制备及<em>在体内</em>使用基于活动的探针对<em>ñ</em> -acylethanolamine酸酰胺酶
Summary
这里，我们描述一个基于活动的探针的制备和使用（ARN14686，十一碳-10- ynyl- -N – [（3 S）-2-代氮杂环丁烷-3-基]氨基甲酸），允许的检测和定量促炎酶ñ-acylethanolamine酸酰胺酶的活性形式（NAAA）， 在体外和离体 。
Abstract
基于活动的蛋白谱（ABPP）是用于通过使用靶向酶的活性位点的化学探针的识别在复杂蛋白质组感兴趣的酶的方法。导入探针A记者标记允许通过在凝胶荧光扫描，蛋白质印迹，荧光显微镜，或液相色谱 – 质谱法检测标记的酶。这里，我们描述了制备和使用所述化合物ARN14686的，基于活动点击化学探针（CC-ABP），其选择性识别酶Ñ-acylethanolamine酸酰胺酶（NAAA）。 NAAA是半胱氨酸水解酶，通过去激活内源性过氧化物酶体增殖物激活受体（PPAR）-α激动剂如十六酰胺乙醇（PEA）和油酰乙醇酰胺（OEA）促进炎症。 NAAA被合成为一个无效的全长酶原，由autoproteolysis在溶酶体的酸性pH激活。定位研究ħ表明NAAA在巨噬细胞等单核细胞衍生的细胞在B淋巴细胞中表达，以及AVE。我们提供的ARN14686如何能够被用来检测和通过蛋白质印迹和荧光显微镜量化活性NAAA 离体啮齿动物组织的例子。
Introduction
常用的方法来研究的表达模式，交互和蛋白质的功能，包括用于鸟枪分析1,2，酵母液相色谱-质谱平台双杂交方法3,4和体外测定法，是有限的，因为它们是无法评估在其天然状态的蛋白质的活性。基于活动的蛋白谱（ABPP）可以用来填补这一空白。在这种方法中，小分子探针能够共价结合到所关注的酶的活性位点被偶联至一个报告基团，其允许目标检测。使用点击化学（CC），记者可以集成到探针或之后发生5,6-靶接合可以引入。后者程序需要使用含有适当的化学基团，探针如末端炔或叠氮化物，其可以与许多记者试剂经由SUC生物正交反应改性H作为铜（Ⅰ）催化胡伊斯根[3 + 2]环加成7-9或Staudinger连接10,11。
最近，我们公开的化合物ARN14686作为第一ABP为在体外和半胱氨酸水解酶，NAAA 12的体内检测。 NAAA催化饱和和单不饱和的FAE，包括油酰乙醇酰胺（OEA）和十六酰胺乙醇（PEA），这是抗炎核受体PPAR-α13-15的内源性激动剂的水解失活。 NAAA在巨噬细胞等单核细胞衍生的细胞主要表达，以及在B淋巴细胞14,16，这表明在先天免疫应答的调节作用。所述酶在粗面内质网中合成的非活性形式和通过自我蛋白酶机构17中的细胞的酸性区室被激活。该裂解自我蛋白酶生成新N端半胱氨酸（C131在小鼠和大鼠，C126在人中），即是亲核体负责FAE水解18,19。 NAAA活动的药理学抑制改变有利于现场应用工程师16,20,21增加细胞水平的FAE合成/降解的平衡。数β内酯和β内酰胺衍生物已显示出抑制NAAA活性高效力和选择性16,22-26。这些抑制剂作用通过催化半胱氨酸16,27,28号第酰化。
（ – [（S）-2-代氮杂环丁烷-3-基]氨基甲酸4- cyclohexylbutyl- N）16化合物ARN14686是基于全身活性，丝氨酸衍生的β内酰胺NAAA抑制剂的化学结构，ARN726设计。 ARN726的4-丁基环己基用C 9饱和脂族链轴承与叠氮化物载运记者标签随后CC缀合末端炔标签替换。我们选择了设计一个两步ABP到minimallý改变原始支架的结构，从而保持了探针NAAA的亲和力。此外，避免引入笨重标签，这样的探针可以是更适合于不是直接ABP 体内治疗。 ARN14686抑制NAAA具有高效力（hNAAA IC 50 = 6纳米，rNAAA IC 50 = 13纳米）通过形成与酶12的催化半胱氨酸的共价加合物。在活老鼠的实验表明，该探针是在捕捉NAAA在肺部表达选择性。采用高探针浓度（10μM 体外 ，10毫克/毫升静脉内，ⅳ）12时酸性神经酰胺，共享33-34％的同一性与NAAA另一个半胱氨酸酰胺酶，也被确定为一个低亲和力靶。我们也使用ARN14686研究活性NAAA在以下完全弗氏佐剂（CFA）29的施用发炎大鼠组织的存在。
在这里，我们列出了preparati协议上ARN14686（ 图1）并将其应用到NAAA激活体外的调查。作为一个例子，我们描述的实验程序来可视化NAAA的CFA给药后的大鼠爪子。在这个实验中，蛋白从爪组织探针静脉注射后萃取，ABP标记蛋白质进行与生物素叠氮化物以CC。生物素化的样品使用链霉珠富集，并执行蛋白质印迹。在另一应用中，我们描述了活性NAAA的荧光显微镜在小鼠肺从探针处理的小鼠的本地化。在这种情况下，将组织切片并部分经受CC为罗丹明加成。工作流方案， 如图2所示。
Protocol
注意：所有的化学反应，应在通风橱和使用实验室工作服，手套和护目镜进行。该反应应在氮气环境中也进行。 伦理声明：我们涉及动物的程序符合有关动物用于实验和其他科学目的（DM 116192）和欧共体法规的保护意大利规定执行（EC的OJ L一分之三百五十八1986年12月18日）。 注：合成[（3 S）-2-代氮杂环丁烷-3-基]乙酸铵是用于大规模收率描述（50克N-CBZ- L-丝氨酸），但是它可以很容易地按…
Representative Results
ARN14686是基于NAAA抑制剂ARN726的支架设计。 ARN726的4-丁基环己基用C 9饱和脂族链带有终端炔标签（ 图1）被取代。炔标签是为了允许使用两步标记过程的添加荧光团或通过CC一个生物素分子引入。这一功能使得ARN14686一个非常通用的工具，以探测NAAA 在体外和体内 。 这里，我们显示ARN14686的两个应…
