Name: analysis of cap-binding proteins in human cells exposed to physiological oxygen conditions
Uploaded: 2016-12-28T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions at JoVE.com

This work was supported by the Natural Sciences and Engineering Council of Canada and the Ontario Ministry of Research and Innovation.

1.准备细胞培养<ol><li>购买人细胞的市售股票。 注：此协议利用HCT116结直肠癌和初级人肾小管上皮细胞（HRPTEC）。 </li><li>使对HCT116的培养500毫升培养基：Dulbecco改良的Eagle培养基（DMEM）中补充有7.5％胎牛血清（FBS）和1％青霉素/链霉素（P / S）/高葡萄糖培养基。 </li><li>作出HRPTEC的培养500毫升培养基：补充有5％FBS，1％的上皮细胞生长添加物上皮细胞培养基和1％P / S。 </li><li>制成500毫升之1×?...

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在暴露于生理氧条件的人细胞帽结合蛋白质的分析可允许的新颖氧 - 调节翻译起始因子的鉴定。这些因素为mRNA或其它帽相关蛋白的5&#39;帽的亲和性可以通过其关联的强度被测量到m 7 GTP联的琼脂糖珠。这种技术的一个需要注意的是它测量蛋白裂解后的帽结合的潜力，但它是该保持蛋白质 - 蛋白质相互作用和翻译后后修饰非变性条件下进行。几个蛋白 - 蛋白相互作用介导的eIF4E家族的5&#39;帽?...

平移控制正在成为基因表达的转录调节同样重要的一步，尤其是在细胞应激1期。翻译调控的一个重点是在（M 7 GTP）5&#39;的mRNA 2帽引发的限速步骤，其中的蛋白质合成的第一个步骤涉及真核细胞起始因子4E（eIF4E的）的结合到7-甲基。 eIF4E的是一个名为eIF4F三聚体复杂，包括eIF4A，一个RNA解旋酶和eIF4G，对其他翻译因子和核糖体40S 3招聘要求支架蛋白的一部分。在正常生理条件下，绝大多数的mRNA经由帽依赖性机制翻译，但在细胞应激期间人类mRNA的约10％含有5&#39;非编码区，可能允许不依赖帽的翻译intiation 1,4。帽依赖性翻译在历史上同义词OU中与eIF4F，但是，eIF4F的特定压力变化已经成为一个热门话题5-8。 各种细胞应激引起雷帕霉素通过复杂1（mTORC1的）的哺乳动物目标受到抑制eIF4E的活动。这种激酶变得在压力下，这导致在其目标之一的增加的活性受损，所述4E结合蛋白（4E-BP）。非磷酸化4E-BP结合eIF4E与阻断其与eIF4G引起帽依赖性翻译9,10的压制交往的能力。有趣的是，一个名为eIF4E2（或4EHP）的eIF4E的同源物具有4E-BP 11低得多的亲和力，或许允许它逃避压力介导的镇压。事实上，最初表征为翻译阻遏由于其缺乏与eIF4G 12相互作用，eIF4E2发起数百包含在其3&#39;端非编码区的RNA缺氧反应元件的mRNA的低氧应激6,13-在翻译。这种激活我s至与eIF4G3，RNA结合蛋白基序4，和低氧诱导因子（HIF）2α构成低氧eIF4F复杂，或eIF4Fħ6,13-相互作用来实现的。如正常条件下的阻遏，eIF4E2结合与GIGYF2和ZNF598 14。这些复合物，在部分地通过琼脂糖联M 7 GTP的亲和树脂识别。这种传统方法15在翻译领域的标准，是最好的，最常用的技术，在下拉隔离帽结合复合物和体外结合试验16-19。作为帽依赖性翻译机器正在成为灵活性和适应性与间变化部分6-8,13，这种方法是一个强大的工具，迅速查明参与应激反应小说帽结合蛋白。此外，eIF4F的变化可能有广泛影响几个真核模型系统出现使用eIF4E2同源的应激反应等如拟南芥 20， 裂殖酵母 21， 果蝇 22和线虫 23。 有证据表明，在eIF4F变化可能不严格限于强调的条件，但参与正常生理学24。氧气供给到组织中（在毛细管两端）或组织中（通过微电极测定）从2-6％在脑中25而变化，在肺26 3-12％，在肠道27，在4％3.5-6％肝脏28，在肾脏29 7-12％，在肌肉30 4％，在骨髓中31 6-7％。细胞线粒体含有比1.3％的氧气少32。这些值比周围的空气，其中细胞常规培养更接近缺氧。这表明什么以前被认为是特定缺氧细胞过程可以是在生理环境相关。有趣的是，eIF4F和eIF4F ^ h </suP&gt;积极参与不同池或mRNA的类的翻译起始于暴露于生理氧或“physioxia”24几个不同的人细胞系。低氧也推动适当的胎儿发育33和电池通常具有较高的增殖率，寿命长，少的DNA损伤和physioxia 34较少的一般应激反应。因此，eIF4F H是可能在生理条件下选择的基因的表达的关键因素。 这里，我们提供了一个协议，以在固定的生理氧条件下或在这是可能的更具代表性的组织微环境的动态波动范围培养的细胞。这种方法的一个优点是，细胞在缺氧工作站内裂解。它是不是经常不清楚如何从缺氧细胞培养细胞裂解转变在其他协议执行。细胞常常首先从一个小缺氧孵化器中删除是前的裂解，但是这种暴露于氧气可能影响生化途径以氧气的细胞反应是快速（一个或两个分）35。某些帽结合蛋白需要用第二碱相互作用或能水解第m 7 GTP，因此一些帽交互件可在纯化过程被遗漏。琼脂糖联以酶促抗帽类似物可以在该协议被取代。探索通过这里描述的方法的活性和eIF4F H和eIF4F的其它变型的组合物将在该细胞中的生理条件或胁迫应答利用复杂的基因表达的机械线索。

