Abstract
Fatty acid amides constitute a large and diverse class of lipid transmitters that includes the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. The magnitude and duration of fatty acid amide signaling are controlled by enzymatic hydrolysis in vivo. Fatty acid amide hydrolase (FAAH) activity in mammals has been primarily attributed to a single integral membrane enzyme of the amidase signature (AS) family. Here, we report the functional proteomic discovery of a second membrane-associated AS enzyme in humans that displays FAAH activity. The gene that encodes this second FAAH enzyme was found in multiple primate genomes, marsupials, and more distantly related vertebrates, but, remarkably, not in a number of lower placental mammals, including mouse and rat. The two human FAAH enzymes, which share 20% sequence identity and are referred to hereafter as FAAH-1 and FAAH-2, hydrolyzed primary fatty acid amide substrates (e.g. oleamide) at equivalent rates, whereas FAAH-1 exhibited much greater activity with N-acyl ethanolamines (e.g. anandamide) and N-acyl taurines. Both enzymes were sensitive to the principal classes of FAAH inhibitors synthesized to date, including O-aryl carbamates and alpha-keto heterocycles. These data coupled with the overlapping, but distinct tissue distributions of FAAH-1 and FAAH-2 suggest that these proteins may collaborate to control fatty acid amide catabolism in primates. The apparent loss of the FAAH-2 gene in some lower mammals should be taken into consideration when extrapolating genetic or pharmacological findings on the fatty acid amide signaling system across species.
Highlights
N-acyl amino acids (e.g. N-acyl taurines (NATs), Ref. 3)
fatty acid amide hydrolase (FAAH) is an integral membrane protein widely distributed in mammalian tissues that belongs to a large family of enzymes that share a highly conserved ϳ130 amino acid motif designated the “amidase signature” (AS) sequence [20]
Based on this sequence resents a version of FAAH-2 lacking the FLAG tag, as only the analysis and the biochemical data presented below, we have 60-kDa protein was detected with anti-FLAG antibodies
Summary
ABPP of FAAH-transfected Cells—Transfected cell proteomes (1 mg/ml) were treated with a rhodamine-conjugated FP probe (FP-rhodamine [34]; 100 nM, 1 h in Buffer 1 at 25 °C). FAAH-2 protein was quantified by summing the intensities of the 58- and 60-kDa bands observed in transfected COS-7 cells. FAAH Substrate Assays—FAAH assays were performed by following the conversion of 14C-labeled substrates to their corresponding fatty acid using a thin layer chromatography (TLC) assay as described previously [27, 35]. Reactions performed in triplicate were initiated by incubating membrane lysates of COS-7 cells expressing FAAH-1 or FAAH-2 with 100 M substrate, and were quenched with 0.5 N HCl at two time points. Identification of a novel AS enzyme FLJ31204 (FAAH-2) in human cancer cell lines by ABPP-MudPIT
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