Lipid droplets are novel sites of N-acylethanolamine inactivation by fatty acid amide hydrolase-2

Abstract

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.

FIGURE 1.

Enzymatic activity and kinetics of FAAH-2. A, hydrolysis of [¹⁴C]AEA (black bars) or [¹⁴C]PEA (gray bars) by HeLa homogenates expressing FAAH or FAAH-2. AEA/PEA hydrolysis by vector-transfected cells was subtracted from all samples. *, p < 0.05; ***, p < 0.001 (n = 3–4). B, [¹⁴C]AEA (0.1–100 μm) hydrolysis by HeLa homogenates expressing FAAH-2. [¹⁴C]AEA was hydrolyzed with an apparent V_max and K_m of 0.71 ± 0.04 nmol/mg/min and 7.9 ± 1.5 μm, respectively (n = 7). C, [¹⁴C]PEA (0.1–100 μm) was hydrolyzed with an apparent V_max and K_m of 1.21 ± 0.1 nmol/mg/min and 4.3 ± 1.4 μm, respectively (n = 7).

FIGURE 2.

Uptake and hydrolysis of AEA and PEA in transfected HeLa cells. A, HeLa cells transfected with FAAH, FAAH-2, or vector controls were incubated with 100 nm [¹⁴C]AEA for 1 or 5 min and uptake (black bars) and hydrolysis (gray bars) determined. Significance was determined between FAAH-transfected controls and FAAH-2/vector-transfected cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (n = 3). B, uptake and hydrolysis of 100 nm [¹⁴C]PEA by HeLa cells transfected with FAAH, FAAH-2, or vector controls. **, p < 0.01; ***, p < 0.001 (n = 3). C, intracellular hydrolysis of [¹⁴C]AEA and [¹⁴C]PEA (both at 100 nm) following uptake at 3 s in HeLa cells transfected with FAAH, FAAH-2 or vector controls. *, p < 0.05, **, p < 0.01 (n = 3).

FIGURE 3.

Subcellular localization of FAAH-2 in COS-7 and HeLa cells. A, COS-7 and HeLa cells were transiently transfected with FAAH2-FLAG, fixed, permeabilized, and stained with anti-FLAG or anti-calreticulin antibodies. The top panel shows FAAH-2, the middle panel represents calreticulin, and the bottom panel shows the merged images. B, COS-7 and HeLa cells expressing FAAH2-FLAG or FAAH2-Myc were incubated with anti-FLAG or anti-Myc antibodies and stained with BODIPY493/503. The top panel shows FAAH-2, the middle panel represents BODIPY493/503, and the bottom panel sows the merged images. C, close-up image of a FAAH2-FLAG expressing HeLa cell stained with BODIPY493/503. D, localization of FAAH-FLAG in COS-7 and HeLa cells treated with BODIPY493/503. E, subcellular localization of FAAH-FLAG and FAAH2-FLAG following an overnight incubation with 400 μm oleic acid. F, distribution of FAAH-FLAG, FAAH2-FLAG, and FAAH2(N)-rΔTMFAAH-FLAG after subcellular fractionation. HeLa cells were co-transfected with ADRP and the indicated plasmids and were grown overnight in media supplemented with 400 μm oleic acid. The post-nuclear supernatants were subjected to ultracentrifugation. Fractions were collected from the top with fraction 1 corresponding to the floating lipid droplet layer, P representing the pellet, and S the unfractionated post-nuclear supernatant. The fractions were resolved by SDS-PAGE and probed with anti-FLAG, anti-calnexin, or anti-ADRP antibodies. G, proteinase K protection analysis of FAAH-FLAG, FAAH2(N)-rΔTMFAAH-FLAG, and FAAH2-FLAG in post-nuclear supernatants of COS-7 cells. Supernatants (60 μg) were either left untreated or were incubated with 500 μg/ml proteinase K for 30 min at 37 °C. The samples were subsequently resolved by SDS-PAGE and probed with anti-FLAG, anti-β-actin, and anti-calreticulin antibodies.

