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
Summary
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.
Abstract
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.
Introduction
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'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'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'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>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。
Protocol
Representative Results
Discussion
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'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'帽?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Natural Sciences and Engineering Council of Canada and the Ontario Ministry of Research and Innovation.
Materials
| γ-aminophenyl-m7GTP agarose C10-linked beads | Jena Bioscience | AC-1555 | Agarose-linked m7GTP |
| 100 mm culture dish | Corning | 877222 | 10-cm culture dish |
| 150 mm culture dish | Thermofisher | 130183 | 15-cm culture dish |
| AEBSF Hydrochloride | ACROS Organics | A0356829 | AEBSF |
| Agarose Beads | Jena Bioscience | AC-0015 | Agarose bead control |
| Bromophenol Blue | Fisher | BP112-25 | Component of SDS-PAGE loading buffer |
| 1.5 mL Centrifuge Tubes | FroggaBio | 1210-00S | Used to centrifuge small volumes |
| 15 mL Conical Centrifuge Tubes | Fisher | 1495970C | Used in culturing primary cells |
| Defined trypsin inhibitor | Fisher | R007100 | DTI |
| Dithiothreital | Fisher | BP172-25 | DTT |
| Epithelial cell medium (complete kit) | ScienCell | 4101 | Includes serum and growth factor supplements) |
| Glycerol | Fisher | BP229-1 | Component of SDS-PAGE loading buffer |
| 100 mM Guanosine 5'-triphosphate, 1 mL | Jena Bioscience | 272076-0251M | GTP |
| HCT116 colorectal carcinoma | ATCC | CCL-247 | Human cancer cell line |
| Human renal proximal tubular epithelial cells | ATCC | PCS-400-010 | HRPTEC |
| Hyclone DMEM/High Glucose | GE Life Sciences | SH30022.01 | Standard media for human cell culture |
| Hyclone Penicillin-Streptomycin solution | GE Life Sciences | SV30010 | Antibiotic component of DMEM |
| H35 HypOxystation | Hypoxygen | N/A | Hypoxia workstation |
| Igepal CA-630 | MP Biomedicals | 2198596 | Detergent component of lysis buffer |
| Monopotassium phosphate | Fisher | P288-500 | KH2PO4 |
| Potassium chloride | Fisher | P217-500 | KCl |
| Magnesium chloride | Fisher | M33-500 | MgCl2 |
| Sodium chloride | Fisher | BP358-10 | NaCl |
| Sodium fluoride | Fisher | 5299-100 | NaF (phosphatase inhibitor component of lysis buffer) |
| Disodium phosphate | Fisher | 5369-500 | Na2HPO4 |
| Premium Grade Fetal Bovine Serum | Seradigm | 1500-500 | FBS |
| Protease Inhibitor Cocktail (100 x) | Cell Signalling | 58715 | Component of lysis buffer |
| Sodium Dodecyl Sulfate | Fisher | BP166-100 | SDS |
| Sodium Orthovanadate | Sigma | 56508 | Na3VO4 |
| Tris Base | Fisher | BP152-5 | Component of buffers |
| 0.05% Trypsin-EDTA (1x) | Life Technologies | 2500-067 | Trypsin used to detach adherent cells |
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