Name: detection of detergent-sensitive interactions between membrane proteins
Uploaded: 2018-03-07T14:45:00
Description: Watch this Scientific Journal Video about Detection of Detergent-sensitive Interactions Between Membrane Proteins at JoVE.com

这项工作得到了研究所资助的研究补助金 DK52057 和 DK107498 从 nih 到 K.V.K. x P 得到了来自 nih 的机构培训补助金2T32DK007201 的支持。

1. 肽的处理
<ol><li>肽的设计与排序<ol>
<li>选择所需的肽序列, 并添加一个标记, 如 c-myc 表位 (EQKLISEED) 在其 N 或 C 终点。</li>
		<li>用肽溶解度计算器 http://pepcalc.com/peptide-solubility-calculator.php 检查水中肽的预测溶解度。如果溶解度低, 尝试添加另一个水溶性标签, 以改变电荷平衡。 注意: 我们使用了与第一个腔圈 (fll) 相对应的肽, 在 N 总站 (Myc-fll-GLUT4) 上标记了 GLUT4 的 c-myc 表位 (粗体): EQKLISEE...

<ol>
	<li>Bogan, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+glucose+transporter+translocation+in+health+and+diabetes.">Regulation of glucose transporter translocation in health and diabetes.</a> Annu Rev Biochem. 81, 507-532 (2012).</li><li>Kandror, K. V., Pilch, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sugar+is+sIRVed:+sorting+Glut4+and+its+fellow+travelers.">The sugar is sIRVed: sorting Glut4 and its fellow travelers.</a> Traffic. 12 (6), 665-671 (2011).</li><li>Pan, X., Zaarur, N., Singh, M., Morin, P., Kandror, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sortilin+and+retromer+mediate+retrograde+transport+of+Glut4+in+3T3-L1+adipocytes.">Sortilin and retromer mediate retrograde transport of Glut4 in 3T3-L1 adipocytes.</a> Mol Biol Cell. 28 (12), 1667-1675 (2017).</li><li>Kim, J., Kandror, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+luminal+loop+confers+insulin+responsiveness+to+the+glucose+transporter+4.">The first luminal loop confers insulin responsiveness to the glucose transporter 4.</a> Mol Biol Cell. 23 (5), 910-917 (2012).</li><li>Shi, J., Kandror, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sortilin+is+essential+and+sufficient+for+the+formation+of+Glut4-storage+vesicles+in+3T3-L1+adipocytes.">Sortilin is essential and sufficient for the formation of Glut4-storage vesicles in 3T3-L1 adipocytes.</a> Dev Cell. 9 (1), 99-108 (2005).</li><li>Coutinho, M. F., Prata, M. J., Alves, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+shortcut+to+the+lysosome:+the+mannose-6-phosphate-independent+pathway.">A shortcut to the lysosome: the mannose-6-phosphate-independent pathway.</a> Mol Genet Metab. 107 (3), 257-266 (2012).</li><li>Willnow, T. E., Petersen, C. M., Nykjaer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VPS10P-domain+receptors+-+regulators+of+neuronal+viability+and+function.">VPS10P-domain receptors - regulators of neuronal viability and function.</a> Nat Rev Neurosci. 9 (12), 899-909 (2008).</li><li>Mazella, J., 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=The+100-kDa+neurotensin+receptor+is+gp95/sortilin,+a+non-G-protein-coupled+receptor.">The 100-kDa neurotensin receptor is gp95/sortilin, a non-G-protein-coupled receptor.</a> J Biol Chem. 273 (41), 26273-26276 (1998).</li><li>Ni, X., Morales, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lysosomal+Trafficking+of+Acid+Sphingomyelinase+is+Mediated+by+Sortilin+and+Mannose+6-phosphate+Receptor.">The Lysosomal Trafficking of Acid Sphingomyelinase is Mediated by Sortilin and Mannose 6-phosphate Receptor.</a> Traffic. 7 (7), 889-902 (2006).</li><li>Westergaard, U. B., 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=Functional+organization+of+the+sortilin+Vps10p+domain.">Functional organization of the sortilin Vps10p domain.</a> J Biol Chem. 279 (48), 50221-50229 (2004).</li><li>Nykjaer, A., 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=Sortilin+is+essential+for+proNGF-induced+neuronal+cell+death.">Sortilin is essential for proNGF-induced neuronal cell death.</a> Nature. 427 (6977), 843-848 (2004).</li></ol>

Sortilin 是一种进化保守的多配体蛋白受体, 涉及两个信号在等离子膜和细胞内排序事件6,7。然而, 寻找正宗 sortilin 的配体 (其中一些是腔内或整体膜蛋白) 是复杂的, 因为 sortilin 与它的一些结合伙伴的相互作用似乎对洗涤剂敏感。因此, 一种简单而广泛的方法来研究蛋白质-蛋白质相互作用, 共同免疫沉淀, 不能轻易地应用。一些研究组使用免疫沉淀来演示 ...

