Name: monitoring eif4f assembly by measuring eif4e-eif4g interaction in live cells
Uploaded: 2020-05-01T13:30:00
Description: Watch this Scientific Journal Video about Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells at JoVE.com

这项工作得到了p53lab（BMSI、A*STAR）和JCO VIP赠款（A*STAR）的核心预算的支持。

HEK293细胞系用于协议，并在杜尔贝科的改良鹰介质中培养，补充10%胎儿牛血清，2米L-谷氨酰胺和100U/mL青霉素/链霉素。细胞在37°C的培养下，在潮湿 的环境中产生5%的二氧化碳。
1. 通过 eIF4E 对 eIF4F 复杂中断进行定量评估：eIF4G604-646 补充分析
<ol><li>eIF4E 的细胞培养和瞬态瞬变：eIF4G604-646 补充测定<ol>
<li>使用新鲜解冻的细胞，所有实验的通道少于20个?...

<ol>
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本文所述方法采用基于路西法酶的补充测定法，通过直接测量活细胞中的eIF4G-eIF4E相互作用，对eIF4F复合组件进行定量监测。我们提供了使用eIF4E-eIF4G补充系统的详细信息，也表明该系统在测量eIF4E-eIF4G相互作用16的药物诱导4EBP1介导分离方面非常准确。为了便于此检测的吞吐量，本文中描述的实验设置已设计为 96 井微板格式的使用。
为了获得最佳结果，在执?...

基因表达控制在正确执行细胞程序（如生长增殖和分化）方面起着至关重要的作用。监管控制机制可以在基因转录水平或mRNA转化水平上进行。在过去十年中，越来越明显的是，通过调节启动过程而不是后来的拉长和终止步骤进行转化控制，可以精细地调节具有广泛生物功能的蛋白质特定子集的合成。
参与生存、抗自噬和抗凋亡反应的 mRNA 的翻译增加与几种癌症有关，并且与异常激活或过度表达翻译启动因子1有因果关系。
eIF4F 综合体是翻译启动的主调节器。通过将上限结构与 mRNA 的 5' 端绑定，eIF4F 正在推动初始 mRNA-里博索姆的招聘，进而提高翻译不力的真核素 mRNA2的 mRNA 翻译效率。据报道，许多癌症模型都存在异常激活RAS/MAPK或AKT/TOR通路的癌症模型，eIF4F的中介翻译，这表明癌细胞会调节eIF4F，以增强自身的抗肿瘤活性。通过抑制eIF4F复合形成来破坏这个进气回路，因此是一个非常有希望的治疗策略3，4。
eIF4F 综合体由 （i） eIF4E 组成， eIF4F 的帽绑定子单位，与 mRNA 的 5' UTR、（ii） eIF4A、RNA 六氯酶和 （iii） eIF4G 的上限结构相互作用，后者是与 eIF4A 和 eIF4E 相互作用的脚手架蛋白，最终招募了 40S 核糖核酸亚单位5。eIF4G 与 eIF4E 关联是功能 eIF4F 复合体组装的限速步骤，由 eIF4E 结合蛋白（4EBP、成员 1、2 和3）6负调节。通过由规范和非规范 eIF4E 绑定序列7、8、9（人类 eIF4E 上的 aa 604-646 区域）组成的界面与 eIF4G 绑定竞争，4EBP 可减少积极参与翻译和防止 eIF4F 复杂形成的 eIF4E 池。这些蛋白质-蛋白质相互作用的相互作用主要受4EBP的拉帕霉素（mTOR）介质磷酸化的哺乳动物目标的调节。在偏头痛刺激下，mTOR直接磷酸化4E-BP蛋白家族的成员，减少他们与eIF4E的关联，从而促进eIF4E-eIF4G相互作用和功能eIF4F复合体10的形成。
尽管在开发针对eIF4F复合完整性的化合物方面付出了巨大努力，但缺乏测量活细胞中eIF4E-eIF4G相互作用直接中断的检测结果，限制了对细胞活性命中化合物的搜索。我们应用了基于共聚苯乙烯模拟（例如，基于纳诺鲁克的补充测定）的路西法酶检测，通过 eIF4E-eIF4G 交互实时监控 eIF4F 完整性的状态。路西法酶补充蛋白系统由18kDa蛋白片段（SubA）和11氨基酸肽片段（SubB）组成，优化为最小的自结和稳定性11。一旦表示为与人类全长eIF4E和eIF4E相互作用域从人类eiF4G1（aa 604-646）的融合产品，两种相互作用的蛋白质将把SubA和SubB片段彼此接近，并诱导形成活性荧光酶，当细胞渗透基质存在时，最终将产生明亮的发光信号（图1）。我们已在其他地方报告了eIF4E：eIF4G604-646 补充系统16的建设和验证情况。
在这里，我们描述了如何应用 eIF4E：eIF4G604-646 补充系统（可应要求提供）来准确测量活细胞中 4EBP1 介质的 eIF4E-eIF4G 中断。此外，我们通过测量目前作为潜在的癌症治疗药物12的临床试验中的几种mTOR抑制剂的效果来证明它的效用。由于目标外效应通常掩盖药物特异性活动，我们还描述了如何通过细胞生存能力正交测量来扩展 eIF4E：eIF4G604-646 系统测量的多功能性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>293FT cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>R70007</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture microplate 96 well, F-Bottom</td>
			<td>greiner bio-one</td>
			<td>655083</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell titer Glo 2.0</td>
			<td>PROMEGA</td>
			<td>G9241</td>
			<td></td>
		</tr>
		<tr>
			<td>Envision Multilabel Reader</td>
			<td>PerkinElmer</td>
			<td>not applcable</td>
			<td></td>
		</tr>
		<tr>
			<td>Finnpipette F2 Multichannel Pipettes 12-channels 30-300 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>4662070</td>
			<td></td>
		</tr>
		<tr>
			<td>Finnpipette F2 Multichannel Pipettes 12-channels 5-50 ml</td>
			<td>Thermo Fisher Scientific</td>
			<td>4662050</td>
			<td></td>
		</tr>
		<tr>
			<td>FUGENE6</td>
			<td>PROMEGA</td>
			<td>E2692</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 3000</td>
			<td>Thermo Fisher Scientific</td>
			<td><a href="https://www.thermofisher.com/order/catalog/product/L3000015" target="_blank">L3000015</a></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoBiT PPI Starter Systems</td>
			<td>PROMEGA</td>
			<td>N2014</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimem I Reduced Serum Mediun, no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>11058021</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Eppendorf</td>
			<td>not appicable</td>
			<td></td>
		</tr>
		<tr>
			<td>&#947;-Aminophenyl-m7GTP (C10-spacer)-Agarose</td>
			<td>Jena Bioscience</td>
			<td>AC-155S</td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring eif4f assembly by measuring eif4e-eif4g interaction in live cells

