Probing Enzymatic Lysine Modifications

描述(申请人提供)：这项提议的目标是开发新的方法来产生适合于探索赖氨酸翻译后修饰(PTM)的生理效应的化学和生化工具。蛋白PTM在表观遗传调控中发挥着重要作用，已知在异常细胞和正常细胞中以不同的速度发生，这意味着它们是癌症等疾病状态的主要参与者。虽然众所周知的PTMS，如乙酰化和甲基化已被广泛研究，但关于更新发现的PTMS，如琥珀酸化和羟丁酰化，知之甚少。因此，大量需要新的技术，以便对其进行有效的评估。虽然存在一些引入非天然氨基酸和修饰氨基酸的方法，但它们都受到产量、位点的多功能性和化学范围的严重限制。本申请中提出的前两个具体目标旨在解决这一问题。目的1.开发半胱氨酸烷基化策略，将位点特异性的酰基赖氨酸模拟物整合到蛋白质靶标中。半胱氨酸烷基化提供了一种将这些模拟物化学引入蛋白质的通用方法，如果成功，我们的方法将适用于模拟其他PTM。含有琥珀酰化模拟物的组蛋白和含有羟丁酰化模拟物的磷酸葡萄糖异构酶(PGI)的产生将作为方法可行性的典型例子。目的2.评价特异性赖氨酸PTMS对细胞功能的影响。利用在目标1中制备的模拟物，将通过动力学分析、Western印迹分析和比率荧光分析来确定组蛋白赖氨酸琥珀酸化对核小体组装的影响以及PGI羟丁酰化对蛋白质活性的影响。蛋白质-蛋白质相互作用的变化也将使用标准的下拉和共IP分析进行评估。目的3.此外，还将使用15-20个合理设计的小分子探针来研究组蛋白赖氨酸甲基化，以抑制赖氨酸特异性脱甲基酶1(LSD1)，LSD1是负责去除组蛋白甲基化标记的酶之一。基于初步的发现，已经提出了一系列基于机理的灭活剂，它们与相关的黄素辅因子共价结合。除了细胞生长和迁移实验外，还可以使用前面描述的动力学分析和Western印迹实验来评估它们对酶活性的影响。这项提案中概述的工作有望为评估蛋白质PTM提供广泛适用的方法，并为表观遗传调控及其与癌症等疾病的关系提供实质性的见解。此外，拟议的研究培训计划将为申请者提供一个显著扩大规模的独特机会 他的实验曲目包括生化和细胞培养技术。约翰霍普金斯大学提供了一个科学丰富和合作的氛围，应该会鼓励申请者的发展。一系列补充教育和教学机会应有助于申请人迈向作为独立调查员的成功职业生涯。

DESCRIPTION (provided by applicant): The goal of this proposal is to develop novel methods to generate chemical and biochemical tools suitable for probing the physiological effects of lysine post-translational modifications (PTMs). Protein PTMs play a significant role in epigenetic regulation and are known to occur at different rates in aberrant cells as opposed to normal cells, implicating them as major players in disease states such as cancer. While well-known PTMs such as acetylation and methylation have been extensively studied, little is known regarding more recently identified PTMs such as succinylation and hydroxybutyrylation. Therefore, there is a substantial need for new techniques that will allow for their efficient evaluation. While a few methods for introducing unnatural and modified amino acids exist, they are all severely limited by yield, versatility of site and chemical scope. The first two specific aims presented in this application are designed to address this issue. Aim 1. Develop cysteine alkylation strategies to incorporate site specific acyl-lysine mimics into protein targets. Cysteine alkylation offers a versatile way to chemically introduce these mimics into proteins and, if successful, our approach would be applicable to mimicking other PTMs. The generation of histones containing a succinylation mimic and phosphoglucose isomerase (PGI) containing a hydroxybutyrylation mimic will serve as representative examples of method feasibility. Aim 2. Evaluate the effect of specific lysine PTMs on cellular functions. Using the mimics prepared in Aim 1, the effect of histone lysine succinylation on nucleosome assembly and of PGI hydroxybutyrylation on protein activity will be determined with kinetic assays, Western blot analysis and ratiometric fluorescence assays. Changes in protein-protein interactions will also be evaluated using standard pull-down and Co-IP assays. Aim 3. In addition, histone lysine methylation will also be investigated using 15-20 rationally designed small molecule probes to inhibit lysine specific demethylase 1 (LSD1), one of the enzymes responsible for removing histone methylation marks. Based on preliminary findings, a series of mechanism based inactivators that covalently bind to the associated flavin cofactor have been proposed. Their effect on enzymatic activity can be evaluated using previously described kinetic assays and Western blot experiments in addition to cell growth and migration assays. The work outlined in this proposal promises to yield broadly applicable methods for evaluating protein PTMs and provide substantial insight into epigenetic regulation and its relation to diseases such as cancer. In addition, the proposed research training plan will provide the applicant with a unique opportunity to significantly expand his experimental repertoire to include biochemical and cell culture techniques. Johns Hopkins University provides a scientifically rich and collaborative atmosphere that should encourage the applicant's development. An array of supplementary educational and teaching opportunities should facilitate the applicant's launch toward a successful career as an independent investigator.