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 8，150 mM NaCl，0.5 mM EDTA）中至每mL 100 OD600 单位。</li>
		<li>通过法国压力池压力机在 10，000 psi 下通过两次通道破碎细胞。</li>
		<li>通过以13，000× g 离心20分钟来除去未破碎的细胞和碎片。保留上清液并丢弃沉淀。</li>
		<li>将上清液以120，000× g 离心60分钟并收集膜囊泡（半透明的棕色颗粒）。弃去上清液。</li>
		<li>在室温（RT）下用1%（w / v）正十二烷基β-D-麦芽糖苷（DDM）在缓冲液WA中溶解膜沉淀中的整合膜蛋白30分钟。以120，000× g 离心并除去不溶性物质30分钟。使用上清液级分进行纯化。</li>
		<li>如Nozeret等人8所述，在亲和色谱柱（见材料表）和S400凝胶过滤柱（见材料表）上纯化Lnt-strep。</li>
		<li>将纯化的酶储存在含有0.05%DDM和10%甘油的缓冲液WA中，温度为-80°C。 注意:该酶可稳定保存5年以上。</li>
	</ol></li>
	<li>炔烃磷脂和生物素化成纤维细胞刺激配体（FSL-1-生物素）底物的制备
	<ol><li>将 100 μL 定制合成的炔烃-POPE（1-十六烷基-15-炔酰基-2-油酰基-sn-甘油-3-磷酸乙醇胺，见 材料表）等分到 1.5 mL 管中。</li>
		<li>用注射器刺穿试管，并在室温下以快速真空蒸发氯仿2小时。将干燥的磷脂样品储存在-20°C。</li>
		<li>使用前将炔烃-POPE 溶解在 0.1% Triton X-100 中的 500 μM 中。如有必要，在室温超声水浴中加入3分钟的超声处理步骤以溶解炔烃-POPE。</li>
		<li>将FSL-1-生物素重悬于水中，浓度为445μM。 注意:两种溶液都可以在-20°C下储存至少2个月。</li>
	</ol></li>
</ol>2. 试管检测
注意:在第1天，在1.5 mL管中设置Lnt反应（步骤2.1），并用链霉亲和素涂覆96孔板（步骤2.2）。
<ol><li>试剂混合物的制备和酶促反应
	<ol><li>对于标准 Lnt 测定，在 Lnt 反应缓冲液（50 mM Tris-HCl pH 7.2、150 mM NaCl、0.1% Triton X-100，含 1% BSA）中以 18 μL 的最终体积在 1.5 mL 管中混合炔烃-POPE（来自 500 μM 储备液的最终 50 μM）和 FSL-1-生物素（来自 445 μM 储备液的终浓度为 50 μM）。执行所有条件一式三份。</li>
		<li>将底物混合物（在步骤2.1.1中制备）在超声水浴中超声处理3分钟，并在加入Lnt酶（步骤2.1.3）之前在37°C下孵育5分钟。 注意:MTSES（（2-磺乙基）甲硫代磺酸钠）是一种硫醇特异性试剂，可抑制Lnt8。向反应管（步骤2.1.1）中加入10mM MTSES（100mM储备溶液在100%DMSO中）用作阴性对照样品。调整Lnt反应缓冲液的体积，使总体积为18 μL。</li>
		<li>加入活性（1 ng/μL，相当于 17.2 nM）或非活性 Lnt （C387S）（2 μL 10 ng/μL 储备液中含有 0.05% DDM）的缓冲液 WA），并通过上下移液与底物混合（步骤 2.1.2）。</li>
		<li>将反应在37°C下在带加热盖的热混合器中孵育16小时。</li>
	</ol></li>
	<li>96孔板的链霉亲和素包被
	<ol><li>在 H2O 中制备 2 mg/mL 的链霉亲和素储备溶液。 注意:该溶液可在-20°C下储存长达6个月。</li>
		<li>从储备溶液中制备10 μg/mL链霉亲和素在水中作为工作溶液。向 96 孔板的每个孔中加入 100 μL 溶液。通过在37°C下孵育过夜而不盖风干孔，将链霉亲和素结合到板上。</li>
	</ol></li>
</ol>注意:在第2天进行点击化学（步骤2.3），将Lnt反应混合物结合到链霉亲和素包被的板（步骤2.4），并检测荧光并分析结果（步骤2.5）。
<ol start="3"><li>咔嗒声化学反应
	<ol><li>制备叠氮基-FAM（5 mM DMSO 溶液）、TCEP（三（2-羧乙基）膦盐酸盐;50 mM 在水中制备）、TBTA（三[（1-苄基-1H-1，2，3-三唑-4-基）甲基]胺;2 mM 叔丁醇:DMSO （4:1））和 CuSO4∙5H 2 O（50 mM 在 H2O 中新鲜制备）的储备溶液。</li>
		<li>在含有步骤2.1.4制备的Lnt反应混合物的1.5 mL管中，按以下顺序添加试剂进行点击化学:0.2 μL原液叠氮-FAM（终浓度50 μM），0.4 μL原液TCEP（终浓度1 mM），0.2 μL原液TBTA（终浓度0.02 mM）。</li>
		<li>涡旋溶液5秒。然后加入 0.4 μL 原液CuSO 4 （终浓度 1 mM）。再次涡旋5秒。将样品在室温下在黑暗中孵育1小时。</li>
	</ol></li>
	<li>Lnt反应混合物与链霉亲和素包被板的结合