Discussion
酶活性是在不同的层次，包括RNA转录，蛋白质合成，蛋白质易位，翻译后修饰，和蛋白质 – 蛋白质相互作用精细调节。通常，酶表达单独不占它的活性。 ABPP的开发是为了研究在其天然状态的蛋白质的活性。两个特征是必需的：共价地结合到感兴趣的酶和记者标签的活性位点的化学探针来检测探针标记的酶。
探针设计与合成的过程的关键点。探针必须具有对其靶足够亲和力和…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank the Nikon Imaging Center at Istituto Italiano di Tecnologia, Genova, Italy (NIC@IIT).
Materials
| 1,1’-sulfonyldiimidazole | Sigma Aldrich | 367818 | Harmful |
| 2-dipyridylcarbonate | Fluorochem | 11331 | Harmful |
| 2-Methylbutan | Sigma Aldrich | M32631 | Flamable, toxic,hazardous to the aquatic environment |
| 4-(Dimethylamino)pyridine | Sigma Aldrich | 107700 | Toxic |
| Acetic acid | Sigma Aldrich | 695092 | Flammable, Corrosive |
| Acetonitrile | Sigma Aldrich | 34998 | Flammable, Toxic |
| Activated charcoal | Sigma Aldrich | 161551 | |
| Ammonium chloride | Sigma Aldrich | A9434 | Harmful |
| Azide-PEG3-Biotin | Jena Biosciences | CLK-AZ104P4 | |
| Azide-PEG3-Fluor 545 | Jena Biosciences | CLK-AZ109 | |
| BCA protein assay kit | Thermo Fisher Scientific | 23227 | |
| Bio-spin columns | Biorad | 732-6204 | |
| Biotin | Sigma Aldrich | B4501 | |
| Blocking buffer | Li-Cor Biosciences | 927-40000 | |
| b-mercaptoethanol | Sigma Aldrich | M6250 | Higly toxic |
| Bovin serum albumine (BSA) | Sigma Aldrich | A7030 | |
| Bromophenol blue | Sigma Aldrich | B0126 | |
| Bruker Avance III 400 | Bruker | ||
| Celite | Sigma Aldrich | 419931 | Health hazard |
| Ceric ammonium nitrate | Sigma Aldrich | 22249 | Oxidizing, Harmful |
| Chloral hydrate | Sigma Aldrich | C8383 | Higly toxic |
| CuSO4.5H2O | Sigma Aldrich | 209198 | Toxic |
| Cyclohexadiene | Sigma Aldrich | 125415 | Flammable, Health hazard |
| Cyclohexane | Sigma Aldrich | 34855 | Flammable, Harmful, Health hazard, Environmental hazard |
| Dichloromethane | Sigma Aldrich | 34856 | Harmful, Health hazard |
| Diethyl ether | Sigma Aldrich | 296082 | Flammable, Harmful |
| Dimethyl sulfoxide (DMSO) | Acros Organics | 348441000 | |
| Dimethyl sulfoxide d6 (DMSO-d6) | Sigma Aldrich | 175943 | |
| Ethanol | Sigma Aldrich | 2860 | Flammable, Harmful |
| Ethyl acetate | Sigma Aldrich | 34858 | Flammable, Harmful |
| Glycerol | Sigma Aldrich | G5516 | |
| Irdye 680-LT Streptavidin | Li-Cor Biosciences | 925-68031 | |
| IRDye680-LT Streptavidin | Licor | 925-68031 | Briefly centrifuge before use to precipitate protein complexes |
| Methanol | Sigma Aldrich | 34966 | Highly toxic |
