Functional Analysis of O-GlcNAc Modifications Using Synthetic Protein Chemistry

 描述(申请人提供)：“使用合成蛋白质化学对O-GlcNAc修饰进行的功能分析”O-GlcNAc修饰(O-GlcN酰化)是一种动态的蛋白质修饰，是哺乳动物胚胎发育所必需的，在糖尿病、神经退行性疾病和癌症等疾病中受到错误调控。虽然大约有1000个潜在的蛋白质被O-GlcNAc修饰，但这些修饰中的绝大多数对蛋白质功能的影响是完全未知的。这种严重的知识缺乏存在的部分原因是传统的方法不能用于研究特定部位的O-GlcN酰化事件。我们研究计划的长期目标是了解O-GlcN酰化对人类疾病的关键蛋白质的影响。这项应用的目标是开发独特的蛋白质工程策略，使具有特定位置的O-GlcNAc修饰的蛋白质的产生成为可能，并应用这些方法来了解O-GlcNacylation对蛋白质a-突触核蛋白的影响，a-突触核蛋白是帕金森病中的一种易于聚集的蛋白质。我们的初步研究表明，利用合成化学方法可以制备出均一的O-GlcNacylated蛋白质。此外，我们还利用人工合成的蛋白质化学方法证明了O-GlcN酰化能阻断α-突触核蛋白的聚集。在这些初步研究的指导下，我们将：1)继续开发制备O-GlcN酰化蛋白的一般合成策略，2)研究O-GlcN酰化阻断α-突触核蛋白聚集的分子机制，3)确定O-GlcN酰化对α-突触核蛋白细胞毒性的影响。这些研究意义重大，因为O-GlcN酰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一个关键的潜在治疗策略。我们的方法也是创新的，因为它使O-GlcN酰化的效果能够在 并可在未来应用于其他关键蛋白质。

 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.