Name: reverse yeast two-hybrid system to identify mammalian nuclear receptor residues that interact with ligands and/or antagonists
Uploaded: 2013-11-15T18:30:00
Description: Watch this Scientific Journal Video about Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists at JoVE.com

这项工作是由卫生研究院（NIH）国家机构支持的补助CA127231及达蒙鲁尼恩基金会临床研究奖（CI 1502）（以SM）。我们要感谢的Zdenek德沃夏克教授帕拉茨基大学奥洛穆茨，捷克共和国的有益见解讨论这个技术移植到他们的机构和协议的标准化。

1。 PXR和SRC-1融合体在酵母载体的构建<ol><li> PCR扩增人类PXR LBD（107-434氨基酸）和人类SRC-1全长（1-1401氨基酸）。 <ol><li>使用pSG5-hPXR 8质粒作为PXR LBD模板，使用PCMX-SRC1质粒为SRC-1的模板。 </li><li>解冻PCR SuperMix，DNA模板和引物（见材料），让他们在冰上。 </li><li>加0.25微克/微升DNA模板1微升，10毫引物对2微升各自的PCR SuperMix 45微升，并添加H 2 O至使总体积为50微升。 </li><li>设置PCR在94?...

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在我们修改的酵母双杂交实验中，我们已经确定了对PXR 6酮康唑相互作用重要的残留物。因为SRC-1是共活化剂（和克隆到pGADNot矢量），我们还测试了SRC-1是否能当克隆到PSH载体系统激活lacZ的表达，以及是否会改变活动模式和/或影响的泄漏酵母双杂交实验。利用我们在ERG3Δ/ ERG11Δ酵母进行双杂交试验我们重新设计的质粒。和以前一样，我们表明lacZ基因表达（蓝色菌落）也诱导ERG3Δ...

在酵母双杂交（Y2H）法被广泛地用于发现蛋白质-蛋白质相互作用以及最近用于发现新颖的小分子干扰蛋白质-蛋白质相互作用复合体7，8，9，10，11。然而，这种分析的传统方法中，用于药物发现的或“命中”，不允许用于检测的化学品化合物的变构相互作用的残基中的蛋白-蛋白的表面，即改变仍然交互，并允许改变的残基11的询问时。事实上，这样的方法（次），如果制定可行，将使为别构相互作用的残基的蛋白质 - 蛋白质相互作用破坏关键的高通量评价一个听话的酵母系统。在药物发现的背景下，最直接的方式来建立化合物的相互作用与蛋白质会涉及结构测定（蛋白-抑制剂复合物如晶化）。这些方法暨bersome，用精心制作的资源和执行的每一个蛋白质结构研究它在技术上并不可行。 听话的基因药物筛选系统已经建立了细菌1，2和类似哺乳动物双杂交等模型系统。然而，这些系统需要最优化和类似Y2H替代系统仍然在药物发现的最测试。有迹象表明，包括灵敏度，并使用单数的方法13的相互作用的可靠性差的限制，然而，一个单一的Y2H测定法可以被修改来回答关于相互作用的残基的具体问题。在核受体的研究领域中，Y2H已被用来定义相互作用蛋白14，然而，这些蛋白质的相互作用很少被用于定义在其中的配体/受体拮抗剂与核受体-蛋白质复合物相互作用的性质。因此，我们的实验室集中精力定义一个方法，尤其是对于受体蛋白质，是不随时服从蛋白质组学为基础的方法，这将发掘新的配体/受体拮抗剂使用基于反向Y2H发现平台交互的残留物。 基于我们以前发现，酮康唑破坏PXR及其活化剂SRC-1，我们开发了一种新的反向酵母双杂交系统，使我们能够对PXR 6定义和查询酮康唑相互作用的残留物。我们的方法是基于酵母的GAL4的蛋白质，它由负责DNA结合和转录激活结构域可分离的属性。的PXR LBD蛋白被表达为融合到LexA的DNA结合结构域（DNA-BD），而全长共活化剂的SRC-1（类固醇受体共激活因子1）的蛋白质表达为融合至GAL4激活结构域（AD ）。 PXR和SRC-1融合蛋白之间的相互作用导致的含有报告基因的β-Lac的Ž已整合到酵母染色体组的GAL4结合位点的转录活化Ë。酮康唑，一个PXR受体拮抗剂，扰乱PXR和SRC-1相互作用15，16，17，我们可以检测PXR的相互作用和SRC-1的染色上筛选菌落用于X-gal的活动之后的存在或不存在酮康唑。 Y2H的原理图示于图1和实验过程总结于图2。

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Yeast Strain CTY10-5d erg3&#916;/erg11&#916;</td>
			<td>Our lab</td>
			<td></td>
			<td>CTY10-5d yeast was double knocked out ERG3 and ERG11 (erg3&#916;/erg11&#916;) genes6 .</td>
		</tr>
		<tr>
			<td>YPD Growth Medium</td>
			<td>BD Biosciences</td>
			<td>630409</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Yeast Nitrogen Base (YNB) w/o Amino Acids and Ammonium Sulfate</td>
			<td>BD Biosciences</td>
			<td>233520</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD Biosciences</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-His/-Leu Complete Supplement Mixture</td>
			<td>MP Biomedicals</td>
			<td>4250-412</td>
			<td></td>
		</tr>
		<tr>
			<td>ONPG (o-Nitrophenyl &#914;-D- Galactopyranoside).</td>
			<td>Sigma-Aldrich</td>
			<td>N1127</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>Luria Broth (LB)</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Gal</td>
			<td>Fisher</td>
			<td>BP-1615</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicated Salmon Sperm DNA boiled (10 mg/ml)</td>
			<td>Life Technology</td>
			<td>156-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Acros Organics</td>
			<td>61177</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketoconazole</td>
			<td>Sigma-Aldrich</td>
			<td>K1003</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide</td>
			<td>Acros Organics</td>
			<td>326871000</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>L4158</td>
			<td></td>
		</tr>
		<tr>
			<td>50% PEG-3350 solution, filter-sterilized</td>
			<td>Sigma-Aldrich</td>
			<td>P-3640</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose Membrane</td>
			<td>Whatman</td>
			<td>10402091</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Petri Dish</td>
			<td>Fisher</td>
			<td>875712</td>
			<td></td>
		</tr>
		<tr>
		</tr>
	</tbody>
</table>

