Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Summary

Here, we describe the preparation and use of an activity-based probe (ARN14686, undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl]carbamate) that allows for the detection and quantification of the active form of the proinflammatory enzyme N-acylethanolamine acid amidase (NAAA), both in vitro and ex vivo.

Abstract

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical probe that targets the enzyme's active sites. A reporter tag introduced into the probe allows for the detection of the labeled enzyme by in-gel fluorescence scanning, protein blot, fluorescence microscopy, or liquid chromatography-mass spectrometry. Here, we describe the preparation and use of the compound ARN14686, a click chemistry activity-based probe (CC-ABP) that selectively recognizes the enzyme N-acylethanolamine acid amidase (NAAA). NAAA is a cysteine hydrolase that promotes inflammation by deactivating endogenous peroxisome proliferator-activated receptor (PPAR)-alpha agonists such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). NAAA is synthesized as an inactive full-length proenzyme, which is activated by autoproteolysis in the acidic pH of the lysosome. Localization studies have shown that NAAA is predominantly expressed in macrophages and other monocyte-derived cells, as well as in B-lymphocytes. We provide examples of how ARN14686 can be used to detect and quantify active NAAA ex vivo in rodent tissues by protein blot and fluorescence microscopy.

Introduction

Commonly used methods to investigate the expression patterns, interactions, and functions of proteins, including liquid chromatography-mass spectrometry platforms for shotgun analysis^1,2, yeast two-hybrid methods^3,4, and in vitro assays, are limited in that they are unable to assess the activity of proteins in their native state. Activity-based protein profiling (ABPP) can be used to fill this gap. In this approach, small-molecule probes capable of covalently binding to the active site of an enzyme of interest are conjugated to a reporter group that allows for target detection. Using click chemistry (CC), the reporter can be integrated i....

Protocol

Caution: All chemistry reactions should be carried out in a ventilated fume hood and with the use of a lab coat, gloves, and protective goggles. The reactions should be also carried out in a nitrogen environment.

Ethical statement: Our procedures involving animals are performed in accordance with the Italian regulations on the protection of animals used for experimental and other scientific purposes (D.M. 116192), and European Economic Community regulations (O.J. of E.C. L 358/1 12/18/1986).

NOTE: Synthesis of [(3S)-2-oxoazetidin-3-yl]ammonium acetate is described for large-scale yields (50 g of N-Cbz-L-Serine), but it can be easily scaled down.

Results

ARN14686 was designed based on the scaffold of the NAAA inhibitor ARN726. The 4-butyl-cyclohexyl group of ARN726 was substituted with a C9 saturated aliphatic chain bearing a terminal alkyne tag (Figure 1). The alkyne tag was introduced in order to allow the use of a two-step labeling procedure to add a fluorophore or a biotin molecule via CC. This feature renders ARN14686 a very versatile tool to probe NAAA in vitro and in vivo.

Discussion

Enzyme activity is finely regulated at different levels, including RNA transcription, protein synthesis, protein translocation, post-translational modification, and protein-protein interaction. Often, enzyme expression alone does not account for its activity. ABPP was developed to study the activity of proteins in their native state. Two features are required: a chemical probe that covalently binds to the active site of an enzyme of interest and a reporter tag to detect the probe-labeled enzyme.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
β-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