We thank Fabrice Agou and Alix Boucharlat from the Chemogenomic and Biological Screening Platform, Center for Technological Resources and Research (C2RT) at Institut Pasteur Paris for helpful suggestions on the protocol, all members of the BGPB Unit for support and scientific discussions, and Simon Legood for critical reading of the manuscript. Work was funded by Global Care Initiatives of the Institute Carnot Infectious diseases and the Institute Carnot Microbes and Health (15 CARN 0017-01 and 16 CARN 0023-01).

1. Enzyme and substrate preparation
<ol>
	<li>Purification of enzyme
	<ol>
		<li>Produce and purify Lnt enzyme from detergent solubilized membranes as described previously4,8. Briefly, induce expression of the lnt-strep gene, encoding Lnt with a C-terminal Strep tag, at OD600 of 0.6 with anhydrous tetracycline (200 ng/mL) at 37 &#176;C for 16 h.</li>
		<li>Harvest cells by centrifugation at 4,000 x g for 10 min and discard the supernatant.</li>
		<li>Resuspend the cell pellet in buffer WA (20 mM Tris-HCl pH 8, 150 mM NaCl, 0.5 mM EDTA) to 100 OD600 units per mL.</li>
		<li>Break the cells by two passages through a French pressure cell press at 10,000 psi.</li>
		<li>Remove unbroken cells and debris by centrifugation at 13,000 x g for 20 min. Keep the supernatant and discard the pellet.</li>
		<li>Centrifuge the supernatant at 120,000 x g for 60 min and collect the membrane vesicles (translucent brown colored pellet). Discard the supernatant.</li>
		<li>Solubilize the integral membrane proteins from the membrane pellet with 1% (w/v) n-Dodecyl &#946;-D-maltoside (DDM) in buffer WA for 30 min at room temperature (RT). Centrifuge and remove insolubilized material at 120,000 x g for 30 min. Use the supernatant fraction for purification.</li>
		<li>Purify Lnt-strep on an affinity chromatography column (see Table of Materials) and an S400 gel filtration column (see Table of Materials) as described by Nozeret et al.8.</li>
		<li>Store the purified enzyme in buffer WA containing 0.05% DDM and 10% glycerol at -80 &#176;C. 
		NOTE: The enzyme is stable for over 5 years.</li>
	</ol>
	</li>
	<li>Preparation of alkyne-phospholipid and biotinylated Fibroblast Stimulating Ligand (FSL-1-biotin) substrates
	<ol>
		<li>Aliquot 100 &#181;L of custom-synthesized alkyne-POPE (1-hexadec-15-ynoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, see Table of Materials) solubilized in chloroform into 1.5 mL tubes.</li>
		<li>Pierce the tubes with a syringe and evaporate the chloroform in a speed-vac at RT for 2 h. Store the dry phospholipid samples at -20 &#176;C.</li>
		<li>Dissolve alkyne-POPE in 0.1% Triton X-100 at 500 &#181;M prior to use. Add a 3 min sonication step in an ultrasonic water bath at RT to solubilize alkyne-POPE if necessary.</li>
		<li>Resuspend FSL-1-biotin in water at 445 &#181;M. 
		NOTE: Both solutions can be stored at -20 &#176;C for at least 2 months.</li>
	</ol>
	</li>
</ol>
2. Tube assay
NOTE: On Day 1, set up the Lnt reaction in 1.5 mL tubes (step 2.1) and coat 96 well plates with streptavidin (step 2.2).
<ol>
	<li>Preparation of reagent mixture and enzymatic reaction
	<ol>
		<li>For a standard Lnt assay, mix alkyne-POPE (final 50 &#181;M from 500 &#181;M stock) and FSL-1-biotin (final concentration of 50 &#181;M from a 445 &#181;M stock) in Lnt reaction buffer (50 mM Tris-HCl pH 7.2, 150 mM NaCl, 0.1% Triton X-100 containing 1% BSA) at a final volume of 18 &#181;L in 1.5 mL tubes. Perform all conditions in triplicate.</li>
		<li>Sonicate the substrate mixture (prepared in step 2.1.1) for 3 min in an ultrasonic water bath and incubate at 37 &#176;C for 5 min prior to the addition of Lnt enzyme (step 2.1.3). 
		NOTE: MTSES (sodium (2-sulfonatoethyl)methanethiosulfonate), a thiol-specific reagent, inhibits Lnt8. Add 10 mM MTSES (100 mM stock solution in 100% DMSO) to the reaction tubes (step 2.1.1) to be used as negative control samples. Adjust the volume of the Lnt reaction buffer so the total volume is 18 &#181;L.</li>
		<li>Add active (1 ng/&#181;L, corresponding to 17.2 nM) or inactive Lnt (C387S) (2 &#181;L of 10 ng/&#956;L stock in buffer WA containing 0.05% DDM) and mix with the substrates (step 2.1.2) by pipetting up and down.</li>
		<li>Incubate the reaction at 37 &#176;C for 16 h in a thermomixer with heated lid.</li>
