聚集的机制，并提供关键数据来支持正在进行的 O-GlcNAc 治疗靶向努力。