组蛋白去甲基化酶LSD2的结构和功能研究

In this application, we propose the structural and functional studies of histone demethylase LSD2. LSD2 (also known as AOF1 and KDM1B) is a flavin adenine dinucleotide-dependent amine oxidase domain containing protein related to the lysine demethylase LSD1 (also known as KDM1). LSD2 functions as a histone H3 lysine 4 demethylase and is required for de novo DNA methylation of some imprinted genes in oocytes, suggesting its important role in epigenetic regulation and development. Genome-wide mapping reveals that LSD2, differ from LSD1, localizes predominantly to the gene bodies of actively transcribed genes, but is absent from promoters. The domain compositation of LSD2 is different from LSD1 and may lead to the difference of their functions. However, very limited structural and related functional studies of LSD2 have been reported up to now. Through Tandem affinity purification and Mass spectrometry analyse, we identified several LSD2 binding proteins and characterized that one of these proteins (named putative oxidoreductase), can interact with LSD2 and enhance the demethylase activity of LSD2. We also got crystals of LSD2 protein and structure determination is underway. Based on these preliminary data, we propose here, a systematic studies of the structure and function of LSD2. We will crystallize and determine the structures of LSD2, LSD2 in complex with histone H3, LSD2 in complex with PO, as well as the ternary complex LSD2/H3/PO. These structural studies will reveal mechanism of substrate recognition and PO mediated LSD2 activity enhancement. We will also try to identify which histone modification, mainly methylation,is specifically recognized by LSD2 and LSD2 associated proteins. The following up structual and functional studies may reveal the mechanism of the localization of LSD2 related complex. Our studies will provide a framework for deciphering the function of LSD2 in epigenetic regulation.

本项目致力于对组蛋白赖氨酸去甲基化酶LSD2的结构与功能研究。LSD2可去组蛋白H3K4me2甲基化修饰，是LSD1的唯一同源蛋白酶。LSD2的缺失导致卵母细胞起源的胚胎死亡及DNA甲基化水平改变，提示其功能的重要性。LSD2的结构域组成，基因组定位及调控的基因都与LSD1不同，目前对LSD2的结构和功能研究都非常有限。申请人通过前期工作发现一系列与LSD2结合的蛋白并从中鉴定出PO（putative oxidoreductase）可以结合LSD2并提高LSD2酶活性，获得了LSD2蛋白的晶体。申请人此基础上提出本项目申请，拟通过解析LSD2与其底物的结构，揭示LSD2识别底物的特异性与催化机制；解析LSD2与PO的蛋白复合物结构，研究体内外LSD2酶活性调控的机制及对LSD2功能的影响，从结构和功能全面阐明LSD2参与表观遗传调控的分子机制。

本项目致力于全面研究组蛋白去甲基化酶LSD2的结构与功能，探索LSD2及其结合蛋白对组蛋白修饰的识别机制的研究，比较全面的阐明LSD2参与表观遗传调控的分子机制。取得成果简述如下：.1．揭示LSD2的底物识别机制。我们发现LSD2对H3（1-26氨基酸）的结合能力以及酶活性与H3（1-21氨基酸）相比，都比较高。因此我们解析了LSD2与H3（1-26）的复合物结构，发现LSD2与底物的结合除了酶活中心外，还有第二个底物识别位点，而LSD1不具有此部分序列，通过体内外结合以及酶活试验证明，第二个结合位点对于LSD2的酶活有重要的作用，该项研究成果发表在Cell Research，2013（徐彦辉为共同通讯作者）。.2. LSD2酶活性受NPAC蛋白激活的分子机制。我们发现NPAC蛋白可以结合并显著提高LSD2的酶活性，进而解析了LSD2-NPAC-H3复合物的结构。发现NPAC结合在第二底物识别位点附近，促进LSD2对底物组蛋白的结合进而促进其酶活性。该项工作发表在 Molecular Cell，2013（徐彦辉为共同通讯作者）。 “Nature中国”将该论文作为“研究亮点”。评价是：“这些结果表明NPAC结合LSD2对其组蛋白去甲基化酶活性是必须的，靶向LSD2及其辅因子有助于开发治疗疾病（如肿瘤）的药物”。.3. LSD2具备E3泛素连接酶活性并抑制肿瘤生长。在LSD2的功能研究中，我们发现，LSD2具备E3泛素连接酶活性。LSD2能够直接泛素化N-乙酰氨基葡萄糖转移酶（OGT），促使其发生降解。LSD2通过其E3泛素连接酶活性（而不是组蛋白去甲基化酶活性）抑制肺癌细胞系A549的细胞生长。我们的研究揭示了LSD2通过其E3泛素连接酶活性抑制细胞生长，并建立了组蛋白去甲基化酶与泛素化讲解途径之间的关联 (Mol Cell, 2015,唯一通讯作者)。Science Signaling杂志将该论文选为编辑推荐，评价是：“这些结果表明LSD2是个双功能蛋白，疾病独立的两种活性，其E3连接酶活性靶向OGT并抑制癌细胞生长”。