Cryo-EM analysis of PI3K signaling complexes in glioblastoma

项目摘要/摘要 胶质母细胞瘤(GBM)是一种致命的疾病，目前还没有已知的治疗方法。在经过大约两年的初步治疗后， 包括手术切除、放疗和化疗，90%以上的GBM患者死于 疾病的发展。磷脂酰肌醇-4，5-二磷酸3-激酶(PI3K)的抑制剂已被使用 临床上以温和的疗效治疗原发和复发的肾小球系膜瘤。我们最近的研究发现，PIK3CB(PI3K β催化亚基编码p110β)，而不是其他PI3K催化亚基，与基底膜有很强的相关性 进步。此外，p110β的耗尽或药物抑制可导致细胞生长抑制/细胞死亡。 高表达该亚单位的GBM细胞。相反，阻断其他PI3K催化亚基则无法做到这一点。因此， PIK3CB/p110β是基底膜的选择性生存因子。我们的结果有力地支持了靶向一种PI3K亚型的观点 以基底膜为主可能是治疗基底膜更有效的方法。虽然过去对PI3K亚型的研究 已经确定了PI3K异构体选择性抑制剂，这些化合物的临床益处是有限的。 缺乏与p110β选择性激活有关的分子细节和天然p110的结构信息 复合体可能是目前治疗效果不佳的原因之一。了解分子/结构 P110β的细节将允许更好地设计更具选择性和更有效的基于p110β的治疗基底膜的疗法。至 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的3D 免疫捕获冷冻EM法研究p110β天然蛋白复合体的构象。低温电子显微镜图像的分辨率 我们以前获得的数据不足以提供p110β/p85复合体的3D构象 细节。进入FEI Titan Krios G2电子显微镜后，呈现了高分辨率的3D构象 可能存在天然的p110β/p85复合体。为了获得更多临床相关的蛋白质结构，初级GBM 来自患者样本的异种大鼠将被使用。在目标2中，我们将检验p110βC2in的假设 改变p110β天然蛋白复合体的3D构象，从而使p110β失活。原代GBM细胞将 用p110βC2in或对照扰乱肽治疗。P110β天然络合物的3D构象将是 通过免疫捕获冷冻-EM揭示。P110-β天然复合体的结构差异 然后将确定P110βC2in。此R21应用程序的结果将产生很大影响，特别是对我们的 PI3K信号转导及对GBM治疗干预的未来研究。P110β的分子细节 低温电子显微镜揭示的天然络合物将有助于我们进一步了解分子机制 P110β在GBM中的潜在选择性激活，并在原子水平上识别p110β的新漏洞。 对含有或不含有p110βC2in的p110β天然复合体的可视化不仅有助于我们了解 多肽不仅起作用，而且还将促进p110βC2in进一步发展为基底膜治疗。

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.