| Methanol | Sigma Aldrich | 34860 | Flammable, Toxic, Health hazard |
| N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride | Sigma Aldrich | E7750 | Harmful, Corrosive |
| N,N-diisopropylethylamine | Sigma Aldrich | D125806 | Flammable, Corrosive, Toxic |
| N,N-dimethylformamide | Sigma Aldrich | 227056 | Flammable, Harmful, Health hazard |
| N-Cbz-L-Serine | Fluorochem | M03053 | Harmful |
| Nikon A1 confocal microscopy | Nikon | Read the user manual | |
| NuPAGE 4-12% Bis-Tris gel | Thermo Fisher Scientific | NP0335BOX | |
| Palladium on carbon | Sigma Aldrich | 330108 | |
| p-anisidine | Sigma Aldrich | A88255 | Toxic, Health hazard, Environmental hazard |
| Paraformaldehyde | sigma Aldrich | 441244 | Toxic, respiratory harmful, corrosive, falmable |
| Poly(ethylene glycol) | Sigma Aldrich | P3265 | |
| ProLong Gold antifade mountant with DAPI | Thermo Fisher Scientific | P36931 | Avoid bubbles formation |
| Protease inhibitor cocktail | Sigma Aldrich | P8340 | |
| Sodium bicarbonate | Sigma Aldrich | S6014 | |
| Sodium dodecyl sulfate (SDS) | Sigma Aldrich | L3771 | Toxic, corrosive, falmmable |
| Sodium hydride | Sigma Aldrich | 452912 | Flammable |
| Sodium sulfate | Sigma Aldrich | 239313 | |
| Starion FLA-9000 immage scanner | FUJIFILM | Read the user manual | |
| Streptavidin agarose | Thermo Fisher Scientific | 20349 | |
| Sucrose | Sigma Aldrich | S7903 | |
| Tert-butanol | Sigma Aldrich | 360538 | Toxic, flammable |
| Tetrahydrofuran | Sigma Aldrich | 186562 | Flammable, Harmful, Health hazard |
| Thiourea | Acros Organics | 424542500 | Toxic, warm at 50 °C to dissolve |
| Tris | Sigma Aldrich | RDD008 | |
| Tris(2-carboxyethyl)phosphine (TCEP) | Sigma Aldrich | C4706 | |
| Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) | Sigma Aldrich | 678937 | |
| Triton-x100 | Sigma Aldrich | X100 | Toxic |
| Tween-20 | Sigma Aldrich | P9416 | |
| Tween-80 | Sigma Aldrich | P1754 | |
| Ultra turrax IKA T18 basic tissue homogenizer | IKA | ||
| Undec-10-yn-1-ol | Fluorochem | 13739 | Harmful |
| Urea | Sigma Aldrich | U5378 | Toxic, warm at 50 °C to dissolve |
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Cite This Article
Romeo, E., Pontis, S., Ponzano, S., Bonezzi, F., Migliore, M., Di Martino, S., Summa, M., Piomelli, D. Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase. J. Vis. Exp. (117), e54652, doi:10.3791/54652 (2016).