	</ol>
	</li>
	<li>Streptavidin coating of 96 well plates
	<ol>
		<li>Prepare a stock solution of streptavidin at 2 mg/mL in H2O. 
		NOTE: This solution can be stored at -20 &#176;C for up to 6 months.</li>
		<li>From the stock solution prepare 10 &#181;g/mL streptavidin in water as a working solution. Add 100 &#181;L of the solution to each well of the 96 well plate. Bind streptavidin to the plate by incubating at 37 &#176;C overnight without a lid to air-dry the wells.</li>
	</ol>
	</li>
</ol>
NOTE: On Day 2 perform click-chemistry (step 2.3), bind Lnt reaction mixture to streptavidin-coated plates (step 2.4), and detect fluorescence and analyze results (step 2.5).
<ol start="3">
	<li>Click-chemistry reaction
	<ol>
		<li>Prepare stock solutions of Azido-FAM (5 mM in DMSO), TCEP (Tris(2-carboxyethyl)phosphine hydrochloride; 50 mM prepared in water), TBTA (Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; 2 mM in tert-butanol:DMSO (4:1)), and CuSO4&#8729;5H2O (50 mM freshly prepared in H2O).</li>
		<li>In the 1.5 mL tubes containing the Lnt reaction mixture prepared in step 2.1.4, perform click-chemistry by adding the reagents in the following order: 0.2 &#181;L of stock Azido-FAM (final concentration 50 &#181;M), 0.4 &#181;L of stock TCEP (final concentration 1 mM), 0.2 &#181;L of stock TBTA (final concentration 0.02 mM).</li>
		<li>Vortex the solution for 5 s. Then add 0.4 &#181;L of stock CuSO4 (final concentration 1 mM). Vortex again for 5 s. Incubate the samples in the dark at RT for 1 h.</li>
	</ol>
	</li>
	<li>Binding of Lnt reaction mixture to streptavidin-coated plates
	<ol>
		<li>Wash the wells of the streptavidin-coated plates after the overnight incubation from step 2.2.2 3x with 200 &#181;L of PBS-T1 (PBS containing 0.05% Tween-20). 
		NOTE: Streptavidin plates can be used immediately or stored dry at 4 &#176;C.</li>
		<li>Transfer 18 &#181;L from the click-chemistry mixture from step 2.3.3 to a well in a 96 well streptavidin-coated plate and add 100 &#181;L of PBS-T1 to bind N-acyl-FSL-1-biotin-FAM product to streptavidin plates.</li>
		<li>Use biotin-fluorescein (0.26 &#181;M from a 3.1 mM stock in DMSO) as positive control for streptavidin binding and fluorescence readout.</li>
		<li>Incubate the plates at RT for 1 h in the dark in a thermomixer, shaking at 300 rpm.</li>
		<li>Perform manual wash steps of the 96 well plates with a multichannel electronic pipette as follows: six washes with 200 &#181;L of PBS-T2 (PBS containing 1% Tween-20), three flushes with a pipette, three washes with 200 &#181;L of PBS, three flushes with a pipette.</li>
	</ol>
	</li>
	<li>Fluorescence detection and analysis
	<ol>
		<li>Add 200 &#181;L of PBS and record the fluorescence at 520 nm in a fluorescence microplate reader. Save results in spreadsheet software. 
		NOTE: Biotin-fluorescein is used as a positive control for streptavidin binding and fluorescence detection at 520 nm. A reproducible readout of 30,000&#8211;40,000 A.U. with 0.26 &#181;M biotin-fluorescein is expected. All samples are analyzed in triplicate, including controls.</li>
		<li>Calculate the standard deviation for each reaction and the calculate P-values for negative controls and positive samples using unpaired t-test using a statistical software.</li>
	</ol>
	</li>
</ol>
3. Multiwell plate assay
NOTE: On Day 1 set up the Lnt reaction in 384 well plates (step 3.1) and coat 384 well plates with streptavidin (step 3.2).
<ol>
	<li>Preparation of reagent mixture and enzymatic reaction 
	NOTE: The quantity of the reagents and enzyme is reduced compared to the tube assay and the Lnt reaction is performed directly in 384 well plates. This allows the use of less material and a reduction of steps that can be further automated.
	<ol>
		<li>Mix the substrates as follows: alkyne-POPE (final concentration 50 &#181;M from 500 &#181;M stock) and FSL-1-biotin (final 50 &#181;M from 500 &#181;M stock) in Lnt reaction buffer (50 mM Tris-HCl pH 7.2, 150 mM NaCl, 0.1% Triton X-100 containing 1% BSA) at a volume of 13.5 &#181;L per reaction per well in a 384 well plate format. Perform all conditions in triplicate. Calculate the total amount of reagents required for the number of reactions to be performed (i.e., 5,184 &#181;L for one 384 well plate).</li>
