Chaperone-Enabled studies of epigenetic regulation enzymes

描述（由申请人提供）：本项目的首要目标是阐明组蛋白修饰酶的催化和调节的分子机制。我们将使用我们的伴侣使能生物学和结构（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）及其互补伙伴赖氨酸脱甲基酶（KDM）通过其作为组蛋白翻译后修饰的“写入器和擦除器”的作用而充当表观遗传信号传导的关键介质。许多最近的研究已经强调了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过程的直接影响，使得这些酶成为药物开发的潜在关键靶标。然而，在获得有关其操作模式的结构-功能关系的基础知识方面的进展一直很缓慢，因为它们是多结构域蛋白质，并且已经被结构和功能分析所取代。为了克服现有的障碍，我们将产生称为“合成亲和结合剂”或sAB的专门试剂，它们将用作蛋白质的伴侣。 结晶，以及用于细胞生物学应用的定制亲和试剂。为了实现我们的目标，我们组建了一个世界级的研究团队，将利用sAB试剂进行结构测定和高水平的生物测定。我们的方法的一个主要重点是确定和结构/生物化学表征的分子复合物中的HMT和KDM的功能，通过使用sAB的稳定和增强复合物的结晶。因此，CEBS的努力将依赖于与大型高通量中心的密切联系，这些中心用于蛋白质生产和多种构象状态下的单个酶、酶-底物复合物和多蛋白复合物的结构测定。我们的方法的一个独特的优势是，我们将提供结晶分子伴侣和相互作用伙伴的信息，以高通量中心，大大增加了结构确定的成功概率。 公共卫生相关性：赖氨酸特异性组蛋白甲基转移酶和组蛋白脱甲基酶是DNA复制和修复以及重组和基因表达和阻遏的表观遗传调控中的关键酶。这些酶的突变与许多疾病有关。这些研究将为药物开发提供生物学原理和结构基础。

DESCRIPTION (provided by applicant): 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-translational 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. PUBLIC HEALTH RELEVANCE: Lysine-specific histone methyltransferases and histone demethylases are critical enzymes in the epigenetic regulation of DNA replication and repair, as well as recombination and gene expression and repression. Mutations in these enzymes have been implicated in a number of diseases. These studies will provide the biological rationale and structural basis for drug development.