The Chemistry and Biology of Protein Glycation

项目摘要 糖基化是一种非酶翻译后修饰(PTM)，通过自发的 还原糖或糖代谢产物与蛋白质上的氨基和胍基的反应。糖基化是一种 许多疾病的标志，包括糖尿病、心血管疾病、癌症、阿尔茨海默氏症和年龄- 相关性黄斑变性。然而，与酶促PTMS相比，糖基化的生物学作用仍然存在。 人们对此知之甚少。这是因为传统的生化和化学生物学工具，它们抑制或剖析 特定的酶活性，不是很适合研究没有酶的PTM。作为一名 结果，新的方法可以克服活细胞中非酶、自发、化学的挑战 都是非常需要的。该项目的总体目标是开发能够精确地 控制泛素的糖基化，并用这些来研究糖基化对泛素的功能影响。 导致蛋白质降解。我们的具体目标是(1)确定促进蛋白质序列的特征 糖基化结果(2)确定影响糖基化结果的蛋白质结构特征，以及(3)确定 糖基化对泛素驱动的蛋白质分解的影响。 我们的中心前提是选择性糖基化是以序列和结构的组合为模板的， 围绕着一个反应性位置。为了支持这一前提，我们的初步和公布的数据表明，主要的 序列可以控制整体糖基化水平和特定的产物分布，而蛋白质结构 雕刻形成的特定糖基化产物。在我们的第一个目标中，我们使用 改变初级序列的组合多肽文库，并检查对糖基化类型和程度的影响 我们在泛素中确定的四个糖化位点。这些实验将识别出 促进不同程度的总糖基化和/或偏向特定糖基化产物的形成。在我们的第二个 目的，我们描述了实验，以进一步定义结构如何影响糖基化结果，使用一系列 破坏泛素内部结构或改变化学微环境的点突变。我们还整合了 这些数据与将用于预测糖基化位点的计算模型相结合。最后，我们将使用这个 定义和验证一系列可预测地调节糖基化的“DialAGE”泛素变体的信息 通过改变对糖基化的敏感性和/或通过促进特定基因的形成在活细胞中的结果 糖基化加合物。在我们的第三个目标中，我们使用这些工具来揭示糖基化的机制 防止活细胞中泛素-蛋白酶体系统的蛋白质降解。具体地说，我们将测试 假设泛素的糖基化本身会损害蛋白质的降解。因此，这项工作将提供长期- 寻找可以探索糖基化功能作用的方法，并将揭示新的治疗靶点 一系列疾病。这些研究将极大地提高我们对糖基化的理解，并将有 直接影响我们对这种独特的PTM如何影响人类健康、衰老和疾病的认识。

Project Summary Glycation is a non-enzymatic posttranslational modification (PTM) that occurs through the spontaneous reaction of reducing sugars or sugar metabolites with amino and guanidino groups on proteins. Glycation is a hallmark of many diseases, including diabetes, cardiovascular disease, cancer, Alzheimer's disease, and age- related macular degeneration. However, compared to enzymatic PTMs, the biological role of glycation remains poorly understood. This is because traditional biochemical and chemical biology tools, which inhibit or profile specific enzyme activities, are not well-suited for the study of a PTM that transpires without an enzyme. As a result, new methods that can overcome the challenge of non-enzymatic, spontaneous, chemistry in living cells are critically needed. The overall objectives of this project are to develop chemical methods that can precisely control the glycation of ubiquitin, and to use these to study the functional consequence of glycation on ubiquitin- driven protein degradation. Our specific aims are to (1) identify features of protein sequence that promote glycation outcomes (2) identify features of protein structure that influence glycation outcomes, and (3) determine the impact of glycation on ubiquitin-driven proteolysis. Our central premise is that selective glycation is templated by the combination of sequence and structure that surrounds a reactive site. In support of this premise, our preliminary and published data show that primary sequence can govern both overall glycation levels and specific product distributions, while protein structure sculpts the specific glycation products that form. In our first aim, we examine the contributions of sequence using combinatorial peptide libraries to vary primary sequence and examine the effect on glycation type and extent for each of the four glycated sites we have identified in ubiquitin. These experiments will identify sequences that promote varying extents of total glycation and/or bias the formation of specific glycation products. In our second aim, we describe experiments to further define how structure contributes to glycation outcomes using a series of point mutations that disrupt structure or alter the chemical microenvironment within ubiquitin. We also integrate these data with computational models that will be used to predict glycation sites. Finally, we will use this information to define and validate a series of “dialAGE” ubiquitin variants that can predictably modulate glycation outcomes in living cells by altering susceptibility to glycation, and/or by promoting the formation of specific glycation adducts. In our third aim, we use these tools to uncover the mechanism through which glycation prevents protein degradation by the ubiquitin-proteasome system in living cells. Specifically, we will test the hypothesis that the glycation of ubiquitin itself impairs protein degradation. This work will therefore provide long- sought methods that can explore the functional role of glycation, and will reveal new therapeutic targets for a range of diseases. These studies will dramatically improve our understanding of glycation and will have an immediate impact on our appreciation for how this unique PTM influences human health, aging, and disease.