		<li>Sonicate the substrate mixture for 3 min in an ultrasonic water bath.</li>
		<li>Aliquot 13.5 &#181;L of the substrate mixture per well in a 384 well plate. 
		NOTE: As a negative control, 10 mM of MTSES (625 mM stock solution in 100% DMSO) can be added per well. Adjust the volume of Lnt buffer (step 3.1.1) to reach a final reaction volume of 13.5 &#181;L.</li>
		<li>Incubate at 37 &#176;C for 5 min prior to the addition of Lnt enzyme (step 3.1.5).</li>
		<li>Add 0.5 ng/&#181;L (corresponding to 8.6 nM) active Lnt enzyme, or inactive variant (C387S) (1.5 &#181;L from 5 ng/&#181;L stock in buffer WA containing 0.05% DDM) in Lnt reaction buffer (50 mM Tris-HCl pH 7.2, 150 mM NaCl, 0.1% Triton X-100, 1% BSA) to each well containing the reaction mixture from step 3.1.3. Mix reagents by pipetting up and down. The total reaction volume is 15 &#181;L.</li>
		<li>Seal the plate with plastic foil for multiwell plates.</li>
		<li>Incubate at 37 &#176;C for 16 h in a thermomixer with heated lid.</li>
	</ol>
	</li>
	<li>Streptavidin coating 384 well plates
	<ol>
		<li>Use 75 &#181;L of streptavidin (10 &#181;g/mL in H2O) from step 2.2.2 to coat wells of a 384 well plate. Let streptavidin bind to the plate by incubating at 37 &#176;C overnight without a lid to air-dry the wells.</li>
	</ol>
	</li>
</ol>
NOTE: On Day 2 perform click-chemistry (step 3.3), bind Lnt reaction mixture to streptavidin-coated plates (step 3.4), detect fluorescence, and analyze results (step 3.5).
<ol start="3">
	<li>Click-chemistry reaction
	<ol>
		<li>Prepare stock solutions of Azido-FAM (1.2 mM in DMSO), TCEP (tris(2-carboxyethyl)phosphine hydrochloride; 24 mM prepared in H2O), TBTA (Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; 0.48 mM in tert-butanol:DMSO (4:1)) and CuSO4&#8729;5H2O (24 mM freshly prepared in H2O).</li>
		<li>Combine the three click-chemistry reagents: a mixture of Azido-FAM (final concentration 50 &#181;M), TCEP (1 mM final), and TBTA (0.02 mM final), each at 0.75 &#181;L in final volume of 2.25 &#181;L per well. Calculate the required volume per 384 well plate (i.e., use 864 &#181;L when all wells of the 384 well plate are used). Mix vigorously.</li>
		<li>Add 2.25 &#181;L of the reagent solution per well directly to the completed Lnt reaction in the 384 well plate from step 3.1.7.</li>
		<li>Mix by pipetting up and down with a multichannel electronic pipette.</li>
		<li>Add CuSO4 (0.75 &#181;L from 24 mM stock for a 1 mM final concentration) and mix by pipetting up and down with a multichannel electronic pipette.</li>
		<li>Incubate at RT for 1 h in the dark.</li>
	</ol>
	</li>
	<li>Binding of Lnt reaction mixture to streptavidin-coated 384 well plates
	<ol>
		<li>Wash the wells of the streptavidin-coated plates after the overnight incubation from step 3.2.1 1x with 85 &#181;L PBS-T1.</li>
		<li>Transfer 11 &#181;L of the click-chemistry reaction from each well of the incubation plate from step 3.3.6 to the streptavidin-coated plate from step 3.4.1 to bind the N-acyl-FSL-1-biotin-FAM product to the streptavidin plates.</li>
		<li>Add 64 &#181;L of buffer PBS-T1.</li>
		<li>Include biotin-fluorescein (0.19 &#181;M from a 3.1 mM stock in DMSO) as a control for binding to streptavidin-coated wells.</li>
		<li>Incubate the 384 well plates at RT for 1 h in the dark in a thermomixer, shaking at 300 rpm.</li>
		<li>Wash the plates using an automated plate washer as follows: 10 washes with 85 &#181;L PBS-T2, plates are shaken between washes; 5 washes with 85 &#181;L PBS, plates are shaken between washes.</li>
	</ol>
	</li>
	<li>Fluorescence detection and analysis
	<ol>
		<li>Add PBS (85 &#181;L) and record fluorescence in a fluorescence microplate reader at 535 nm.</li>
		<li>Analyze data as described (step 2.5.2).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The protocol for the Lnt assay described here, based on fluorescence detection of the triacylated product, is sensitive and reproducible. The specific and efficient binding of biotin to streptavidin is a key element in the assay. Alkyne-POPE substrate left after completion of the Lnt reaction is also fluorescently labeled with FAM but is efficiently removed after binding onto the streptavidin plates by multiple wash steps. Furthermore, addition of DMSO does not affect Lnt activity and has no impact on the assay. Both sub...

