Name: click-chemistry based fluorometric assay for apolipoprotein n-acyltransferase from enzyme characterization to high-throughput screening
Uploaded: 2020-05-13T13:30:00
Description: Watch this Scientific Journal Video about Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening at JoVE.com

我们感谢来自化学基因组和生物筛选平台，巴黎巴斯德研究所技术资源和研究中心（C2RT）的Fabrice Goou和Alix Boucharlat对协议的有用建议，BGPB部门的所有成员的支持和科学讨论，以及Simon Legood对手稿的批判性阅读。这项工作由卡诺传染病研究所和卡诺微生物与健康研究所的全球护理倡议资助（15 CARN 0017-01和16 CARN 0023-01）。

1. 酶和底物制备
<ol><li>酶的纯化
	<ol><li>如前所述，从洗涤剂溶解膜中产生和纯化Lnt酶4，8。简而言之，诱导lnt-strep基因的表达，用C末端链球菌标签编码Lnt，OD600为0.6，无水四环素（200ng / mL）在37°C下16小时。</li>
		<li>通过以4，000× g 离心10分钟收获细胞并弃去上清液。</li>
		<li>将细胞沉淀重悬于缓冲液WA（20 mM Tris-HCl pH...

<ol>
	<li>Buddelmeijer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+mechanism+of+bacterial+lipoprotein+modification--how,+when+and+why.">The molecular mechanism of bacterial lipoprotein modification--how, when and why.</a> FEMS Microbiology Reviews. 39 (2), 246-261 (2015).</li><li>Kovacs-Simon, A., Titball, R. W., Michell, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoproteins+of+bacterial+pathogens.">Lipoproteins of bacterial pathogens.</a> Infections and Immunity. 79 (2), 548-561 (2010).</li><li>Jackowski, S., Rock, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+fatty+acids+from+the+1-position+of+phosphatidylethanolamine+to+the+major+outer+membrane+lipoprotein+of+Escherichia+coli.">Transfer of fatty acids from the 1-position of phosphatidylethanolamine to the major outer membrane lipoprotein of Escherichia coli.</a> Journal of Biological Chemistry. 261, 11328-11333 (1986).</li><li>Hillmann, F., Argentini, M., Buddelmeijer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+and+phospholipid+specificity+of+apolipoprotein+N-acyltransferase.">Kinetics and phospholipid specificity of apolipoprotein N-acyltransferase.</a> Journal of Biological Chemistry. 286 (32), 27936-27946 (2011).</li><li>Rangan, K. J., Yang, Y. Y., Charron, G., Hang, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Visualization+and+Large-Scale+Profiling+of+Bacterial+Lipoproteins+with+Chemical+Reporters.">Rapid Visualization and Large-Scale Profiling of Bacterial Lipoproteins with Chemical Reporters.</a> Journal of American Chemical Society. 132 (31), 10628-10629 (2010).</li><li>Thiele, C., 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=Tracing+fatty+acid+metabolism+by+click-chemistry.">Tracing fatty acid metabolism by click-chemistry.</a> ACS Chemical Biology. 7 (12), 2004-2011 (2012).</li><li>Ganesan, L., Shieh, P., Bertozzi, C. R., Levental, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click-Chemistry+Based+High+Throughput+Screening+Platform+for+Modulators+of+Ras+Palmitoylation.">Click-Chemistry Based High Throughput Screening Platform for Modulators of Ras Palmitoylation.</a> Science Reports. 7, 41147 (2017).</li><li>Nozeret, K., Boucharlat, A., Agou, F., Buddelmeijer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+fluorescence-based+assay+to+monitor+enzymatic+activity+of+the+essential+integral+membrane+protein+Apolipoprotein+N-acyltransferase+(Lnt).">A sensitive fluorescence-based assay to monitor enzymatic activity of the essential integral membrane protein Apolipoprotein N-acyltransferase (Lnt).</a> Science Reports. 9 (1), 15978 (2019).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> Journal of Biomolecules Screening. 4 (2), 67-73 (1999).</li><li>Mao, G., 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=Crystal+structure+of+E.+coli+lipoprotein+diacylglyceryl+transferase.">Crystal structure of E. coli lipoprotein diacylglyceryl transferase.</a> Nature Communication. 7, 10198 (2016).</li><li>Vogeley, L., 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=Structural+basis+of+lipoprotein+signal+peptidase+II+action+and+inhibition+by+the+antibiotic+globomycin.">Structural basis of lipoprotein signal peptidase II action and inhibition by the antibiotic globomycin.</a> Science. 351 (6275), 876-880 (2016).</li><li>Wiktor, M., 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=Structural+insights+into+the+mechanism+of+the+membrane+integral+N-acyltransferase+step+in+bacterial+lipoprotein+synthesis.">Structural insights into the mechanism of the membrane integral N-acyltransferase step in bacterial lipoprotein synthesis.</a> Nature Communication. 8, 15952 (2017).</li><li>Noland, C. L., 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=Structural+insights+into+lipoprotein+N-acylation+by+Escherichia+coli+apolipoprotein+N-acyltransferase.">Structural insights into lipoprotein N-acylation by Escherichia coli apolipoprotein N-acyltransferase.</a> Proceedings of the National Academy of Sciences USA. 114 (30), 6044-6053 (2017).</li><li>Lu, G., 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=Crystal+structure+of+E.+coli+apolipoprotein+N-acyl+transferase.">Crystal structure of E. coli apolipoprotein N-acyl transferase.</a> Nature Communication. 8, 15948 (2017).</li></ol>

