Structural basis of signaling by ING2 PHD

描述（由申请人提供）：生长抑制剂（ING 2）肿瘤抑制因子与肿瘤发生、DNA修复、生长调节和细胞凋亡有关。ING 2通过增强p53的乙酰化来负调节细胞增殖，p53是一种主要的肿瘤抑制因子，在大约一半的人类癌症中发生突变。我们的初步研究表明，ING 2的PHD指特异性识别组蛋白尾部结构域和肌醇六磷酸（IP 6）信使，揭示了IP 6介导的信号传导和染色质调控之间的新联系。然而，ING 2功能的分子机制尚未建立。组蛋白和IP 6识别的结构基础尚未探索，IP 6相互作用对ING 2靶向核小体的影响也未知。 该项目的重点是组蛋白和IP 6结合的结构表征，PHD结构域的新功能。待检验的假设是：（1）通过PHD与组蛋白尾部的相互作用，ING 2靶向核小体，和（2）通过IP 6结合负调节NG 2的核小体募集。 结合到H4组蛋白尾肽和IP 6的ING 2 PHD的原子分辨率结构将通过多维异谱NMR或通过X射线晶体学来确定。将突变结合位点残基，并通过NMR和下拉实验在体外和通过荧光显微镜在体内测试突变蛋白。将使用电泳迁移率变动分析研究ING 2与核小体的缔合。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IP 6结合对p53活化和凋亡的功能意义。 本研究的结果将为全面了解肿瘤抑制因子ING 2靶向染色质、与IP相互作用以及调节p53功能的分子机制提供帮助。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ING 2-p53通路，并可能有助于确定新的诊断标志物和靶点，以预防和治疗癌症。

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.