eIF4F复合物的形成已被证明是信号通路收敛的关键下游节点，这些信号通路经常在人类中经历致癌活化。eIF4F 是一个上限绑定复合体，涉及翻译启动的 mRNA - 相关招聘阶段。在许多癌症的细胞和临床前模型中，eIF4F 的放松管制导致参与癌症增殖和生存的特定 mRNA 子集的翻译增加。eIF4F 是一个由上限绑定子单位 eIF4E、六角酶 eIF4A 和脚手架子单元 eIF4G 构建的异质三元复合体。活性eIF4F复合物组装的关键在于eIF4E和eIF4G蛋白质之间的蛋白质-蛋白质相互作用。在本文中，我们描述了一个测量 eIF4F 组件的协议，该程序可监控活细胞中的 eIF4E-eIF4G 交互状态。eIF4e：4G 基于细胞的蛋白质-蛋白质相互作用检测还允许准确可靠地评估 eIF4F 复杂完整性的药物诱导变化。我们设想，这种方法可以应用于验证市售化合物的活性，或进一步筛选有效破坏eIF4F复合物形成的新化合物或方式。

为了验证eIF4E：eIF4G604-646 补充系统的灵敏度，使用mTOR抑制剂对eIF4F复合组件进行了4EBP1介停抑制评估。通过抑制4EBP蛋白家族的mTORC1激酶依赖磷酸化，mTOR抑制增强4EBP1与eIF4E的关联，因此，eIF4F拆解15。测试了两类机械上不同的mTOR激酶抑制剂：拉帕洛格（如拉帕霉素），它们是mTORC1的同位素抑制剂，但不是mTORC2和ATP基于竞争的抑制剂（例如PP242），?...

Watch this Scientific Journal Video about Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells at JoVE.com

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

在这里，我们提出了一个协议，以测量eIF4E-eIF4G在活细胞中的相互作用，使用户能够评估药物引起的扰动eIF4F复杂动态的筛选格式。

通过测量活细胞中的 eIF4E-eIF4G 交互来监控 eIF4F 装配

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic ...

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