此处描述的Lnt测定方案基于三酰化产物的荧光检测，具有灵敏度和可重现性。生物素与链霉亲和素的特异性和有效结合是测定中的关键要素。Lnt反应完成后留下的炔烃-POPE底物也用FAM进行荧光标记，但在通过多个洗涤步骤结合到链霉亲和素板上后被有效去除。此外，添加DMSO不会影响Lnt活性，对测定没有影响。底物和酶都具有高度亲脂性，需要在混合胶束中进行反应条件。依赖可溶性酶和底物进行?...

细菌脂蛋白的特征在于其氨基末端的共价结合脂肪酸，通过这些脂肪酸将它们锚定在膜1，2中。蛋白质的成熟部分在结构和功能上高度多样化，从而解释了脂蛋白在细菌细胞包膜中各种生物过程中的作用。
脂蛋白在插入细胞质膜后被磷脂衍生的脂肪酸修饰。前脂肪蛋白包含一个标志性基序，即脂盒，它包含一个不变的半胱氨酸残基，该残基变成酰化和成熟蛋白质中的第一个氨基酸。该途径的第一步是由前脂蛋白磷脂酰甘油：:d iacyl甘油转移酶（Lgt）催化的，其通过二酰基甘油和半胱氨酸之间的硫醚连接将二酰基甘油基团从磷脂酰甘油转移到前脂蛋白上。信号肽酶II（Lsp）从二酰基甘油前脂蛋白中切割信号肽，产生载脂蛋白，该载脂蛋白通过其二酰基甘油部分锚定在膜中。第三步也是最后一步由载脂蛋白N-酰基转移酶（Lnt）催化，该酶将脂肪酸从磷脂的 sn-1位置添加到载脂蛋白上，从而产生三酰化成熟脂蛋白（图1）3。Lnt反应是两步乒乓反应，其中形成稳定的硫酯酰基酶中间体。溶血磷脂副产物在反应的第二步载脂蛋白底物酰化之前释放。
磷脂底物特异性在Lnt测定中测定，基于N-酰基二酰基甘油肽在高百分比Tris-Tricine尿素SDS-PAGE 4上的迁移率位移。具有小极性头基饱和[sn-1]和非饱和[sn-2]的磷脂是优选底物4。凝胶移位测定不适用于载脂蛋白N-酰基转移酶的广泛动力学研究，也不适用于HTS鉴定抑制分子。使用炔烃脂肪酸的点击化学已成功用于研究细菌中的脂蛋白修饰5和真核生物6中的脂肪酸代谢。最近，据报道，Ras 棕榈酰化的体外测定可鉴定抑制剂7。
在这里描述的方法中，洗涤剂中纯化的活性Lnt与混合胶束中的底物一起孵育以形成炔基 - 三酰化肽，随后通过荧光光谱法检测。

