title: Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase.
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.
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia.
authors.forename: Elisa
authors.initials: E
authors.lastname: Romeo
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia.
authors.forename: Silvia
authors.initials: S
authors.lastname: Pontis
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia.
authors.forename: Stefano
authors.initials: S
authors.lastname: Ponzano
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia.
authors.forename: Fabiola
authors.initials: F
authors.lastname: Bonezzi
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia.
authors.forename: Marco
authors.initials: M
authors.lastname: Migliore
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia.
authors.forename: Simona
authors.initials: S
authors.lastname: Di Martino
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia.
authors.forename: Maria
authors.initials: M
authors.lastname: Summa
authors.affiliation: Drug Discovery and Development, Istituto Italiano di Tecnologia; Departments of Anatomy and Neurobiology, Pharmacology, and Biological Chemistry, University of California, Irvine School of Medicine; piomelli@uci.edu.
authors.forename: Daniele
authors.initials: D
authors.lastname: Piomelli
mesh_terms: D000581:Amidohydrolases; D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology; D015374:Biosensing Techniques; D057075:Enzyme Assays; D006801:Humans; D007249:Inflammation / Q000201:enzymology; D008264:Macrophages / Q000201:enzymology
chemical_list: D000581:Amidohydrolases