Functional Analysis of O-GlcNAc Modifications Using Synthetic Protein Chemistry

使用合成蛋白质化学对 O-GlcNAc 修饰进行功能分析

• 批准号: 9321152
• 负责人: Matthew Robert Pratt
• 金额: $ 32.59万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9321152
• 关键词: Affect; Alzheimer' s Disease; Biochemical; Biological Assay; Biophysics; Carbon; Cell Culture Techniques; Cells; Chemicals; Complex; Data; Development; Diabetes Mellitus; Disease; Embryonic Development; Enzymes; Event; Functional disorder; Future; Generations; Glucosamine; Goals; Health; In Vitro; Individual; Insecta; Knowledge; Letters; Lewy Bodies; Link; Literature; Malignant Neoplasms; Mammals; Methods; Mitochondrial Proteins; Modeling; Modification; Molecular; Monosaccharides; Nerve Degeneration; Neurons; Nuclear Proteins; Parkinson Disease; Parkinson' s Dementia; Physiological; Positioning Attribute; Post-Translational Protein Processing; Preparation; Protein Chemistry; Protein Engineering; Proteins; Publications; Reaction; Reagent; Research; Route; Science; Site; Synthesis Chemistry; Testing; Therapeutic; Toxic effect; Ubiquitination; alpha synuclein; analog; biophysical properties; collaborative environment; experimental study; extracellular; human disease; in vivo; innovation; mouse model; peptide O-linked N-acetylglucosamine-beta-N-acetylglucosaminidase; prevent; prion-like; programs; protein aggregate; protein function; public health relevance; synthetic protein; tau Proteins; transmission process

 DESCRIPTION (provided by applicant): "Functional Analysis of O-GlcNAc Modifications using Synthetic Protein Chemistry" O-GlcNAc modification (O-GlcNAcylation) 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 1000 potential proteins are modified by O-GlcNAc, the effects of the vast majority of these modifications on protein function are completely unknown. This critical lack of knowledge exists in-part because traditional methods are deficient for the study of site-specific O-GlcNAcylation events. The long-term goal of our research program is to understand the consequences of O-GlcNAcylation on proteins that are key to human disease. The objectives of this application are to develop protein engineering strategies that uniquely enable the generation of proteins with site-specific O-GlcNAc modifications and to apply these methods to understand the effects of O-GlcNAcylation on the protein a-synuclein, the aggregation-prone protein in Parkinson's disease. Our preliminary studies demonstrate that homogeneously O-GlcNAcylated proteins can be prepared using synthetic chemistry. Furthermore, we have used synthetic protein chemistry to demonstrate that O-GlcNAcylation blocks a-synuclein aggregation. Guided by these preliminary studies, we will: 1) continue to develop general synthetic-strategies for the preparation of O-GlcNAcylated proteins, 2) investigate the molecular mechanism by which O-GlcNAcylation blocks a-synuclein aggregation and 3) determine the effects of O-GlcNAcylation on the cellular toxicity of a-synuclein. These studies are significant, as the effects of O-GlcNAcylation are almost completely unknown. Additionally, blocking a-synuclein aggregation is a key potential therapeutic strategy in Parkinson's disease. Our approach is also innovative as it enables the effects of O-GlcNAcylation to be directly tested in a site-specific fashion and can be applied to other critical proteins in the future.

 描述（由申请人提供）：“Functional Analysis of O-GlcNAc Modifications using Synthetic Protein Chemistry”O-GlcNAc修饰（O-GlcNAc Acylation）是哺乳动物胚胎发育所绝对需要的动态蛋白质修饰，并且在包括糖尿病、神经变性和癌症的疾病中被错误调节。虽然大约1000种潜在的蛋白质被O-GlcNAc修饰，但这些修饰中的绝大多数对蛋白质功能的影响完全未知。这种知识的严重缺乏部分是因为传统方法对于位点特异性O-GlcNAc化事件的研究是不足的。我们研究计划的长期目标是了解O-GlcNAc酰化对人类疾病关键蛋白质的影响。本申请的目的是开发蛋白质工程策略，其独特地使得能够产生具有位点特异性O-GlcNAc修饰的蛋白质，并应用这些方法来理解O-GlcNAc化对蛋白质α-突触核蛋白（帕金森病中的聚集倾向蛋白）的影响。我们的初步研究表明，均匀的O-GlcNAc酰化的蛋白质可以使用合成化学制备。此外，我们使用合成蛋白质化学来证明O-GlcNAcylation可以阻止a-突触核蛋白聚集。在这些初步研究的指导下，我们将：1）继续开发用于制备O-GlcNAc酰化蛋白的一般合成策略，2）研究O-GlcNAc酰化阻断α-突触核蛋白聚集的分子机制，3）确定O-GlcNAc酰化对α-突触核蛋白的细胞毒性的影响。这些研究是重要的，因为O-GlcNAc化的影响几乎完全未知。此外，阻断α-突触核蛋白聚集是帕金森病的关键潜在治疗策略。我们的方法也是创新的，因为它使O-GlcNAc酰化的效果能够直接在一个实验室中测试。 位点特异性的方式，并可以在未来应用于其他关键蛋白质。