<table>
	<tbody>
		<tr>
			<td>&#196;kta Purifier FPLC system</td>
			<td>GE Healthcare</td>
			<td>NA</td>
			<td>Purity: NA 
			Lnt purification</td>
		</tr>
		<tr>
			<td>alkyne-POPE</td>
			<td>Avanti Polar Lipids</td>
			<td>900414P</td>
			<td>Purity: &#62;99% 
			Lnt substrate</td>
		</tr>
		<tr>
			<td>Azido-FAM</td>
			<td>Lumiprobe</td>
			<td>A4130</td>
			<td>Purity: 100% (pure) 
			Click reagent</td>
		</tr>
		<tr>
			<td>BioPhotometer Plus</td>
			<td>Eppendorf</td>
			<td>N/A</td>
			<td>Purity: NA 
			OD 600 nm</td>
		</tr>
		<tr>
			<td>BioTek ELX 405 Select plate washer</td>
			<td>BioTek</td>
			<td>N&#176; serie 115800, n&#176; materiel 405 Select</td>
			<td>Purity: NA 
			Wash steps</td>
		</tr>
		<tr>
			<td>biotin-fluorescein</td>
			<td>Sigma</td>
			<td>53608</td>
			<td>Purity: &#8805;90% 
			Fluorescence control</td>
		</tr>
		<tr>
			<td>Copper(II) sulfate pentahydrate</td>
			<td>Sigma</td>
			<td>C3036</td>
			<td>Purity: &#8805;98% 
			Click reagent</td>
		</tr>
		<tr>
			<td>DDM</td>
			<td>Anatrace</td>
			<td>D310A</td>
			<td>Purity: &#8805; 99% &#946;+&#945;; &#60; 15% &#945; 
			Detergent for Lnt purification</td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Invitrogen</td>
			<td>D12435</td>
			<td>Purity: anhydrous 
			Solbilization Click reagent</td>
		</tr>
		<tr>
			<td>Electronic pipet Voyager 8 channels 0.5-12.5 uL</td>
			<td>INTEGRA</td>
			<td>4721</td>
			<td>Purity: NA 
			Handling reagents</td>
		</tr>
		<tr>
			<td>French Pressure Cell</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purity: NA 
			Cell disruption</td>
		</tr>
		<tr>
			<td>FSL-1-biotin</td>
			<td>EMC microcollections</td>
			<td>L7030</td>
			<td>Purity: NA 
			Lnt substrate</td>
		</tr>
		<tr>
			<td>Greiner Bio-One 384-well standard CELLSTAR polystyrene microplate</td>
			<td>Greiner</td>
			<td>781091</td>
			<td>Purity: NA 
			Black with transparent bottom (up or bottom reading)</td>
		</tr>
		<tr>
			<td>Greiner Bio-One 96-well sterile polystyrene plate, high binding</td>
			<td>Greiner</td>
			<td>655097</td>
			<td>Purity: NA 
			Black with transparent bottom (up or bottom reading)</td>
		</tr>
		<tr>
			<td>Microplate reader Infinite M1000 pro</td>
			<td>Tecan</td>
			<td>N/A</td>
			<td>Purity: NA 
			Fluorescence detection</td>
		</tr>
		<tr>
			<td>MTSES (sodium (2-sulfonatoethyl)methanethiosulfonate)</td>
			<td>Anatrace</td>
			<td>S110MT</td>
			<td>Purity: ~100% 
			Thiol specific inhibitor</td>
		</tr>
		<tr>
			<td>Optically Clear Adhesive Seal Sheets</td>
			<td>Thermo Scientific</td>
			<td>AB-1170</td>
			<td>Purity: NA 
			Foil to seal multi-well plate</td>
		</tr>
		<tr>
			<td>Sephacryl S400 HR 16/60 gel filtration column</td>
			<td>GE Healthcare</td>
			<td>GE28-9356-04</td>
			<td>Purity: NA 
			Lnt purification</td>
		</tr>
		<tr>
			<td>StrepTactin Sepharose 50 %</td>
			<td>IBA Biotechnology</td>
			<td>2-1201-010</td>
			<td>Purity: NA 
			Lnt purification</td>
		</tr>
		<tr>
			<td>streptavidin</td>
			<td>Sigma</td>
			<td>S4762-1MG</td>
			<td>Purity: &#8805;13 units/mg protein 
			Biotin binding</td>
		</tr>
		<tr>
			<td>TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine</td>
			<td>Sigma</td>
			<td>678937</td>
			<td>Purity: 97% 
			Click reagent</td>
		</tr>
		<tr>
			<td>TCEP (tris(2-carboxyethyl)phosphine hydrochloride</td>
			<td>Sigma</td>
			<td>75259</td>
			<td>Purity: &#8805;98% 
			Click reagent</td>
		</tr>
		<tr>
			<td>TECAN Infinite F500</td>
			<td>Tecan</td>
			<td>N/A</td>
			<td>Purity: NA 
			Fluorescence detection</td>
		</tr>
		<tr>
			<td>TECAN Infinite M1000 pro</td>
			<td>Tecan</td>
			<td>N/A</td>
			<td>Purity: NA 
			Fluorescence detection</td>
		</tr>
		<tr>
			<td>Thermomixer C</td>
			<td>Eppendorf</td>
			<td>5382000015</td>
			<td>Purity: NA 
			Heated lid</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>93443</td>
			<td>Purity: 10% in H2O 
			Lnt reaction buffer</td>
		</tr>
		<tr>
			<td>Ultra centrifuge</td>
			<td>Beckman LC</td>
			<td>N/A</td>
			<td>Purity: NA 
			Cell fractionation</td>
		</tr>
	</tbody>
</table>

