Structural basis of signaling by ING2 PHD

ING2 PHD 信号传输的结构基础

• 批准号: 7658826
• 负责人: TATIANA G KUTATELADZE
• 金额: $ 19.61万
• 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 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7658826
• 关键词: Acetylation; Address; Affect; Affinity; Apoptosis; Binding; Binding Proteins; Binding Sites; Biological Assay; C-terminal; Cell Proliferation; Chemicals; Chromatin; Chromatin Remodeling Factor; Complex; DNA Repair; Electrophoretic Mobility Shift Assay; Fluorescence Microscopy; Fluorescence Spectroscopy; Green Fluorescent Proteins; Growth; Growth Inhibitors; Heteronuclear NMR; Histone H4; Histones; Human; Inositol; Kinetics; Ligand Binding; Link; Malignant Neoplasms; Mediating; Modeling; Molecular; Molecular Conformation; Mutate; Mutation; NMR Spectroscopy; Nature; Nucleosomes; PHD Finger; Pathway interactions; Peptides; Phosphatidylinositols; Phytic Acid; Polyphosphates; Property; Proteins; Regulation; Research; Research Personnel; Resolution; Signal Pathway; Signal Transduction; Solutions; Specificity; Structure; Surface Plasmon Resonance; TP53 gene; Tail; Testing; Thermodynamics; Tumor Suppressor Proteins; X-Ray Crystallography; base; histone modification; in vitro testing; in vivo; mutant; novel; novel diagnostics; prevent; programs; recombinant peptide; repaired; research study; three dimensional structure; tumorigenesis

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）抑制肿瘤的抑制剂与肿瘤生成，DNA修复，生长调控和凋亡有关。 ING2通过增强p53的乙酰化（一种主要的肿瘤抑制剂）来负调节细胞增殖，该抑制剂在大约一半的人类癌症中被突变。我们的初步研究表明，ING2的PhD手指特异性地识别了组蛋白尾域和肌醇六磷酸二磷酸（IP6）Messenger，揭示了IP6介导的信号传导与染色质调节之间的新联系。然而，尚未建立基于ING2功能的分子机制。组蛋白和IP6识别的结构基础尚未探索，IP6相互作用对ING2靶向核小体的影响尚不清楚。 该项目着重于组蛋白和IP6结合的结构表征，这是PHD域的新功能。要测试的假设是：（1）通过PHD与组蛋白尾巴的相互作用，ING2针对核小体，（2）LNG2的核小体募集受IP6结合对核心体募集负调节。 与H4组蛋白尾肽和IP6结合的ING2 PHD的原子分辨率结构将由多维异核NMR或X射线晶体学确定。结合位点残基将被突变，突变蛋白将通过NMR和下拉实验在体外测试，并通过荧光显微镜在体内进行测试。 ING2与核小体的关联将使用电泳迁移率分析进行研究。为了确定特异性，将通过NMR，表面等离子体共振和荧光光谱分析与未修饰和改良的组蛋白尾肽以及其他IPS的相互作用。将研究组蛋白和IP6结合在p53激活和凋亡中的功能显着性。 这项研究中产生的结果将对肿瘤抑制剂ING2的分子机制具有全面的了解，以染色质，与IPS相互作用并调节p53的功能。这些研究将有助于更深入地了解如何对关键的ING2-P53途径进行治疗，并可能有助于确定预防和治疗癌症的新诊断标记和靶标。