Bacterial lipoproteins are characterized by covalently bound fatty acids at their amino-termini through which they are anchored into membranes1,2. The mature part of the protein is highly diverse in structure and function, thereby explaining the role of lipoproteins in various biological processes in the bacterial cell envelope.
Lipoproteins are modified by phospholipid-derived fatty acids after insertion into the cytoplasmic membrane. The prolipoproteins contain a signature motif, the lipobox, which contains an invariant cysteine residue that becomes acylated and the first amino acid in the mature protein. The first step of this pathway is catalyzed by prolipoprotein phosphatidylglycerol::diacylglyceryl transferase (Lgt), which transfers the diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein via a thioether link between diacylglyceryl and cysteine. Signal peptidase II (Lsp) cleaves the signal peptide from diacylglyceryl prolipoprotein, resulting in an apolipoprotein that is anchored into the membrane through its diacylglyceryl moiety. The third and last step is catalyzed by apolipoprotein N-acyltransferase (Lnt), which adds a fatty acid from the sn-1 position of phospholipid onto apolipoprotein, resulting in triacylated mature lipoprotein (Figure 1)3. The Lnt reaction is a two-step ping-pong reaction where a stable thioester acyl enzyme intermediate is formed. The lysophospholipid byproduct is released prior to the acylation of the apolipoprotein substrate in the second step of the reaction.
The phospholipid substrate specificity is determined in a Lnt assay based on the mobility shift of N-acyl diacylglyceryl peptide on a high percentage Tris-Tricine Urea SDS-PAGE4. Phospholipids with small polar headgroups, saturated [sn-1] and nonsaturated [sn-2], were preferred substrates4. The gel shift assay is not appropriate for extensive kinetic studies of apolipoprotein N-acyltransferase nor for HTS to identify inhibitory molecules. Click-chemistry using alkyne fatty acids has been successfully used to study lipoprotein modification in bacteria5 and fatty acid metabolism in eukaryotes6. Recently, an in vitro assay of Ras palmitoylation was reported to identify inhibitors7.
In the method described here, purified active Lnt in detergent is incubated with substrates in mixed micelles to form alkyne-triacylated peptide that is subsequently detected by fluorescence spectrometry.

