Chaperone-Enabled studies of epigenetic regulation enzymes

该项目的总体目标是阐明催化和 组蛋白修饰酶的调节。我们将使用我们的伴侣启用生物学和结构 （CEBS）技术平台来研究一组重要但具有挑战性的表观遗传调节酶。 赖氨酸特异性组蛋白甲基转移酶（HMT）及其互补配偶体赖氨酸 脱甲基酶（KDM）通过它们作为“编写者和转录者”的作用作为表观遗传信号传导的关键介质发挥作用。 组蛋白的“擦除器”。最近的许多研究强调， 组蛋白的赖氨酸甲基化的重要性导致对DNA复制，修复， 重组、基因沉默、印记和RNA过程使这些酶成为潜在的关键靶标 用于药物开发。然而，在获得关于结构-功能的基础知识方面的进展 由于它们是多结构域蛋白质， 在结构和功能分析上都是不合理的。为了克服现有的障碍，我们将 产生被称为“合成亲和结合剂”或sAB的专门试剂，其将被用作以下的分子伴侣： 结晶，以及用于细胞生物学应用的定制亲和试剂。完成我们 目标我们组建了一支世界一流的研究人员团队，将为两者开发sAB试剂 结构测定和高水平生物测定。我们的方法的一个主要重点是确定和 结构/生物化学表征其中HMT和KDM通过以下方式起作用的分子复合物： 使用稳定和增强复合物结晶的sAB。因此，CEBS的努力将依赖于密切的 与用于蛋白质生产和个体结构测定的大型高通量中心的联系 多种构象状态的酶、酶-底物复合物和多蛋白复合物。一 我们的方法的独特优势是，我们将提供结晶分子伴侣和以下信息 互动合作伙伴的高通量中心，大大增加了成功的概率结构 保持战略定力

The overarching goal of this project is to elucidate the molecular mechanism governing the catalysis and regulation of histone modification enzymes. We will use our Chaperone-Enabled Biology and Structure (CEBS) technology platform to study an important, but challenging group of epigenetic regulating enzymes. The lysine-specific histone methyltransferases (HMT) and their complementary partners, lysine demethylases (KDM) function as key mediators of epigenetic signaling through their actions as "writers and erasers" of post-trahslational modifications on histone proteins. Numerous recent studies have highlighted the importance of lysine methylation of histones leading to direct impact on DNA replication, repair, recombination, gene silencing, imprinting and RNA processes making these enzymes potential key targets for drug development. However, progress in gaining fundamental knowledge about structure-function relationships governing their modes of operation has been slow because they are multidomain proteins and have been recalcitrant to both structural and functional analyses. To overcome the existing barriers, we will generate specialized reagents called "synthetic affinity binders" or sABs that will be used as chaperones for crystallization, as well as customized affinity reagents for cell biologically applications. To accomplish our objectives we have assembled a world-class team of investigators that will exploit sAB reagents for both structure determination and high level biological assays. A major emphasis of our approach is to identify and structurally/biochemically characterize the molecular complexes in which the HMTs and KDMs function by using sABs that stabilize and enhance crystallization of the complexes. Thus, CEBS effort will rely on close ties to the large high throughput centers for protein production and structure determination of individual enzymes in multiple conformational states, enzyme-substrate complexes and multiprotein complexes. A unique strength of our approach is that we will provide crystallization chaperones and the information on interaction partners to the high throughput centers to greatly increase the probability of success of structure determination.