Name: study of protein-protein interactions in autophagy research
Uploaded: 2017-09-09T13:00:00
Description: Watch this Scientific Journal Video about Study of Protein-protein Interactions in Autophagy Research at JoVE.com

这项工作得到了土耳其科学和技术研究理事会 (研究) 1001 赠款 107T153, 萨班吉大学的支持。赛博和 NMK 的支持下, 研究 BIDEB 2211 奖学金的博士学位研究。

1. 免疫荧光 <ol> <li> 在 DMEM 高糖培养基中维持 HEK293T 人胚肾细胞, 在 DMEM 低血糖培养基中 N2A 小鼠 neuroblast 细胞, 在 5% CO 2 加湿培养箱中, 37 和 #176;用10% 热灭活胎牛 (FBS) 血清、抗生素 (50 U/毫升青霉素、50和 #181、g/毫升链霉素) 和 l-谷氨酰胺 (2 mM) 补充培养基. </li>
	<li> 使用0.25% 胰蛋白酶分离细胞。首先删除细胞培养的培养基, 然后用10毫升无菌磷酸缓冲盐 (PBS) 冲洗细胞, 在37和 #176 中孵育1...

<ol>
	<li>Oral, O., Akkoc, Y., Bayraktar, O., Gozuacik, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+and+pathological+significance+of+the+molecular+cross-talk+between+autophagy+and+apoptosis.">Physiological and pathological significance of the molecular cross-talk between autophagy and apoptosis.</a> Histol Histopathol. 31, 479-498 (2016).</li><li>Korkmaz, G., Tekirdag, K. A., Ozturk, D. G., Kosar, A., Sezerman, O. U., Gozuacik, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MIR376A+is+a+regulator+of+starvation-induced+autophagy.">MIR376A is a regulator of starvation-induced autophagy.</a> PLoS One. 8, e82556 (2013).</li><li>Itah, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+cavitation+kills+prostate+cells+and+ablates+benign+prostatic+hyperplasia+tissue.">Hydrodynamic cavitation kills prostate cells and ablates benign prostatic hyperplasia tissue.</a> Exp Biol Med (Maywood). 238, 1242-1250 (2013).</li><li>Gozuacik, D., Kimchi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+as+a+cell+death+and+tumor+suppressor+mechanism).">Autophagy as a cell death and tumor suppressor mechanism).</a> Oncogene. 23, 2891-2906 (2004).</li><li>Erzurumlu, Y., Kose, F. A., Gozen, O., Gozuacik, D., Toth, E. A., Ballar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+IBMPFD-related+P97/VCP+mutation+with+differential+binding+pattern+and+subcellular+localization.">A unique IBMPFD-related P97/VCP mutation with differential binding pattern and subcellular localization.</a> Int J Biochem Cell Biol. 45, 773-782 (2013).</li><li>Kuzuoglu-Ozturk, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy-related+gene,+TdAtg8,+in+wild+emmer+wheat+plays+a+role+in+drought+and+osmotic+stress+response.">Autophagy-related gene, TdAtg8, in wild emmer wheat plays a role in drought and osmotic stress response.</a> Planta. 236, 1081-1092 (2012).</li><li>Longatti, A., Tooze, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+trafficking+and+autophagosome+formation.">Vesicular trafficking and autophagosome formation.</a> Cell Death Differ. 16, 956-965 (2009).</li><li>Erbil, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RACK1+Is+an+Interaction+Partner+of+ATG5+and+a+Novel+Regulator+of+Autophagy.">RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy.</a> J Biol Chem. 291, 16753-16765 (2016).</li><li>Gozuacik, D., Yagci-Acar, H. F., Akkoc, Y., Kosar, A., Dogan-Ekici, A. I., Ekici, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticancer+use+of+nanoparticles+as+nucleic+acid+carriers.">Anticancer use of nanoparticles as nucleic acid carriers.</a> J Biomed Nanotechnol. 10, 1751-1783 (2014).</li><li>Phizicky, E. M., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-protein+interactions:+methods+for+detection+and+analysis.">Protein-protein interactions: methods for detection and analysis.</a> Microbiol Rev. 59, 94-123 (1995).</li><li>Uetz, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+analysis+of+protein-protein+interactions+in+Saccharomyces+cerevisiae.">A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae.</a> Nature. 403, 623-627 (2000).</li></ol>

沉淀和免疫荧光技术是研究蛋白质-蛋白质相互作用的关键。虽然这两种技术是常用的和完善的, 但应考虑在使用这些技术时确定实验质量的几个标准。
首先, 在这些测试中使用的主要抗体应该是特定的蛋白质的兴趣。为了确保这一点, 使用 shRNA knockdowns 或敲除细胞测试的特异性抗体的问题。此外, 使用积极的控制, 或治疗细胞与已知的蛋白质诱导的兴趣。批次的抗体的变种也存...

