Structural basis of signaling by ING2 PHD

描述（由申请人提供）：生长抑制剂（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的乙酰化来负性调节细胞增殖，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，ING2的PHD指特异性识别组蛋白尾部结构域和肌醇六磷酸（IP6）信使，揭示了IP6介导的信号传导与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识别的结构基础尚不清楚，IP6相互作用对ING2靶向核小体的影响尚不清楚。

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.