<table border="1">
	<tbody>
		<tr>
			<td>5&#39;-ACCGGATCCCGATGAAGA AGGAGATGATCATGTCC-3&#39;</td>
			<td>our lab</td>
			<td></td>
			<td>PXR LBD forward primer for pSH2-1</td>
		</tr>
		<tr>
			<td>5&#39;-AGAGTCGACTCAGCTA CCTGTGATGCC -3&#39;</td>
			<td>our lab</td>
			<td></td>
			<td>PXR LBD reverse primer for pSH2-1</td>
		</tr>
		<tr>
			<td>5&#39;-TATAGC GGCCGCATGAGTG GCCTCGGGGACAGTTCATCC -3&#39;</td>
			<td>our lab</td>
			<td></td>
			<td>SRC-1 forward primer for pGADNOT</td>
		</tr>
		<tr>
			<td>5&#39;-GCGGTCGACTTATTCAGTCA GTAGCTG -3&#39;</td>
			<td>our lab</td>
			<td></td>
			<td>SRC-1 reverse primer for pGADNOT</td>
		</tr>
		<tr>
			<td>Platinum PCR Supermix</td>
			<td>Invitrogen</td>
			<td>11306-016</td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>our lab</td>
			<td>R0136</td>
			<td></td>
		</tr>
		<tr>
			<td>SalI</td>
			<td>our lab</td>
			<td>R0138</td>
			<td></td>
		</tr>
		<tr>
			<td>NotI</td>
			<td>our lab</td>
			<td>R0189</td>
			<td></td>
		</tr>
	</tbody>
</table>

reverse yeast two-hybrid system to identify mammalian nuclear receptor residues that interact with ligands and/or antagonists

由于药物代谢和炎症反应的重要调节器，孕烷X受体（PXR），它在疾病的病理生理过程中起重要作用联系起来代谢和炎症（ 如脂肪肝）1,2。出现了在激动剂配体为PXR的识别很大的进展，但是，也有类药拮抗剂和PXR 3,4,5其结合位点的有限的描述。一个关键的障碍已经无法有效地净化全长蛋白与拮抗剂的结构研究尽管PXR克隆和特征1998。我们实验室开发了一种新的高通量酵母基于双杂交法来定义的拮抗剂，酮康唑的，上PXR 6结合的残基。我们的方法包括创建突变库，将营救的单一突变对PXR的AF-2面有望与酮康唑交互的效果。事态抢救或“功能获得的”塞科第二突变可制成使得关于酮康唑对PXR的遗传相互作用和表面残基的结论是可行的。因此，我们开发PXR突变体及其共激活因子，SRC-1相互作用的高通量双杂交酵母屏幕。使用这种方法，其中的酵母进行了修改，以适应抗真菌药，酮康唑的研究中，我们能够证明对PXR的特定突变富集的克隆不能结合到酮康唑。通过反向逻辑，我们的结论是原来的残留物是直接的互动残酮康唑。此法是一种新型的，听话的基因检测，以屏幕上的核受体拮抗剂的表面结合位点。该测定法可以适用于任何药物，无论其细胞毒性潜力的酵母以及细胞蛋白（s）表示，不能使用标准结构生物学或基于蛋白质组学方法，研究。潜在的缺陷包括解释数据的（互补的方法很有用），可靠ANCE单Y2H的方法，在处理酵母或进行酵母双杂交试验的专业知识，并检测优化。

我们进行了一个实验，看看我们是否可以检测PXR和类固醇受体共激活因子-1（SRC-1）的协会的比色读数。由于酵母具有显著甾醇的生产，它先前已经表明，在酵母中的lacZ的表达，而不需要额外的外源性配体所诱导。我们发现，lacZ的表达（蓝色菌落）也感应出的酵母菌株转化的PXR和SRC-1，但是，没有诱导LacZ的表达（白色菌落）在酵母转化的空载体，PXR的或单独的SRC-1 （ 图3）。以确定酮...

Watch this Scientific Journal Video about Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists at JoVE.com

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

酮康唑结合并拮抗孕烷X受体（PXR）激活。 PXR突变体的酵母高通量筛选定义一个独特的区域为酮康唑约束力。这种酵母为基础的遗传学方法发现与具有表面结合位点相关联的配体小说核受体相互作用。

反向酵母双杂交系统，以确定哺乳动物核受体残留的互动与配体和/或拮抗剂

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Research

JoVE Journal

Biology

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

蛋白质组学鉴定蛋白质，P2X2配体门控的阳离子通道交互

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

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 ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

在哺乳动物细胞通过显微注射mRNA的核出口动力学分析

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

酵母核内体系统的超微结构分析的纳米金标记

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

应用酵母雌激素筛检优选本科教学实验室的个人护理产品中的雌激素配体

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine ...

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

一个简单的荧光记者的检测方法, 用于识别蓖麻毒素所需的细胞成分 a 链 (贸易协定) 贩运酵母

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...