Structural basis of signaling by ING2 PHD

描述(申请人提供)：生长抑制因子(ING2)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。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基因，在大约一半的人类癌症中发生突变。我们的初步研究表明，ING2的PhD指特异识别组蛋白尾部结构域和六磷酸肌醇(IP6)信使，揭示了IP6介导的信号转导和染色质调节之间的新联系。然而，ING2功能的分子机制尚未建立。组蛋白和IP6识别的结构基础尚不清楚，IP6相互作用对ING2靶向核小体的影响也不清楚。 本项目的重点是组蛋白和IP6结合的结构特征，这是PHD结构域的新功能。需要检验的假设是：(1)ING2通过PHD与组蛋白尾巴的相互作用靶向核小体；(2)IP6结合负调控核小体招募lNG2。 结合H4组蛋白尾肽和IP6的ING2 PhD的原子分辨结构将通过多维异核核磁共振或X射线结晶学确定。结合部位残基将发生突变，突变的蛋白将在体外通过核磁共振和下拉实验进行检测，并在体内通过荧光显微镜进行检测。ING2与核小体的关联将用凝胶迁移率改变分析来研究。为了确定特异性，将通过核磁共振、表面等离子体共振和荧光光谱分析未修饰和修饰的组蛋白尾肽以及与其他IP的相互作用。我们将探讨组蛋白和IP6结合对P53激活和细胞凋亡的功能意义。 这项研究的结果将为全面理解肿瘤抑制基因ING2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依据。这些研究将有助于更深入地理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和靶点来预防和治疗癌症。

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.