Lipid droplets are novel sites of N-acylethanolamine inactivation by fatty acid amide hydrolase-2.
Anandamide (AEA) and other bioactive N-acylethanolamines (NAEs) are primarily inactivated by the enzyme fatty acid amide hydrolase (FAAH). Recently, FAAH-2 was discovered in humans, suggesting an additional enzyme can mediate NAE inactivation in higher mammals. Here, we performed a biochemical characterization of FAAH-2 and explored its capacity to hydrolyze NAEs in cells. In homogenate activity assays, FAAH-2 hydrolyzed AEA and palmitoylethanolamide (PEA) with activities approximately 6 and approximately 20% those of FAAH, respectively. In contrast, FAAH-2 hydrolyzed AEA and PEA in intact cells with rates approximately 30-40% those of FAAH, highlighting a potentially greater contribution toward NAE catabolism in vivo than previously appreciated. In contrast to endoplasmic reticulum-localized FAAH, immunofluorescence revealed FAAH-2 was localized on lipid droplets. Supporting this distribution pattern, the putative N-terminal hydrophobic region of FAAH-2 was identified as a functional lipid droplet localization sequence. Lipid droplet localization was essential for FAAH-2 activity as chimeras excluded from lipid droplets lacked activity and/or were poorly expressed. Lipid droplets represent novel sites of NAE inactivation. Therefore, we examined substrate delivery to these organelles. AEA was readily trafficked to lipid droplets, confirming that lipid droplets constitute functional sites of NAE inactivation. Collectively, these results establish FAAH-2 as a bone fide NAE-catabolizing enzyme and suggest that NAE inactivation is spatially separated in cells of higher mammals.
Kaczocha
Martin
M
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794, USA. MKaczoch@notes.sunysb.edu
Glaser
Sherrye T
ST
Chae
Janiper
J
Brown
Deborah A
DA
Deutsch
Dale G
DG
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism; D019556:COS Cells; D063385:Cannabinoid Receptor Modulators / Q000378:metabolism; D002250:Carbon Radioisotopes; D002451:Cell Compartmentation / Q000502:physiology*; D002458:Cell Fractionation; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000201:enzymology; D063388:Endocannabinoids; D004789:Enzyme Activation / Q000502:physiology; D004983:Ethanolamines / Q000378:metabolism*; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D053284:Polyunsaturated Alkamides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D014162:Transfection; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D001095:Arachidonic Acids; D063385:Cannabinoid Receptor Modulators; D002250:Carbon Radioisotopes; D063388:Endocannabinoids; D004983:Ethanolamines; D053284:Polyunsaturated Alkamides; C022203:N-acylethanolamines; C516565:FAAH2 protein, human; D000099252:Fatty Acid Amide Hydrolases; C078814:anandamide