<table><tbody><tr><td>&amp;Auml;kta Purifier FPLC system</td><td>GE Healthcare</td><td>NA</td><td>Purity: NA&lt;br/&gt; Lnt purification</td></tr><tr><td>alkyne-POPE</td><td>Avanti Polar Lipids</td><td>900414P</td><td>Purity: &gt;99%&lt;br/&gt; Lnt substrate</td></tr><tr><td>Azido-FAM</td><td>Lumiprobe</td><td>A4130</td><td>Purity: 100% (pure)&lt;br/&gt; Click reagent</td></tr><tr><td>BioPhotometer Plus</td><td>Eppendorf</td><td>N/A</td><td>Purity: NA&lt;br/&gt; OD 600 nm</td></tr><tr><td>BioTek ELX 405 Select plate washer</td><td>BioTek</td><td>N&amp;deg; serie 115800, n&amp;deg; materiel 405 Select</td><td>Purity: NA&lt;br/&gt; Wash steps</td></tr><tr><td>biotin-fluorescein</td><td>Sigma</td><td>53608</td><td>Purity: &amp;ge;90%&lt;br/&gt; Fluorescence control</td></tr><tr><td>Copper(II) sulfate pentahydrate</td><td>Sigma</td><td>C3036</td><td>Purity: &amp;ge;98%&lt;br/&gt; Click reagent</td></tr><tr><td>DDM</td><td>Anatrace</td><td>D310A</td><td>Purity: &amp;ge; 99% &amp;beta;+&amp;alpha;; &amp;lt; 15% &amp;alpha;&lt;br/&gt; Detergent for Lnt purification</td></tr><tr><td>Dimethylsulfoxide (DMSO)</td><td>Invitrogen</td><td>D12435</td><td>Purity: anhydrous&lt;br/&gt; 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&lt;br/&gt; Handling reagents</td></tr><tr><td>French Pressure Cell</td><td>N/A</td><td>N/A</td><td>Purity: NA&lt;br/&gt; Cell disruption</td></tr><tr><td>FSL-1-biotin</td><td>EMC microcollections</td><td>L7030</td><td>Purity: NA&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; 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&lt;br/&gt; Fluorescence detection</td></tr><tr><td>MTSES (sodium (2-sulfonatoethyl)methanethiosulfonate)</td><td>Anatrace</td><td>S110MT</td><td>Purity: ~100%&lt;br/&gt; Thiol specific inhibitor</td></tr><tr><td>Optically Clear Adhesive Seal Sheets</td><td>Thermo Scientific</td><td>AB-1170</td><td>Purity: NA&lt;br/&gt; 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&lt;br/&gt; Lnt purification</td></tr><tr><td>StrepTactin Sepharose 50 %</td><td>IBA Biotechnology</td><td>2-1201-010</td><td>Purity: NA&lt;br/&gt; Lnt purification</td></tr><tr><td>streptavidin</td><td>Sigma</td><td>S4762-1MG</td><td>Purity: &amp;ge;13 units/mg protein&lt;br/&gt; 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%&lt;br/&gt; Click reagent</td></tr><tr><td>TCEP (tris(2-carboxyethyl)phosphine hydrochloride</td><td>Sigma</td><td>75259</td><td>Purity: &amp;ge;98%&lt;br/&gt; Click reagent</td></tr><tr><td>TECAN Infinite F500</td><td>Tecan</td><td>N/A</td><td>Purity: NA&lt;br/&gt; Fluorescence detection</td></tr><tr><td>TECAN Infinite M1000 pro</td><td>Tecan</td><td>N/A</td><td>Purity: NA&lt;br/&gt; Fluorescence detection</td></tr><tr><td>Thermomixer C</td><td>Eppendorf</td><td>5382000015</td><td>Purity: NA&lt;br/&gt; Heated lid</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>93443</td><td>Purity: 10% in H2O&lt;br/&gt; Lnt reaction buffer</td></tr><tr><td>Ultra centrifuge</td><td>Beckman LC</td><td>N/A</td><td>Purity: NA&lt;br/&gt; Cell fractionation</td></tr></tbody></table>

