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

Vi takker Fabrice Agou og Alix Boucharlat fra Chemogenomic and Biological Screening Platform, Center for Technological Resources and Research (C2RT) ved Institut Pasteur Paris for nyttige forslag til protokollen, alle medlemmer af BGPB-enheden for støtte og videnskabelige diskussioner og Simon Legood for kritisk læsning af manuskriptet. Arbejdet blev finansieret af Global Care Initiatives fra Institute Carnot Infectious Diseases og Institute Carnot Microbes and Health (15 CARN 0017-01 og 16 CARN 0023-01).

1. Enzym- og substratpræparat
<ol><li>Oprensning af enzym
	<ol><li>Fremstil og rens Lnt-enzym fra opløste membraner i vaskemiddel, som tidligere beskrevet 4,8. Kort fortalt induceres ekspression af lnt-strep-genet, der koder for Lnt med et C-terminal Strep-tag, ved OD600 på 0,6 med vandfri tetracyclin (200 ng/ml) ved 37 °C i 16 timer.</li>
		<li>Høst celler ved centrifugering ved 4.000 x g i 10 min og kassér s...

<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>

Protokollen for Lnt-analysen, der er beskrevet her, baseret på fluorescensdetektion af det triacylerede produkt, er følsom og reproducerbar. Den specifikke og effektive binding af biotin til streptavidin er et centralt element i analysen. Alkyne-POPE-substrat, der er tilbage efter afslutningen af Lnt-reaktionen, er også fluorescerende mærket med FAM, men fjernes effektivt efter binding på streptavitinpladerne ved flere vasketrin. Desuden påvirker tilføjelsen af DMSO ikke Lnt-aktiviteten og har ingen indflydelse p?...

Bakterielle lipoproteiner er kendetegnet ved kovalent bundne fedtsyrer ved deres amino-termini, hvorigennem de er forankret i membraner 1,2. Den modne del af proteinet er meget forskelligartet i struktur og funktion, hvilket forklarer lipoproteinernes rolle i forskellige biologiske processer i bakteriecellekuverten.
Lipoproteiner modificeres af phospholipidafledte fedtsyrer efter indsættelse i den cytoplasmatiske membran. Prolipoproteinerne indeholder et signaturmotiv, lipoboxen, som indeholder en invariant cysteinrest, der bliver acyleret og den første aminosyre i det modne protein. Det første trin i denne vej katalyseres af prolipoprotein phosphatidylglycerol::d iacylglyceryltransferase (Lgt), som overfører diacylglycerylgruppen fra phosphatidylglycerol til prolipoproteinet via en thioetherforbindelse mellem diacylglyceryl og cystein. Signalpeptidase II (Lsp) spalter signalpeptidet fra diacylglycerylprolipoprotein, hvilket resulterer i et apolipoprotein, der er forankret i membranen gennem dets diacylglyceryldel. Det tredje og sidste trin katalyseres af apolipoprotein N-acyltransferase (Lnt), som tilføjer en fedtsyre fra sn-1-positionen af phospholipid på apolipoprotein, hvilket resulterer i triacyleret modent lipoprotein (figur 1)3. Lnt-reaktionen er en to-trins pingpong-reaktion, hvor der dannes et stabilt thioester-acylenzymmellemprodukt. Lysophospholipidbiproduktet frigives før acylationen af apolipoproteinsubstratet i reaktionens andet trin.
Fosfolipidsubstratspecificiteten bestemmes i et Lnt-assay baseret på mobilitetsskiftet af N-acyl diacylglycerylpeptid på en høj procentdel Tris-Tricine Urea SDS-SIDE4. Fosfolipider med små polære hovedgrupper, mættet [sn-1] og umættet [sn-2], var foretrukne substrater 4. Gelskiftassayet er ikke egnet til omfattende kinetiske undersøgelser af apolipoprotein N-acyltransferase eller for HTS til at identificere hæmmende molekyler. Klikkemi ved hjælp af alkynfedtsyrer er med succes blevet brugt til at studere lipoproteinmodifikation i bakterier5 og fedtsyremetabolisme i eukaryoter6. For nylig blev der rapporteret om et in vitro-assay af Ras palmitoylation for at identificere hæmmere7.
I den her beskrevne metode inkuberes oprenset aktiv Lnt i vaskemiddel med substrater i blandede miceller til dannelse af alkyntriacyleret peptid, der efterfølgende detekteres ved fluorescensspektrometri.

<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

Lipoproteiner fra proteobakterier modificeres posttranslationelt af fedtsyrer afledt af membranphospholipider ved virkningen af tre integrerede membranenzymer, hvilket resulterer i triacylaterede proteiner. Det første trin i lipoproteinmodifikationsvejen involverer overførsel af en diacylglycerylgruppe fra phosphatidylglycerol til prolipoproteinet, hvilket resulterer i diacylglycerylprolipoprotein. I det andet trin spaltes signalpeptidet af prolipoprotein og danner et apolipoprotein, som igen modificeres af en tredje fedtsyre afledt af et phospholipid. Dette sidste trin katalyseres af apolipoprotein N-acyltransferase (Lnt). Lipoproteinmodifikationsvejen er afgørende i de fleste γ-proteobakterier, hvilket gør det til et potentielt mål for udviklingen af nye antibakterielle midler. Beskrevet her er et følsomt assay for Lnt, der er kompatibelt med high-throughput screening af små hæmmende molekyler. Enzymet og substraterne er membranindlejrede molekyler; Derfor er udviklingen af en in vitro-test ikke ligetil. Dette omfatter oprensning af det aktive enzym i nærværelse af vaskemiddel, tilgængeligheden af alkyn-phospholipider og diacylglycerylpeptidsubstrater og reaktionsbetingelserne i blandede miceller. For at bruge aktivitetstesten i en HTS-opsætning (High-Throughput Screening) foretrækkes direkte udlæsning af reaktionsproduktet frem for koblede enzymatiske reaktioner. I dette fluorometriske enzymassay gengives alkyntriacyleret peptidprodukt fluorescerende gennem en klikkemireaktion og detekteres i et multiwell-pladeformat. Denne metode kan anvendes til andre acyltransferaser, der anvender fedtsyreholdige substrater, herunder phospholipider og acyl-CoA.

I Lnt-reaktionen overføres sn-1-fedtsyren fra phospholipider til et diacylglycerylpeptid, hvilket resulterer i modent triacyleret peptid8. In vitro Lnt-assayet, der er beskrevet her, er designet til at anvende phospholipider indeholdende en alkynfedtsyre (alkyn-POPE) og FSL-1-biotin som substrater, hvilket resulterer i dannelsen af alkyn-FSL-1-biotin. Ved en klikkemireaktion med azido-FAM skal dette produkt fluorescerende mærkes og detekteres ved fluorescensspektrometri (<strong class="...

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

Præsenteret her er et følsomt fluorescensassay til overvågning af apolipoprotein N-acyltransferaseaktivitet ved anvendelse af diacylglycerylpeptid og alkyn-phospholipider som substrater med klikkemi.

Klikkemibaseret fluorometrisk assay for apolipoprotein N-acyltransferase fra enzymkarakterisering til screening med høj kapacitet

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