The Chemistry and Biology of Protein Glycation

项目摘要 糖基化是一种非酶促的翻译后修饰（PTM），通过自发的糖基化反应发生。 还原糖或糖代谢物与蛋白质上的氨基和胍基的反应。糖化是一种 许多疾病的标志，包括糖尿病、心血管疾病、癌症、阿尔茨海默病和年龄- 黄斑变性然而，与酶促PTM相比，糖化的生物学作用仍然存在， 不太了解。这是因为传统的生物化学和化学生物学工具， 特定的酶活性，不太适合于没有酶的情况下蒸发的PTM的研究。作为 结果，新的方法可以克服活细胞中非酶，自发，化学的挑战， 是非常需要的。该项目的总体目标是开发化学方法， 控制泛素的糖基化，并使用这些来研究泛素糖基化的功能后果- 驱动蛋白质降解。我们的具体目标是（1）确定蛋白质序列的特征， 糖化结果（2）识别影响糖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水解的影响。 我们的中心前提是，选择性糖基化是由序列和结构的组合， 围绕着一个反应位点为了支持这一前提，我们的初步和公布的数据显示， 序列可以控制整体糖化水平和特定产物分布，而蛋白质结构 塑造形成的特定糖化产物。在我们的第一个目标中，我们使用 组合肽文库以改变一级序列并检查对糖基化类型和程度的影响， 我们在遍在蛋白中鉴定的四个糖化位点中的每一个。这些实验将识别出 促进不同程度的总糖化和/或偏向特定糖化产物的形成。在我们的第二 目的，我们描述了实验，以进一步定义结构如何有助于糖化结果使用一系列的， 破坏泛素结构或改变其化学微环境的点突变。我们还整合了 这些数据与计算模型，将用于预测糖化位点。最后，我们将使用 定义和验证一系列可预测调节糖化的“dialAGE”泛素变体的信息 通过改变对糖基化的敏感性，和/或通过促进特异性糖基化的形成， 糖化加合物。在我们的第三个目标中，我们使用这些工具来揭示糖化的机制， 阻止活细胞中泛素-蛋白酶体系统的蛋白质降解。具体来说，我们将测试 泛素自身的糖基化损害蛋白质降解的假说。因此，这项工作将提供长期的- 寻找方法，可以探索糖化的功能作用，并将揭示新的治疗目标， 一系列疾病。这些研究将极大地提高我们对糖基化的理解， 直接影响我们对这种独特的PTM如何影响人类健康，衰老和疾病的理解。

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.