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

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

In the Lnt reaction the sn-1 fatty acid from phospholipids is transferred onto a diacylglyceryl peptide, resulting in mature triacylated peptide8. The in vitro Lnt assay described here is designed to use phospholipids containing an alkyne fatty acid (alkyne-POPE) and FSL-1-biotin as substrates, resulting in the formation of alkyne-FSL-1-biotin. Upon a click-chemistry reaction with azido-FAM, this product should become fluorescently labeled and detected by fluorescence spectrometry (<stron...

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

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

click-chemistry-based-fluorometric-assay-for-apolipoprotein-n

Research

JoVE Journal

Biochemistry

1.9K Views.  Institut Pasteur, Unité Biologie et génétique de la paroi bactérienne, Paris, France ; CNRS, UMR 2001 « Microbiologie intégrative et Moléculaire », Paris, France ; INSERM, Équipe Avenir, Paris, France. Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

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

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

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

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

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

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

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

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Bioengineering

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in addition to its implications in cancer and neurodegenerative diseases. Recent developments in fatty acylation detection methods have enabled efficient and non-hazardous detection of fatty acylated proteins, particularly through the use of click chemistry with bio-orthogonal labeling. However, click chemistry detection can be limited by the poor solubility and potential toxic effects of adding long chain fatty acids to cell culture. Described here is a labeling approach with optimized delivery using saponified fatty acids in combination with fatty-acid free BSA, as well as delipidated media, which can improve detection of hard to detect fatty acylated proteins. This effect was most pronounced with the alkynyl-stearate analog, 17-ODYA, which has been the most commonly used fatty acid analog in click chemistry detection of acylated proteins. This modification will improve cellular incorporation and increase sensitivity to acylated protein detection. In addition, this approach can be applied in a variety of cell types and combined with other assays such as pulse-chase analysis, stable isotope labeling with amino acids in cell culture, and mass spectrometry for quantitative profiling of fatty acylated proteins.

The study of fatty acylation has important implications in cellular protein interactions and diseases. Presented here is a modified protocol to improve click chemistry detection of fatty acylated proteins, which can be applied in various cell types and combined with other assays, including pulse-chase and mass spectrometry.

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in ...

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 enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

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

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Immunology and Infection

Development of a Click-Based Assay for Screening HAT1 Enzyme Activity

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

Author Spotlight: Developing Acetyl-Click Assay for HAT1 Inhibitor Screening

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.