	<ol><li>用200μLPBS-T1（PBS含有0.05%吐温-20）从步骤2.2.2孵育3x过夜后洗涤链霉亲和素包被板的孔。 注意:链霉亲和素板可以立即使用或在4°C干燥储存。</li>
		<li>将步骤 2.3.3 中的点击化学混合物中的 18 μL 转移到 96 孔链霉亲和素包被板中的孔中，并加入 100 μL PBS-T1 以将 N-酰基-FSL-1-生物素-FAM 产物结合到链霉亲和素板上。</li>
		<li>使用生物素-荧光素（来自 DMSO 中 3.1 mM 储备液的 0.26 μM）作为链霉亲和素结合和荧光读数的阳性对照。</li>
		<li>将板在室温下在温混合器中在黑暗中孵育1小时，以300rpm振荡。</li>
		<li>使用多通道电动移液器对96孔板进行手动洗涤步骤，如下所示:用200μLPBS-T2（PBS含有1%吐温-20）洗涤6次，用移液器冲洗3次，用200μLPBS洗涤三次，用移液器冲洗三次。</li>
	</ol></li>
	<li>荧光检测和分析
	<ol><li>加入 200 μL PBS，并在荧光酶标仪中记录 520 nm 处的荧光。将结果保存在电子表格软件中。 注意:生物素荧光素用作链霉亲和素结合和520nm荧光检测的阳性对照。预计使用 0.26 μM 生物素荧光素可获得 30，000–40，000 A.U. 的可重现读数。所有样品一式三份分析，包括对照。</li>
		<li>使用统计软件使用非配对t检验计算每个反应的标准偏差，并计算阴性对照和阳性样品的P值。</li>
	</ol></li>
</ol>3. 多孔板检测
注意:在第1天，在384孔板中设置Lnt反应（步骤3.1），并用链霉亲和素涂覆384孔板（步骤3.2）。
<ol><li>试剂混合物的制备和酶促反应 注意:与试管测定相比，试剂和酶的数量减少，Lnt反应直接在384孔板中进行。这允许使用更少的材料并减少可以进一步自动化的步骤。<ol><li>在Lnt反应缓冲液（50mM Tris-HCl pH 7.2，150mM NaCl，0.1%Triton X-100，含1%BSA）中以13.5μL的体积在Lnt反应缓冲液（50mM Tris-HCl pH 7.2，150mM NaCl，0.1%Triton X-100，含1%BSA）中以13.5μL的体积混合炔烃-POPE（来自500μM储备液的终浓度50μM）和FSL-1-生物素（来自500μM储备液的最终浓度）。执行所有条件一式三份。计算要执行的反应次数所需的试剂总量（即，一个 384 孔板为 5，184 μL）。</li>
		<li>在超声波水浴中超声处理底物混合物3分钟。</li>
		<li>在 384 孔板中每孔等分试样 13.5 μL 底物混合物。 注意:作为阴性对照，每孔可添加10mM的MTSES（100%DMSO中的625mM储备溶液）。调节Lnt缓冲液的体积（步骤3.1.1）以达到13.5μL的最终反应体积。</li>
		<li>在加入Lnt酶之前在37°C孵育5分钟（步骤3.1.5）。</li>
		<li>向含有步骤 3.1.3 中反应混合物的每个孔中加入 0.5 ng/μL（相当于 8.6 nM）活性 Lnt 酶或非活性变体 （C387S）（1.5 μL，来自含有 0.05% DDM 的缓冲液 WA 中的 5 ng/μL 原液）。通过上下移液混合试剂。总反应体积为15μL。</li>
		<li>用塑料箔密封板，用于多孔板。</li>
		<li>在37°C下在带加热盖的热混合器中孵育16小时。</li>
	</ol></li>
	<li>链霉亲和素包衣 384 孔板<ol><li>使用步骤 2.2.2 中的 75 μL 链霉亲和素（10 μg/mL 在 H2O 中）包被 384 孔板的孔。让链霉亲和素结合到板上，在37°C下孵育过夜，没有盖子风干孔。</li>
	</ol></li>
</ol>注意:在第2天进行点击化学（步骤3.3），将Lnt反应混合物结合到链霉亲和素包被的板（步骤3.4），检测荧光并分析结果（步骤3.5）。
<ol start="3"><li>咔嗒声化学反应<ol><li>制备叠氮基-FAM（1.2 mM DMSO 溶液）、TCEP（三（2-羧乙基）膦盐酸盐;在 H 2 O 中制备的 24 mM）、TBTA（三[（1-苄基-1H-1，2，3-三唑-4-基）甲基]胺;0.48 mM 叔丁醇:DMSO （4:1））和 CuSO4∙5H 2 O（24 mM 在 H2O 中新鲜制备）的储备溶液。</li>
		<li>组合三种点击化学试剂:叠氮基-FAM（终浓度 50 μM）、TCEP（1 mM 最终浓度）和 TBTA（0.02 mM 最终浓度）的混合物，每种试剂的混合物为 0.75 μL，最终体积为每孔 2.25 μL。计算每个 384 孔板所需的体积（即，当使用 384 孔板的所有孔时，使用 864 μL）。用力混合。</li>
		<li>每孔加入 2.25 μL 试剂溶液，直接加入步骤 3.1.7 中完成的 Lnt 反应中。</li>
		<li>用多通道电动移液器上下移液混合。</li>
		<li>加入CuSO4 （从24 mM原液中加入0.75 μL，终浓度为1 mM），并用多通道电动移液器上下移液混合。</li>