GLUT4 是一种葡萄糖转运蛋白, 主要表现在脂肪和骨骼肌细胞中, 它介导胰岛素对膳食后血糖清除的影响1。GLUT4 是一种非常稳定的蛋白质, 它是在平移后的水平上调节的。在没有胰岛素的情况下, GLUT4 在很大程度上被排除在血浆膜上 (因此葡萄糖的基底通透性较低), 主要在小胰岛素应答性囊泡 (IRVs) 和反式高尔基网络 (TGN) 的细胞内进行定位, 这可能代表IRV 捐赠舱在胰岛素的管理, IRVs 保险丝与等离子膜, 并提供 GLUT4 到其功能的地方。这增加了血浆膜的渗透率为葡萄糖, 因此葡萄糖吸收从血液入脂肪细胞和骨骼肌肌细胞膜上升10到40倍。胰岛素退出后, GLUT4 被内化成早期/排序内涵体, 然后检索到 TGN 的 IRVs 重新形成。GLUT4 路径中的两个排序步骤,即从外围早期内涵体到核周 TGN 的检索, 以及 IRVs 捐赠膜 TGN 的形成是由 Vps10p 家庭成员 sortilin 启用的, 它表示类型 i跨膜蛋白和分选受体。根据一个模型, sortilin 工作作为跨膜支架蛋白: 它结合 GLUT4 在内涵体和 TGN 的流明, 并招募 retromer 或网格蛋白适配器的细胞质侧的供体膜通过其C总站2 ,3. 这促进了 GLUT4 在胞内隔间植物常常将 GLUT4 的水泡载体的分布。
sortilin 的细胞质尾部与 retromer 和各种适配器蛋白的相互作用得到了很好的证明。然而, sortilin 对 GLUT4 (及其它的其他一些蛋白质配体) 的约束是难以证明的。特别是, 试图共同 immunoprecipitate sortilin 和 GLUT4 是没有成功的可能是由于洗涤剂敏感的性质的相互作用的这两种蛋白质。此外, 作为一种典型的转运蛋白, GLUT4 有12个跨膜域和6个腔圈, 任何组合可能代表一个 sortilin 绑定站点。同时, 大量的间接证据, 如细胞内的大量联合定位, 与膜透性 DSP 的交联, 以及酵母两种杂交系统的相互作用, 表明 sortilin 可以绑定到 GLUT4。此外, 使用后一种方法结合丙氨酸扫描诱变, 我们以前确定, Vps10p 领域的 sortilin 主要绑定到第一个腔内循环的 GLUT4。然而, 哺乳动物细胞中这种相互作用的证据却不见了。在这里, 我们已经分离了他标记的 sortilin 从转染的 3T3-L1 细胞使用钴树脂, 并证明它可以与化学合成肽相对应的第一腔内循环的 GLUT4 在 ph 值6和 ph 值 8, 类似酸性环境在TGN 膜腔内细胞膜腔和中性环境。在控制实验中, 未发现从非转染细胞中提取的萃取物在同一珠子上加载的肽结合。

