Structural basis of signaling by ING2 PHD

ING2 PHD 信号传输的结构基础

• 批准号: 7036477
• 负责人: TATIANA G KUTATELADZE
• 金额: $ 20.23万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-06 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7036477
• 关键词: histones; neoplasm /cancer; nucleosomes; peptides

DESCRIPTION (provided by applicant): The inhibitor of growth (ING2) tumor suppressor is implicated in oncogenesis, DNA repair, growth regulation and apoptosis. ING2 negatively regulates cell proliferation by enhancing acetylation of p53, a major tumor suppressor, which is mutated in about half of all human cancers. Our preliminary studies indicate that the PHD finger of ING2 specifically recognizes the histone tail domains and inositol hexakisphosphate (IP6) messenger revealing a novel link between IP6-mediated signaling and chromatin regulation. However, the molecular mechanisms underlying ING2 function have not been established. The structural basis of the histone and IP6 recognition remains unexplored and the effect of IP6 interaction on ING2 targeting to nucleosomes is not known. This project focuses on structural characterization of the histone and IP6 binding, novel functions of the PHD domain. The hypotheses to be tested are: (1) ING2 is targeted to nucleosomes through the interaction of PHD with histone tails and (2) nucleosome recruitment of lNG2 is negatively regulated by IP6 binding. The atomic-resolution structures of ING2 PHD bound to the H4 histone tail peptide and IP6 will be determined by multidimensional heteronuclear NMR or by X-ray crystallography. The binding site residues will be mutated and the mutant proteins will be tested in vitro by NMR and pull-down experiments and in vivo by fluorescence microscopy. The association of ING2 with nucleosomes will be investigated using electrophoretic mobility shift assays. To determine the specificity, interactions with unmodified and modified histone tail peptides and with other IPs will be analyzed by NMR, surface plasmon resonance and fluorescence spectroscopy. Functional significance of the histone and IP6 binding for p53 activation and apoptosis will be investigated. The results generated in this research will offer comprehensive understanding of the molecular mechanisms by which tumor suppressor ING2 is targeted to chromatin, interacts with IPs and regulates function of p53. These studies will aid in deeper understanding of how the critical ING2-p53 pathways can be therapeutically manipulated and may help to identify new diagnostic markers and targets to prevent and treat cancer.

描述（由申请人提供）：生长抑制剂（ING2）肿瘤抑制因子涉及肿瘤发生、DNA修复、生长调节和细胞凋亡。 ING2 通过增强 p53 的乙酰化来负向调节细胞增殖，p53 是一种主要的肿瘤抑制因子，在大约一半的人类癌症中 p53 发生突变。我们的初步研究表明，ING2 的 PHD 指特异性识别组蛋白尾结构域和肌醇六磷酸 (IP6) 信使，揭示了 IP6 介导的信号传导和染色质调控之间的新联系。然而，ING2 功能的分子机制尚未确定。组蛋白和 IP6 识别的结构基础仍未被探索，IP6 相互作用对 ING2 靶向核小体的影响尚不清楚。 该项目重点关注组蛋白和 IP6 结合的结构表征以及 PHD 结构域的新功能。要测试的假设是：(1) ING2 通过 PHD 与组蛋白尾部的相互作用靶向核小体，(2) IP6 结合对 ING2 的核小体募集进行负调控。 与 H4 组蛋白尾肽和 IP6 结合的 ING2 PHD 的原子分辨率结构将通过多维异核 NMR 或 X 射线晶体学测定。结合位点残基将发生突变，突变蛋白将通过 NMR 和 Pull-down 实验进行体外测试，并通过荧光显微镜进行体内测试。将使用电泳迁移率变动分析来研究 ING2 与核小体的关联。为了确定特异性，将通过 NMR、表面等离子共振和荧光光谱分析与未修饰和修饰的组蛋白尾肽以及与其他 IP 的相互作用。将研究组蛋白和 IP6 结合对 p53 激活和细胞凋亡的功能意义。 这项研究产生的结果将有助于全面了解肿瘤抑制因子 ING2 靶向染色质、与 IP 相互作用以及调节 p53 功能的分子机制。这些研究将有助于更深入地了解如何在治疗上操纵关键的 ING2-p53 通路，并可能有助于确定新的诊断标记物和靶点来预防和治疗癌症。