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Cryo-EM analysis of PI3K signaling complexes in glioblastoma

胶质母细胞瘤中 PI3K 信号复合物的冷冻电镜分析

基本信息

批准号：
10056207
负责人：
Deborah F Kelly
金额：
$ 18.7万
依托单位：
VIRGINIA POLYTECHNIC INST AND ST UNIV
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-01 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10056207
关键词：
3-Dimensional Amino Acid Motifs BRCA1 gene Binding Brain C2 Domain Catalytic Domain Cell Death Inhibition Cell Survival Cells Chemicals Clinical Complex Cryoelectron Microscopy Development Diagnosis Disease Disease Progression Drug Targeting Electron Microscope Excision Fluorescence Fluorescence-Activated Cell Sorting Future Gene Family Genes Glioblastoma Growth Image Immunodeficient Mouse Laboratories Malignant Neoplasms Methods Molecular Molecular Conformation Molecular Structure Mus Mutation Operative Surgical Procedures Outcome PIK3CA gene PIK3CB gene PTEN gene Patients Peptides Pharmacologic Substance Phosphatidylinositol 4,5-Diphosphate Phosphotransferases Play Progressive Disease Protein Isoforms Protein Sequence Analysis Proteins Radiation Recurrence Research Resolution Role Serum Signal Transduction Specimen Starvation Structure Testing Therapeutic Therapeutic Intervention Time Titan Visualization Work base cancer therapy chemotherapy clinically relevant curative treatments design genetic regulatory protein inhibitor/antagonist innovation insight kinase inhibitor malignant breast neoplasm mutant novel peptide drug peptidomimetics prevent protein complex protein structure side effect success therapeutic development tumor tumor growth

项目摘要

PROJECT SUMMARY/ABSTRACT Glioblastoma (GBM) is a lethal disease for which there is no known cure. Following ~2 years of initial treatment, which includes surgical resection, radiation, and chemotherapy, more than 90% of GBM patients succumb to disease progression. Inhibitors of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) have been used clinically to treat original and recurrent GBMs with modest benefits. Our recent research finds that PIK3CB (PI3K catalytic subunit β, encoding p110β), but not other PI3K catalytic subunits, shows a strong association with GBM progression. Moreover, depletion or pharmaceutical inhibition of p110β induces growth inhibition/cell death in GBM cells highly expressing this subunit. In contrast, blocking other PI3K catalytic subunits fails to do so. Hence, PIK3CB/p110β is a selective survival factor for GBM. Our results strongly support that targeting one PI3K isoform that is dominant in GBM may be a more effective approach to treat GBM. While past research on PI3K isoforms has identified PI3K isoform-selective inhibitors, the clinical benefits of these chemical compounds are limited. The lack of molecular details pertaining to p110β selective activation and structural information of native p110 complexes likely contributes to the poor outcomes of current therapies. Understanding the molecular/structural details of p110β will permit a better design of more selective and effective p110β-based therapies for GBM. To this end, we will complete the following two specific aims. In aim 1, we will acquire high-resolution 3D conformations of p110β native protein complexes using immuno-capture cryoEM. Resolution of cryo-EM images we acquired previously was not high enough to provide 3D conformations of p110β/p85 complexes at a greater detail. Access to a FEI Titan Krios G2 electron microscope has rendered high resolution 3D conformations of native p110β/p85 complexes possible. To acquire more clinically relevant protein structures, primary GBM xenograts derived from patient specimens will be used. In aim 2, we will test the hypothesis that p110βC2in changes 3D conformations of p110β native protein complexes, thus inactivating p110β. Primary GBM cells will be treated with p110βC2in or a control scramble peptide. 3D conformations of p110β native complexes will be revealed by immuno-capture cryo-EM. Structural differences between p110β native complexes with or without p110βC2in will then be determined. Results from this R21 application will be highly impactful, particularly to our future research in PI3K signaling and on the therapeutic intervention for GBM. The molecular details of p110β native complexes revealed by cryo-EM will encourage us to further our understanding of molecular mechanisms underlying selective activation of p110β in GBM and to identify novel vulnerabilities of p110β at atomic levels. Visualization of p110β native complexes with or without p110βC2in will not only help us understand how this peptide acts but also will facilitate a further development of p110βC2in into a GBM treatment.

项目总结/摘要胶质母细胞瘤（GBM）是一种致命的疾病，目前尚无治愈方法。经过约2年的初步治疗，包括手术切除、放疗和化疗，超过90%的GBM患者死于疾病进展。已经使用磷脂酰肌醇-4，5-二磷酸3-激酶（PI 3 K）的抑制剂在临床上治疗原发性和复发性GBM具有适度的益处。我们最近的研究发现，PIK 3CB（PI 3 K P110β催化亚基），而非其他PI 3 K催化亚基，显示与GBM的强相关性进展此外，p110β的耗尽或药物抑制诱导了在小鼠中的生长抑制/细胞死亡。 GBM细胞高度表达该亚基。相反，阻断其他PI 3 K催化亚基不能做到这一点。因此，我们认为， PIK 3CB/p110β是GBM的选择性生存因子。我们的结果强烈支持靶向一种PI 3 K亚型可能是治疗GBM更有效的方法。虽然过去对PI 3 K亚型的研究虽然已经鉴定了PI 3 K亚型选择性抑制剂，但是这些化合物的临床益处是有限的。缺乏与p110β选择性激活相关的分子细节和天然p110的结构信息复合物可能导致当前疗法的不良结果。了解分子/结构 p110β的详细信息将允许更好地设计更具选择性和有效的基于p110β的GBM疗法。到为此，我们将完成以下两个具体目标。在目标1中，我们将获得高分辨率3D 使用免疫捕获cryoEM的p110β天然蛋白复合物的构象。冷冻EM图像的分辨率我们之前获得的结果不足以提供p110β/p85复合物的3D构象，详细使用FEI Titan Krios G2电子显微镜，可能存在天然p110β/p85复合物。为了获得更多临床相关的蛋白质结构，原发性GBM 将使用来自患者样本的异种大鼠。在目的2中，我们将检验p110β C2在改变p110β天然蛋白复合物的3D构象，从而使p110β失活。原代GBM细胞将用p110β C2 in或对照乱序肽处理。p110β天然复合物的3D构象将是通过免疫捕获冷冻EM显示。有或没有p110β天然复合物之间的结构差异然后测定p110β C2 in。这一R21应用程序的结果将具有高度影响力，特别是对我们的 PI 3 K信号转导和GBM治疗干预的未来研究。p110β的分子细节冷冻电镜揭示的天然复合物将鼓励我们进一步了解分子机制 GBM中p110β潜在的选择性激活，并在原子水平上识别p110β的新漏洞。可视化p110β天然复合物与或不与p110β C2 in将不仅有助于我们理解这是如何发生的， p110β C2 in不仅能发挥作用，还能促进p110β C2 in进一步发展为GBM治疗。