Name: method for identifying small molecule inhibitors of the protein-protein interaction between hcn1 and trip8b
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This work was supported by National Institutes of Health Grant R21MH104471 and R01MH106511 (D.M.C.), Brain Research Foundation SG 2012-01 (D.M.C.), Northwestern University Clinical and Translational Sciences Institute 8UL1TR000150 (Y.H.), Chicago Biomedical Consortium HTS-004 (Y.H. and D.M.C.), and National Institutes of Health Grant 2T32MH067564 (K.L.). A part of this work was performed by the Northwestern University Medicinal and Synthetic Chemistry Core (ChemCore) at the Center for Molecular Innovation and Drug Discovery (CMIDD), which is funded by the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust and Cancer Center Support Grant P30 CA060553 from the National Cancer Institute awarded to the Robert H. Lurie Comprehensive Cancer Center. The high throughput screen work was performed in the High Throughput Analysis Laboratory which is also a core facility of the Robert H. Lurie Comprehensive Cancer Center.

1. TRIP8b（241-602）蛋白的纯化<ol><li>变换含TRIP8b（241-602）中的细菌蛋白表达载体pGS21 3到感受态质粒大肠杆菌用于根据制造商的说明蛋白的表达。板300微升上卢里亚肉汤（LB）文化 - 琼脂用5微克/毫升氯霉素和氨苄西林。孵育在37℃的板16小时。 </li><li>第二天，挑取单个菌落接种50毫升LB用50微克/毫升氯霉素和氨苄西林。孵育在37℃下培养16小时（振荡）。 </li><li> 50ml培养添加到1...

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因为其在MDD 24的治疗目标电位的，出现了在拮抗HCN通道功能在中枢神经系统4药理学方法相当大的兴趣。然而，这些努力已经被HCN通道在心脏起搏中的重要作用和心律不齐25的风险停滞不前。我们推断，破坏HCN和其大脑特定辅助亚基，TRIP8b 8之间的相互作用可能是足以产生，而不会影响心脏HCN通道3抗抑郁样作用。这一假说是由缺乏的小鼠表现出TRIP8b抗抑郁样...