Macroautophagy (自噬) 是一种细胞应激机制, 其特征是在称为噬囊泡的双膜囊泡中的大体积细胞质、蛋白质和胞器的螯合。通过双膜外层的融合, 噬囊泡及其货物被传递到溶酶体并在其中降解1。自噬发生在所有细胞类型和所有生物体的低基底水平, 执行稳态功能, 如蛋白质降解和器官 (如, 线粒体) 周转。在导致细胞压力的条件下, 如饥饿, 自噬迅速上调并允许细胞维持能量水平和基本代谢1,2,3。
从酵母中克隆出大约30自噬基因, 他们的蛋白质产品在噬过程的各个阶段都扮演了一个角色, 包括囊泡形核、扩张、囊泡融合到晚期 endosome/溶, 以及货物降解4,5. 基因这些基因的大多数已经被确定, 并且在各种生物体中的研究证实了它们的细胞功能的保存情况6。在过去十年的研究表明, 几个自噬相关的蛋白质复合物和蛋白质-蛋白质相互作用存在, 他们控制自噬通路的复杂和控制的方式。交叉路口, 备份, 反馈和前馈机制存在, 他们允许细胞协调自噬与其他相关事件 (如水泡分泌物, 溶生物, 内涵排序和传输7,等)在一个无偏酵母-两个混合屏幕使用自噬蛋白 ATG5 作为诱饵, (ATG5 是一个关键的吞噬蛋白参与 E2-like 共轭系统, 介导 LC3 lipidation 在饥饿诱导自噬), 我们已经确定受体激活c-激酶 1 (RACK1;GNB2L1) 作为一个强大的交互和一个新的自噬成分8。重要的是, 屏幕显示, ATG5-RACK1 相互作用是不可缺少的自噬诱导的经典吞噬诱导 (即, 饥饿和 mTOR 抑制)。
Immunofluorescence-based 方法通常用于监测蛋白质-蛋白质相互作用。这些技术主要是抗体, 有助于可视化交互和确认细胞定位。在这种技术中, 荧光标记共轭抗体是特定的蛋白质的兴趣通常用于特定的染色。每种蛋白质都可以贴上不同荧光染料的抗体。使用蛋白质特异抗体, 在图像被合并时信号的重叠表明在共焦显微镜下蛋白质的共存。该技术适用于细胞甚至组织。免疫荧光技术提供了有关相互作用动力学的线索, 帮助确定蛋白质复合体的大小和分布, 同时跟踪不同条件下细胞形态学的一般变化9。沉淀是另一种常用的抗体技术, 允许分析给定蛋白质之间的相互作用10。利用这一技术, 感兴趣的蛋白质从细胞或组织提取物中分离出来, 使用特定的抗体, 导致蛋白质沉淀在一个复杂的或与感兴趣的蛋白质接触。共沉淀, 其中蛋白质和其 co-interactor 的检测, 不仅揭示了两个蛋白质之间的相互作用, 但可以测量其在不同情况下的相互作用强度11。
本协议详细描述了用于确认和描述 ATG5-RACK1 和 RACK1-LC3 交互的关键技术。重点是免疫荧光和沉淀技术, 重点是在自噬研究的关键步骤和陷阱, 以及故障排除建议。

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			<td>BI03-020-1B</td>
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			<td style="width:133px;">6916</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Nitocellulose membrane</td>
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		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:235px;">X-ray Films</td>
			<td style="width:181px;">Fujifilm</td>
			<td style="width:133px;">47410 19289</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Protease inhibitor</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:133px;">P8340</td>
			<td></td>
		</tr>
	</tbody>
</table>

study of protein-protein interactions in autophagy research

蛋白质-蛋白质相互作用对于理解细胞信号的叶栅和识别新的通路成分和蛋白质动力学是非常重要的。大多数细胞活动需要蛋白质之间的物理相互作用。为了分析和映射这些相互作用, 开发了各种实验技术以及生物信息学工具。自噬是一种细胞回收机制, 它允许细胞处理不同的压力源, 包括营养匮乏, 化学物质和缺氧。为了更好地了解自噬相关的信号事件, 并发现新的因素, 调节蛋白质复合体自噬, 我们进行蛋白质-蛋白质相互作用的屏幕。验证这些筛选结果需要使用免疫荧光和沉淀技术。在这个系统中, 我们发现的特定的自噬相关蛋白-蛋白质相互作用在 Neuro2A (N2A) 和 HEK293T 细胞系中进行了测试。在这个形象化的实验文件中解释了所使用的技术程序的细节。

在图 1中显示了使用此协议获得的共存结果的示例。我们最近的一篇论文中的一个数字显示为8。在这里, 内源性 RACK1 蛋白呈绿色染色, 内源性 LC3 染红。在合并的图片中观察到的黄色点表示绿色和红色信号之间的重叠点。因而黄色点代表这二个蛋白质的部份共存。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55881...

Watch this Scientific Journal Video about Study of Protein-protein Interactions in Autophagy Research at JoVE.com

Study of Protein-protein Interactions in Autophagy Research

本文介绍了两种抗体蛋白-蛋白质相互作用的研究方法: 免疫荧光和沉淀。这些技术适于研究蛋白质之间的物理相互作用, 以发现细胞信号通路的新成分, 以及了解蛋白质动力学。

自噬研究中蛋白质-蛋白质相互作用的研究

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

Research

JoVE Journal

Biology

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

基于分体式泛素膜酵母双杂交（神话）系统：一个强大的工具用于识别蛋白质 - 蛋白质相互作用

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

微图案的表面上的蛋白质-蛋白质相互作用的体内检测

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Imaging Protein-protein Interactions in vivo

Imaging Protein-protein Interactions in vivo

成像蛋白质 - 蛋白质相互作用在体内</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

液相亲和捕获试验用磁珠研究蛋白质相互作用：脊髓灰质炎病毒的Nanobody范例

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

蛋白质 - 蛋白质相互作用的双分子荧光互补的烟草原生质体和叶片可视化

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

结合单分子操纵和成像蛋白-DNA相互作用的研究

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...