<table border="0" cellpadding="0" cellspacing="0" width="901">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:380px;">&#947;-aminophenyl-m7GTP agarose C10-linked beads</td>
			<td style="width:151px;">Jena Bioscience</td>
			<td style="width:165px;">AC-1555</td>
			<td style="width:205px;">Agarose-linked m7GTP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm culture dish</td>
			<td>Corning</td>
			<td>877222</td>
			<td>10-cm culture dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150 mm culture dish</td>
			<td>Thermofisher</td>
			<td>130183</td>
			<td>15-cm culture dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AEBSF Hydrochloride</td>
			<td>ACROS Organics</td>
			<td>A0356829</td>
			<td>AEBSF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose Beads</td>
			<td>Jena Bioscience&#160;</td>
			<td>AC-0015</td>
			<td>Agarose bead control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromophenol Blue</td>
			<td>Fisher</td>
			<td>BP112-25</td>
			<td>Component of SDS-PAGE loading buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL Centrifuge Tubes</td>
			<td>FroggaBio</td>
			<td>1210-00S</td>
			<td>Used to centrifuge small volumes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL Conical Centrifuge Tubes</td>
			<td>Fisher</td>
			<td>1495970C</td>
			<td>Used in culturing primary cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Defined trypsin inhibitor</td>
			<td>Fisher</td>
			<td>R007100</td>
			<td>DTI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreital</td>
			<td>Fisher</td>
			<td>BP172-25</td>
			<td>DTT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epithelial cell medium (complete kit)</td>
			<td>ScienCell</td>
			<td>4101</td>
			<td>Includes serum and growth factor supplements)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher</td>
			<td>BP229-1</td>
			<td>Component of SDS-PAGE loading buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mM Guanosine 5&#39;-triphosphate, 1 mL</td>
			<td>Jena Bioscience</td>
			<td>272076-0251M</td>
			<td>GTP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCT116 colorectal carcinoma</td>
			<td>ATCC</td>
			<td>CCL-247</td>
			<td>Human cancer cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human renal proximal tubular epithelial cells</td>
			<td>ATCC</td>
			<td>PCS-400-010</td>
			<td>HRPTEC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone DMEM/High Glucose</td>
			<td>GE Life Sciences</td>
			<td>SH30022.01</td>
			<td>Standard media for human cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone Penicillin-Streptomycin solution</td>
			<td>GE Life Sciences</td>
			<td>SV30010</td>
			<td>Antibiotic component of DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">H35 HypOxystation</td>
			<td>Hypoxygen</td>
			<td>N/A</td>
			<td>Hypoxia workstation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Igepal CA-630</td>
			<td>MP Biomedicals</td>
			<td>2198596</td>
			<td>Detergent component of lysis buffer</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Monopotassium phosphate</td>
			<td>Fisher</td>
			<td>P288-500</td>
			<td>KH2PO4</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Potassium chloride</td>
			<td>Fisher</td>
			<td>P217-500</td>
			<td>KCl</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium chloride</td>
			<td>Fisher</td>
			<td>M33-500</td>
			<td>MgCl2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Fisher</td>
			<td>BP358-10</td>
			<td>NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium fluoride</td>
			<td>Fisher</td>
			<td>5299-100</td>
			<td>NaF (phosphatase inhibitor component of lysis buffer)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Disodium phosphate</td>
			<td>Fisher</td>
			<td>5369-500</td>
			<td>Na2HPO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Grade Fetal Bovine Serum</td>
			<td>Seradigm</td>
			<td>1500-500</td>
			<td>FBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail (100 x)</td>
			<td>Cell Signalling</td>
			<td>58715</td>
			<td>Component of lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Dodecyl Sulfate</td>
			<td>Fisher</td>
			<td>BP166-100</td>
			<td>SDS</td>
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		<tr height="26">
			<td height="26" style="height:26px;">Sodium Orthovanadate</td>
			<td>Sigma</td>
			<td>56508</td>
			<td>Na3VO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Base</td>
			<td>Fisher</td>
			<td>BP152-5</td>
			<td>Component of buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.05% Trypsin-EDTA (1x)</td>
			<td>Life Technologies</td>
			<td>2500-067</td>
			<td>Trypsin used to detach adherent cells</td>
		</tr>
	</tbody>
</table>