超极化激活环核苷酸门控（HCN）频道在心脏和中枢神经系统，其中它们起到调节膜兴奋1中起重要作用表示。 HCN通道有牵连抑郁症（MDD）2，这导致几个组提议限制性HCN通道功能的药理学可以有效地作为一种新型治疗MDD 3的发病机制。然而，直接针对HCN通道是因为它们在心脏动作电位4重要的作用并不可行。伊伐布雷定，唯一的FDA批准的HCN通道拮抗剂，用于心脏衰竭，以产生一个心动过缓作用5的治疗。因此，有必要为限制HCN通道在中枢神经系统专门功能的药理学试剂。 Rab8b相互作用蛋白（TRIP8b）三十四肽重复含，是一种大脑特定FIC HCN通道控制HCN通道6,7的表面表达和定位的辅助亚基。 TRIP8b基因敲除导致而不影响心脏HCN 8表达降低大脑HCN通道7。有趣的是，TRIP8b基因敲除小鼠花强迫游泳任务，尾吊任务7，两种常用的筛选试验的抗抑郁疗效9-11时间少动。这些结果表明，而不是直接靶向HCN通道与HCN通道功能的小分子拮抗剂，扰乱TRIP8b和HCN之间的相互作用可以是足以产生抗抑郁样行为。 TRIP8b结合氰化氢在两个不同的结合位点。 HCN的环核苷酸结合结构域（CNBD）与TRIP8b位于N端的到的TRIP8b 12,13的TPR结构域的保守结构域相互作用。虽然所涉及在此CNBD的残相互作用已经映射14，即所涉及的TRIP8b的区域还没有被缩小之外的80个氨基酸的片段13。 TRIP8b的三十四肽重复（TPR）结构域和HCN的C末端三肽（&#39;SNL&#39;在HCN1，HCN2和HCN4，而是“ANM”在HCN3）3,12之间发生的第二交互作用。本C尾相互作用的最近解析晶体结构15揭示实质结构相似性的过氧化物酶体导入受体之间的相互作用，peroxin 5（PEX5），和其相互作用伙伴，含有1型的过氧化物酶体靶向序列（PTS1）16。 虽然两者相互作用位点所必需的HCN通道功能，TRIP8b的TPR结构域和HCN1的C-末端三肽之间的相互作用作为主导结合位点和调节HCN表面表达。因此，这种相互作用被选为本研究中的定位点。为原稿，当提及到TRIP8b和HCN之间的相互作用制成的其余部分，它是这种相互作用正在提及。这种相互作用是由TRIP8b的对应于含有它的保守C端结合的HCN的C-末端尾部（残基的第1a-4同种型TRIP8b的241-602）3所需的TPR结构域的高可溶性片段概括。 为了开发高通量筛选，以确定能够破坏这种互动的小分子，荧光偏振（FP）为基础的测定采用17。荧光偏振是基于与偏振光，并测量发射的荧光18的偏振度的荧光团标记的配体的激励。在结合伴侣的存在，所述荧光配体的旋转运动被约束和偏振光发射19。在没有一个结合伴侣，T他的配位体的旋转运动导致去极化光的发射。 在封闭的协议，提出了N末端标记（的6xHis）TRIP8b使用镍氮基三乙酸（的Ni-NTA）珠粒纯化（241-602）的方法。一个类似的协议是用来净化该协议的步骤7中使用的谷胱甘肽-S-转移酶（GST）-taggedçHCN1端40个氨基酸（HCN1 C40）。出于空间考虑，省略该步骤的详细描述。 在步骤2到协议的7，高通量筛选工作流提出了（ 见图1）。蛋白质-蛋白质相互作用的高通量筛选出了名的困难的目标，并建议读者去寻找关于这个专题的20个额外的资源。 步骤2和步骤3表征纯化TRIP8b（241-602） 的体外构建亲和力FO岭异硫氰酸荧光素（FITC）-tagged对应HCN1（HCN1 FITC）的C-末端尾部11个氨基酸的肽。基于所述TRIP8b-HCN络合物15的晶体结构该11氨基酸片段是足以产生与TRIP8b（241-602）的结合。在步骤2中，相互作用的K个d由滴定TRIP8b（241-602）插入HCN1 FITC的固定浓度测定。在步骤3，在步骤2中使用的氰化氢肽的未标记的版本滴定到两个TRIP8b（241-602）和HCN1 FITC的固定浓度如果FITC标记具有结合干扰检查。这些实验都是在选择高通量筛选使用TRIP8b（241-602）和HCN1 FITC的适当浓度是必不可少的。 高通量筛选的前提是能够扰乱TRIP8b（241-602）和HCN1 FITC之间的相互作用将产生一个分解的小分子rease在偏振光。在步骤4中，测定的Z因子计算21 TRIP8b（241-602）和HCN1 FITC的给定浓度，以确保该测定是适当的高通量筛选（步骤5）。步骤6和7是验证实验，以确认在初级高通量筛选中鉴定的命中通过破坏TRIP8b（241-602）和HCN1 FITC之间，而不是通过非特异性机制的交互作用。在步骤6中，carboxytetramethylrhodamine（TAMRA）标记的HCN1肽（HCN1 TAMRA）是在其它方面相同的荧光偏振测定法用于筛选危及使用FITC标记的FP试验的荧光化合物。步骤7使用较大HCN1 C端片段（HCN1 C40）并采用基于珠子的亲近测定，这是基于从供体珠子单线态氧的“隧道”由相互作用的蛋白质带到彼此接近的受体珠22。