		<li>在室温下在黑暗中孵育1小时。</li>
	</ol></li>
	<li>Lnt反应混合物与链霉亲和素包被的384孔板结合
	<ol><li>用85μL PBS-T1从步骤3.2.1 1x孵育过夜后洗涤链霉亲和素包被的板的孔。</li>
		<li>将步骤3.3.6孵育板每个孔中的11μL点击化学反应转移到步骤3.4.1的链霉亲和素包被的板中，以将N-酰基-FSL-1-生物素-FAM产物结合到链霉亲和素板上。</li>
		<li>加入 64 μL 缓冲液 PBS-T1。</li>
		<li>包括生物素荧光素（来自DMSO中3.1mM储备液的0.19μM）作为与链霉亲和素包被孔结合的对照。</li>
		<li>将384孔板在室温下在室温下在温混合器中孵育1小时，以300rpm振荡。</li>
		<li>使用自动洗板机洗涤板，如下所示:用85μL PBS-T2洗涤10次，两次洗涤之间摇动板;用 85 μL PBS 洗涤 5 次，两次洗涤之间摇动板。</li>
	</ol></li>
	<li>荧光检测和分析
	<ol><li>加入PBS（85μL）并在荧光酶标仪中记录535nm的荧光。</li>
		<li>按所述分析数据（步骤2.5.2）。</li>
	</ol></li>
</ol>

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作者没有什么可透露的。

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>&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

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。

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

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 次观看.  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. 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。

视频: 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.

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

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

科研

生物化学

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.

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 ...

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.

一种快速定量荧光方法蛋白靶向小分子药物筛选

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

生物工程

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.

优化了炔基脂肪酸类似物的掺入，用于使用点击化学检测脂肪酰化蛋白

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.

一种高通量酶偶联活性测定法，用于探测小分子与 dNTP 酶 SAMHD1 的相互作用

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;

基于荧光的连续核酸内切酶偶联DNA甲基化测定，用于筛选DNA甲基转移酶抑制剂

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.

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

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

免疫与感染

开发基于点击的检测方法，用于筛选 HAT1 酶活性

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.

作者聚焦:开发用于 HAT1 抑制剂筛选的乙酰点击检测

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.

用于测量组蛋白乙酰转移酶 1 乙酰化的乙酰基点击化学测定法

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

基于点击化学的载脂蛋白N-酰基转移酶荧光测定从酶表征到高通量筛选

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