analysis of cap-binding proteins in human cells exposed to physiological oxygen conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

在一个m 7 GTP亲和柱的响应eIF4E与eIF4E2氧帽结合力分析 图1和2表示响应于氧的波动在两个人细胞系中两个主要的帽结合蛋白的典型M 7 GTP的亲和纯化的蛋白质印迹：原代人肾小管上皮F中igure 1和大肠癌细胞（HRPTEC）（ HCT116）在图2中。细胞维持在所指示的氧气供应24?...

Watch this Scientific Journal Video about Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions at JoVE.com

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

The overall goal of this experiment is to capture cap-binding proteins in conditions of low oxygen. This method utilizes an agarose linked seven methyl-guanosine cap analog to investigate cap-binding factors and their interacting partners. This method will help answer key questions in the field of protein synthesis such as identifying novel oxygen regulated factors involved in cap dependent translation.The main advantage of this technique is that cells are kept in a hypoxia workstation up until the point of lysis. The implications of this technique extend towards cancer therapy because cancer cells within a tumor are exposed to very low levels of oxygen and translational control plays a key role in tumor progression. We first had the idea for this method when we read a recent publication from Corona's colleagues reporting that tissue oxygenation is actually closer to what we call hypoxia rather than the normaxia that cells are routinely cultured in.Begin this procedure will cell culture as described in the text protocol. After counting the cells using a hemocytometer feed 150 millimeter cultured dishes containing 15 milliliters of complete medium to obtain a cell density of 2500 to 5000 cells per square centimeter. Use two 150 millimeter dishes for each cap-binding assay.Place the seeded culture dishes in the incubator at 37 degrees celsius and a five percent carbon dioxide atmosphere. After the cells have incubated at least 24 hours place the cells into a hypoxia workstation for the desired time. Then, set the workstation to the appropriate physioxia.Pre-warm one X phosphate buffered saline, or PBS, and trypsin in a 37 degree celsius water bath for 30 minutes. Aliquot 980 microliters of lysis buffer into a 1.5 microliter centrifuge tube for each cap-binding assay being performed. 10 microliters of 100X protease inhibitor cocktail and 10 microliters of 4-benzenesulfonyl fluoride hydrocloride to the 980 microliters of lysis buffer on ice.Discard the media from each 150 millimeter dish in a waste container within the hypoxia workstation. Then, wash the cells with three to four milliliters of warm one X PBS and discard the liquid. Add one milliliter of warm 05 percent trypsin EDTA to each dish and let the dishes sit for two minutes or until the cells are no longer adhered to the plate.Using a pipette transfer the cells of one plate to a 1.5 milliliter micro-centrifuge tube. Repeat as needed so that each plate of cells is transferred to a separate 1.5 milliliter micro-centrifuge tube. Centrifuge the micro-centrifuge tubes at 6000 times g for 90 seconds to pellet the cells.Following centrifugation aspirate the trypsin using a pipette without disturbing the pellet. Wash the cells with warm one X PBS by gently pipetting 200 microliters of PBS into the tube just above the pellet. Then aspirate the PBS without disturbing the pellet.Next, pipette 500 microliters from the one milliliter prepared lysis buffer solution onto the first cellular pellet. Re-suspend the pellet entirely by pipetting up and down. Combine the re-suspended cells with the second cell pellet and re-suspend the second pellet by pipetting up and down.Repeat this process until all pellets have been combined and re-suspended for each sample. Next, combine the lysate with the remaining 500 microliters of lysis buffer in a 1.5 liter micro-centrifuge tube. Remove the samples from the hypoxia workstation.Lyse the cells using gentle agitation by rotating the samples at four degrees celsisus for 1.5 to two hours. Following incubation, centrifuge the lysed cells at 1200 times g for 15 minutes at four degrees celsius to remove cellular debris. Transfer the lysate to a new 1.5 milliliter micro-centrifuge tube and discard the pellet.Reserve a portion of the supernatant to be used as whole cell lysate input control for western blot analysis. To prepare for the cap-binding assay use scissors to remove the tip of the pipette to facilitate collection