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			<td>Ni-NTA agarose&#160;</td>
			<td>Qiagen</td>
			<td>30210</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis cassette&#160;</td>
			<td>ThermoFisher</td>
			<td>66456</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl b-D-1-thiogalactopyranoside&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>I5502-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>384 Well Black plate&#160;</td>
			<td>Corning</td>
			<td>3820</td>
			<td></td>
		</tr>
		<tr>
			<td>Proxiplate&#160;</td>
			<td>Perkin-Elmer&#160;</td>
			<td>6008289</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GST Acceptor beads&#160;</td>
			<td>Perkin-Elmer&#160;</td>
			<td>6760603C</td>
			<td></td>
		</tr>
		<tr>
			<td>NiChelate</td>
			<td>Perkin-Elmer&#160;</td>
			<td>AS101D</td>
			<td></td>
		</tr>
		<tr>
			<td>pGS21-a</td>
			<td>Genscript</td>
			<td>SD0121</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>10837091001</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Kit</td>
			<td>ThermoFisher</td>
			<td>23200</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein concentrator</td>
			<td>ThermoFisher</td>
			<td>88527</td>
			<td></td>
		</tr>
		<tr>
			<td>Perkin Elmer Enspire Multimode Plate reader</td>
			<td>Perkin-Elmer&#160;</td>
			<td>#2300-001M</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 (DE3) Competent Cells</td>
			<td>Agilent</td>
			<td>200131</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for identifying small molecule inhibitors of the protein-protein interaction between hcn1 and trip8b

超极化激活环核苷酸门控（HCN）频道整个大脑，在那里它们起作用来调节神经元的兴奋遍在表达。海马CA1区锥体细胞这些通道的亚细胞分布由三十四肽含有重复-Rab8b相互作用蛋白（TRIP8b），辅助亚基调节。 HCN的遗传敲除孔形成亚基或TRIP8b，既导致增加抗抑郁样行为，这表明限制HCN通道的功能可以是作为用于抑郁症（MDD）的治疗是有用的。尽管显著治疗意义，HCN通道也都在心脏，在那里它们调节节律性表达。规避与阻断心脏HCN通道相关联的脱靶的问题，我们的实验室最近提出靶向为了专门破坏HCN通道功能在大脑HCN和TRIP8b之间的蛋白质 - 蛋白质相互作用。TRIP8b结合HCN孔形成在两个不同的相互作用位点亚基，尽管这里的重点是TRIP8b的三十四肽重复（TPR）结构域和HCN1的C-末端尾部之间的相互作用。在这个协议中，一种方法的用于纯化TRIP8b并执行高通量筛选，以鉴定HCN和TRIP8b之间的相互作用的小分子抑制剂的扩展的说明中，进行说明。用于高通量筛选的方法，利用荧光偏振（FP）为基础的测定以监测大TRIP8b片段荧光团标记十一结合氨基酸对应于HCN1 C端尾酸肽。基于在发射的光的偏振的变化，以确定该方法允许&#39;命中&#39;的化合物。然后进行验证试验，以确保&#39;打&#39;的化合物不是人为的。

以避免与我们先前的出版物的版权问题，一个TAMRA标记的探针HCN1 TAMRA用于生成图2和3。需要注意的是，这种替代并没有使在结果有明显的差别，并且这些协议是相同的上述与HCN1 FITC。来评估与HCN1 TAMRA，TRIP8b（241-602）的相互作用滴定成使用在步骤2（ 图2）中概述的协议HCN1 TAMRA的固定浓度。接着，进行在...

Watch this Scientific Journal Video about Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b at JoVE.com

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

HCN通道和其辅助亚基之间的相互作用已被确定为在抑郁症的治疗靶点。这里，用于识别这种蛋白质 - 蛋白质相互作用的小分子抑制剂荧光基于偏振的方法，提出。

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

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

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

通过免疫共沉淀在核质馏分蛋白质相互作用的可视化和<em&gt;原位</em&gt;接近结扎分析

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

用生物层干涉法捕获小分子离子通道蛋白的相互作用动力学

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

滴定 ELISA 法测定受体配体相互作用的离解常数

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

单分子水平标记均匀性的全蛋白质定量

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

用于筛选小分子抑制剂的NADH耦合ATPase分析的半高通量自适应

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

用利甘德分析（DRACALA）的微分径向毛细管作用识别小配体的结合蛋白

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...