click-chemistry based fluorometric assay for apolipoprotein n-acyltransferase from enzyme characterization to high-throughput screening

来自变形杆菌的脂蛋白在三种整合膜酶的作用下被源自膜磷脂的脂肪酸进行翻译后修饰，从而产生三酰化蛋白。脂蛋白修饰途径的第一步涉及将二酰基甘油基团从磷脂酰甘油转移到前脂蛋白上，从而产生二酰基甘油前脂蛋白。在第二步中，前脂蛋白的信号肽被裂解，形成载脂蛋白，而载脂蛋白又被源自磷脂的第三种脂肪酸修饰。最后一步由载脂蛋白N-酰基转移酶（Lnt）催化。脂蛋白修饰途径在大多数γ变形杆菌中是必不可少的，使其成为开发新型抗菌剂的潜在靶标。这里描述的是Lnt的灵敏测定，它与小抑制分子的高通量筛选兼容。酶和底物是膜嵌入的分子;因此，体外测试的开发并不简单。这包括在洗涤剂存在下纯化活性酶，炔磷脂和二酰基甘油肽底物的可用性，以及混合胶束中的反应条件。此外，为了在高通量筛选（HTS）设置中使用活性测试，直接读取反应产物优于偶联酶促反应。在该荧光酶测定中，炔烃-三酰化肽产物通过点击化学反应呈现荧光，并以多孔板形式进行检测。该方法适用于使用含脂肪酸底物的其他酰基转移酶，包括磷脂和酰基辅酶A。

在Lnt反应中，来自磷脂的 sn-1脂肪酸被转移到二酰基甘油肽上，产生成熟的三酰化肽8。此处描述的体外Lnt测定旨在使用含有炔烃脂肪酸（炔烃-POPE）和FSL-1-生物素的磷脂作为底物，从而形成炔烃-FSL-1-生物素。在与叠氮基-FAM进行点击化学反应时，该产品应具有荧光标记并通过荧光光谱法检测（图2）。
优化反应条件，以在酶标仪中...

Watch this Scientific Journal Video about Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening at JoVE.com

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

这里介绍的是一种灵敏的荧光测定法，用于使用二酰基甘油肽和炔烃磷脂作为底物，通过点击化学来监测载脂蛋白N-酰基转移酶活性。

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