of the bead slurry. For each sample transfer 50 microliters of the blank agarose bead control slurry and 50 microliters of the m7GTP agarose C10-linked beads slurry to separate 1.5 milliliter micro-centrifuge tubes.Then, pellet the beads by centrifuging the slurry at 500 times g for 30 seconds. Remove the supernatant carefully and re-suspend the beads in 500 microliters of TBS. After repeating these steps pellet the beads at 500 times g for 30 seconds and remove the supernatant.Next, transfer the supernatant containing the lysate to the 1.5 milliliter micro-centrifuge tube containing the blank agarose beads. Incubate for 10 minutes at four degrees celsius with gentle agitation. After 10 minutes, pellet the blank agarose beads by centrifuging at 500 times g for 30 seconds.Following centrifugation transfer the lysate that doesn't bind to the beads to the 1.5 milliliter micro-centrifuge tube containing the m7GTP agarose C10-linked beads. Wash the blank agarose beads by re-suspending the beads in 500 microliters of lysis buffer, pelleting the beads by centrifuging at 500 time g for 30 seconds, and discarding the supernatant. Repeat this four times for a total of five washes.Re-suspend the blank agarose beads in one X SDS-PAGE sample buffer. And boil the beads for 90 seconds at 95 degrees celsius. Store the sample at 20 degrees celsius for future western blot analysis to observe weather the protein of interest binds non-specifically to the bead.Incubate the lysate with the m7GTP agarose C10-linked beads with gentle agitation for one hour at four degrees celsuis to capture cap-binding proteins. Following agitation, pellet the m7GTP agarose C10-linked beads by centrifuging at 500 times g for 30 seconds. After discarding the supernatant, wash the m7GTP agarose C10-linked beads as before.Re-suspend the beads in 600 microliters of lysis buffer and add GTP to a final concentration of one millimolar. Incubate the m7GTP agarose C10-linked beads plus one millimolar GTP with gentle agitation for one hour at four degrees celsius. This will dissociate proteins that non-specifically interact with m7GTP.Pellet the m7GTP agarose C10-linked beads by centrifuging at 500 time g for 30 seconds. Transfer the supernatant to a new 1.5 milliliter micro-centrifuge tube, and add 200 microliters of four X SDS-PAGE sample buffer. After washing the beads as before, re-suspend the beads in one X SDS-PAGE sample buffer and boil at 95 degrees celsius for 90 seconds.Shown here are representative western blots of a typical m7GTP affinity purification of two major cap-binding proteins in response to oxygen fluctuations in primary human renal proximal tubule epithelial cells. The cell lysates were exposed to eight percent, five percent, three percent, or one percent oxygen for 24 hours. The lanes include 10 percent of the whole cell lysate as the input, a GTP washed to measure the specificity for m7GTP, and the proteins bound to m7GTP beads after the GTP wash.Data from at least three independent experiments were quantified by Image J and expressed as relative density units relative to 10 percent input of the whole cell lysate. Results indicate that human cells cultured under physiological oxygen utilize two cap-binding proteins to recruit distinct mRNAs for translation. After watching this video you should have a good understanding of how to capture cap-binding proteins in low oxygen conditions.While attempting this procedure it's important to remember to keep cells in the hypoxia workstation up until the point of lysis, as oxygen can rapidly alter the biochemical properties of these translation initiation factors. Following this procedure other methods like polysome fractionation can be performed to answer additional questions such as which cap-binding proteins associate with active translation under conditions of low oxygen.

Research

JoVE Journal

Genetics

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科研

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CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

人视网膜色素上皮细胞的分化、维持和分析: 一种BEST1突变的碟形模型

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

人类胰腺胰岛的单细胞RNA测序与分析

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

在人类多能干细胞中使用CRISPR-CAS9生成定义的基因组修饰

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

CRISPR/Cas9核糖核酸蛋白在急性骨髓性白血病细胞中的RUNX1电子消光器的转录作用研究

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...