Biophysical basis for enzyme mediated deglycation in protein repair

项目概要/摘要 生命的各个领域的生物体都用令人难以置信的多样性来装饰它们的蛋白质分子。 化学修饰对蛋白质的修饰对其功能至关重要，影响蛋白质的结构，稳定性， 和互动伙伴。许多蛋白质和酶读，写，并删除这些修改 与人类疾病密切相关，从神经系统疾病到癌症，再到2型糖尿病。虽然这些 蛋白质和途径可以成为治疗这些疾病的靶点，我们缺乏一个高分辨率， 理解细胞如何安装、识别和利用某些翻译后修饰， 特别是泛素化和自发的非酶修饰。我们的实验室正在研究 蛋白质-蛋白质相互作用动态调节翻译后修饰以改变蛋白质组景观 影响人类疾病。 蛋白质糖基化是一种未被充分研究的翻译后修饰， 连接到伯胺上。这一过程在正常生理条件下自发发生， 衰老和疾病如糖尿病，阿尔茨海默病， 骨关节炎和动脉粥样硬化。早期的糖基化事件是可逆的，代表了少数蛋白质之一， 细胞内的修复机制。去糖基化是由一种不寻常的“杂合”激酶/去糖酶介导的， 果糖胺-3-激酶（FN 3 K）。FN 3 K促进蛋白质连接的聚糖的去除， 磷酸化连接的糖并使糖-蛋白质键不稳定。FN 3 K和FN 3 K同源物是 在生命之树的所有树枝上都能找到。细胞内蛋白质的糖基化还没有得到很好的研究， FN 3 K和FN 3 K相关蛋白的保守性强调了这些酶的重要生物学作用。在 在这个项目中，我的实验室将采用多学科的方法，包括结构生物学的技术和专业知识， 酶学和系统生物学，以解决关于FN 3 K介导的尖锐的机制问题， 蛋白质修复我们假设，对FN 3 K机制的进一步理解将揭示新的生物学机制。 深入了解这个古老的修复过程，我们可以利用这种洞察力来更好地诊断和治疗疾病， 与糖基化升高有关为了将我们的贡献与其他人的贡献区分开来，我们将整合 简化主义和全球方法，以更深入、更全面地了解法规， 糖基化蛋白的修复。在五年的供资期内，该项目的目标是：㈠确定 FN 3 K介导的蛋白质修复的结构和生物物理基础（ii）系统地表征结合 FN 3 K和FN 3 K-RP在不同底物上的动力学和酶活性;（iii）鉴定位点特异性FN 3 K 去糖基化位点及其与其他PTM的潜在串扰。这项工作的顺利完成将 建立控制蛋白质去糖基化修复过程的分子机制，并最终提供 需要在生物医学研究上取得突破

Project Summary/Abstract Organisms across all domains of life decorate their protein molecules with an incredible diversity of chemical modifications. Modifications on proteins are critical for their function, affecting protein structure, stability, and interaction partners. Many of the proteins and the enzymes that read, write, and erase these modifications are closely tied to human diseases ranging from neurological disorders to cancer to type 2 diabetes. While these proteins and pathways can be targets to treat these diseases, we lack a high-resolution, mechanistic understanding of how the cell installs, recognizes, and leverages certain post-translational modifications, specifically ubiquitination and spontaneous, non-enzymatic modifications. Our lab is working to understand how protein-protein interactions dynamically regulate post-translational modifications to alter proteome landscape and impact human disease. Protein glycation is an understudied post-translational modification that arises when a sugar covalently attaches to a primary amine. This process occurs spontaneously under normal physiological conditions and is a bio-marker in aging and the development, or worsening, of diseases such as diabetes, Alzheimer's disease, osteoarthritis, and atherosclerosis. Early glycation events are reversible and represent one of the few protein repair mechanisms in the cell. Deglycation is mediated by an unusual “hybrid” kinase/deglycase called Fructosamine-3-kinase (FN3K). FN3K facilitates the removal of protein-linked glycans by directly phosphorylating the attached sugar and destabilizing the sugar-protein linkage. FN3K and FN3K homologs are found in all branches of the tree of life. The glycation of intracellular proteins is not well studied, yet the conservation of FN3K and FN3K-related proteins underscores an important biological role for these enzymes. In this project, my lab will use a multidisciplinary approach, including techniques and expertise in structural biology, enzymology, and systems biology, to address sharply focused mechanistic questions regarding FN3K-mediate protein repair. We hypothesize that an improved mechanistic understanding of FN3K will reveal new biological insight into this ancient repair process, and that we can leverage this insight to better diagnose and treat diseases associated with elevated glycation. In order to distinguish our contributions from those of others, we will integrate reductionist and global approaches to develop a deeper and more complete understanding of the regulation and repair of glycated proteins. Over the five-year funding period, the goals of this project are to: (i) determine the structural and biophysical basis for FN3K-mediated protein repair (ii) systematically characterize the binding kinetics and enzymatic activity of FN3K and FN3K-RP on diverse substrates; (iii) identify sites-specific FN3K deglycation sites and their potential cross-talk with other PTMs. The successful completion of this work will establish the molecular mechanisms that govern the protein deglycation repair process and will ultimately provide needed breakthroughs in biomedical research.