<table border="0" cellpadding="0" cellspacing="0" width="608">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protease inhibitor cocktail</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">P8849</td>
			<td style="width:167px;">for use in purification of histidine tag protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phosphate-Buffered Saline&#160;</td>
			<td style="width:107px;">Corning</td>
			<td style="width:119px;">21-040-CV</td>
			<td style="width:167px;">PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1mL Insulin Syringe U-100</td>
			<td style="width:107px;">BD</td>
			<td style="width:119px;">329652</td>
			<td style="width:167px;">26G x 1/2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BCA Protein Assay Kit</td>
			<td style="width:107px;">Pierce</td>
			<td style="width:119px;">23228</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wash buffer</td>
			<td style="width:107px;">Boston BioProducts</td>
			<td style="width:119px;">BP-234</td>
			<td style="width:167px;">For His-tag protein purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HisPur Cobalt Resin</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:119px;">89964</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Tricine sample Buffer</td>
			<td style="width:107px;">Bio-Rad</td>
			<td style="width:119px;">161-0739</td>
			<td style="width:167px;">with SDS, bMercaptaethanol should be added</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Elution&#160; Buffer</td>
			<td style="width:107px;">Boston BioProducts</td>
			<td style="width:119px;">BP-236</td>
			<td style="width:167px;">For His-tag protein purificationwith 250mM Imidazole</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Mini-Protean Tris-Tricine precast gels 10-20%&#160;</td>
			<td style="width:107px;">Bio-Rad</td>
			<td style="width:119px;">456-3115</td>
			<td style="width:167px;">For seperation of peptide and small protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris/Tricine/SDS running buffer&#160;</td>
			<td style="width:107px;">Bio-Rad</td>
			<td style="width:119px;">161-0744</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transfer Buffer</td>
			<td style="width:107px;">Boston BioProducts</td>
			<td style="width:119px;">BP-190</td>
			<td style="width:167px;">Add 20% methanol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nitrocellulose membrane&#160; 0.45 mm</td>
			<td style="width:107px;">Bio-Rad</td>
			<td style="width:119px;">1620115</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">A9647</td>
			<td style="width:167px;">BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti Myc antibody</td>
			<td style="width:107px;">Cell Signaling</td>
			<td style="width:119px;">2272</td>
			<td style="width:167px;">Rabbit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peptide</td>
			<td style="width:107px;">Genscipt</td>
			<td style="width:119px;"></td>
			<td>customize ordering</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Precision Plus Protein Dual color Standarts</td>
			<td style="width:107px;">BioRad</td>
			<td style="width:119px;">161-0374</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of detergent-sensitive interactions between membrane proteins

我们探索蛋白质与蛋白质相互作用的能力是理解细胞中调控连接的关键。然而, 在许多情况下, 检测蛋白质与蛋白质之间的相互作用与重大的实验挑战有关。特别地, 排序感受器与他们的蛋白质货物在膜隔间的腔内相互作用经常以洗涤剂敏感的方式, 使这些蛋白质的共同免疫沉淀不可用。分类受体 sortilin 对葡萄糖转运体 GLUT4 的结合可以作为膜蛋白间弱腔相互作用的一个例子。在这里, 我们描述了一个快速, 简单, 廉价的检测, 以验证 sortilin 和 GLUT4 之间的相互作用。为此, 我们设计和化学合成了与 GLUT4 内腔部分的潜在 sortilin 结合表位对应的 c-myc 标记肽。Sortilin 标记六 histidines 的哺乳动物细胞, 并从细胞裂解物使用钴珠分离。在不同 pH 值下, 用多肽溶液在珠上固定 Sortilin, 用洗脱材料进行了分析。这种检测可以很容易地适应研究其他洗涤剂敏感的蛋白质-蛋白质相互作用。

裂解物是由 3T3 L1 细胞稳定转染的 Sortilin-c-myc/他的5和从野生 3T3 L1 细胞, 用作负控制.两个裂解物在 ph 值6或 ph 值8和彻底洗涤的钴珠孵化。然后在 Myc-fll-Glut4 的溶液中孵化出固定蛋白的珠子。经过仔细的洗涤, 与珠子绑定的蛋白质与0.25 米咪唑洗脱。样品被接受了, 连同原始的裂解物, 到 SDS 页在一个 10-20% Tricine 梯度凝胶跟随西部印迹与抗 c-myc 抗体, 允许检?...

Watch this Scientific Journal Video about Detection of Detergent-sensitive Interactions Between Membrane Proteins at JoVE.com

Detection of Detergent-sensitive Interactions Between Membrane Proteins

我们描述了一个协议, 以检测在膜蛋白之间的洗涤剂敏感的相互作用, 使用结合的排序受体, sortilin, 到第一个腔内循环的葡萄糖转运蛋白, GLUT4, 作为一个例子。

膜蛋白对洗涤剂敏感相互作用的检测

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

科研

生物化学

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

方法可视化和用透射电子显微镜分析膜蛋白的相互作用

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Evaluation of ProteinÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“Protein Interactions using an On-Membrane Digestion Technique

使用膜内消化技术评估蛋白质与蛋白质相互作用

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

淀粉样蛋白纤维蛋白的相互作用和脑线粒体的膜渗透

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

阿西尔-PEGyl交换凝胶移位测定，用于脑膜蛋白棕榈酸化定量测定

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

实时测量核苷酸与完整功能性膜蛋白的结合

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support