FIGURE 4.

Expression and activities of cytoplasmically and luminally oriented FAAH-2 variants. A, constructs used in the study. ΔTMFAAH and ΔTMFAAH2 indicate the respective proteins lacking their putative N-terminal TM regions, N(TM) represents the N-terminal TM region of the indicated protein, CAL is the calreticulin signal sequence, and RFP represents red fluorescent protein. FLAG and Myc are the epitope tags used. B, relative expression of FAAH-FLAG, FAAH2-FLAG, and cytoplasmically and luminally facing FAAH and FAAH-2 variants. Transfected HeLa homogenates (20 μg) were separated by SDS-PAGE and probed with anti-FLAG and anti-β-actin antibodies. C, enzymatic activities of cell lysates containing the aforementioned proteins. Activity of CAL-ΔTMFAAH-FLAG was compared with FAAH-FLAG (black bars). For the rest of the proteins, activities were compared with FAAH-2 (blue bars). Inset: activities of FAAH-2 and its corresponding chimeras. **, p < 0.01 and ***, p < 0.001 (n = 4–6).

FIGURE 5.

Membrane orientation and localization of cytoplasmically and luminally oriented ER FAAH-2 chimeras. A, membrane orientation of FAAH-2 variants. Membrane fractions of COS-7 cells transfected with the indicated proteins were treated with 500 μg/ml proteinase K in the presence or absence of 1% Triton X-100 for 30 min at 37 °C. Control samples were left untreated. The samples were resolved by SDS-PAGE and probed with FLAG or calreticulin antibodies. B, immunolocalization of cytoplasmically and luminally oriented FAAH and FAAH-2 chimeras. COS-7 cells expressing CAL-ΔTMFAAH2-FLAG, CAL-ΔTMFAAH-FLAG, or FAAH(N)-ΔTMFAAH2-FLAG were probed with anti-FLAG antibodies and stained with BODIPY 493/503. The top panel represents the FAAH/FAAH-2 variants, the middle panel shows BODIPY 493/503, and the bottom panel shows the merged image. C, PNGase F sensitivity of FAAH chimeras. Membrane fractions of HeLa cells expressing FAAH-FLAG, FAAH2(N)-rΔTMFAAH-FLAG, CAL-ΔTMFAAH-FLAG, or CAL-ΔTMFAAH(N/Q)-FLAG were incubated in the presence or absence of PNGase F as described under “Experimental Procedures.” The proteins were subsequently separated by SDS-PAGE and probed with anti-FLAG and anti-β-actin antibodies. Note that CAL-ΔTMFAAH(N/Q)-FLAG was poorly expressed.

FIGURE 6.

The N terminus of FAAH-2 is a lipid droplet localization sequence. A, COS-7 cells transfected with RFP or FAAH2(N)-RFP were fixed and stained with BODIPY493/503. The top panel shows RFP, the middle panel depicts BODIPY493/503, and the bottom panel shows the merged images. Note that RFP aggregates were present in every FAAH2(N)-RFP-expressing cell examined. B, co-localization of FAAH2(N)-rΔTMFAAH-FLAG with BODIPY493/503 in COS-7 and HeLa cells. C, expression of rFAAH-FLAG, rFAAH-FLAG(low), FAAH2(N)-rΔTMFAAH-FLAG, and FAAH2-FLAG in HeLa cells. rFAAH-FLAG(low) represents rat FAAH-FLAG expressed at levels comparable to FAAH2(N)-rΔTMFAAH-FLAG. The proteins were resolved by SDS-PAGE and probed with anti-FLAG or anti-β-actin antibodies. D, similar (p > 0.05) enzymatic activities of rFAAH-FLAG(low) and FAAH2(N)-rΔTMFAAH-FLAG in homogenates of HeLa cells (n = 3). E, uptake (black bar) and hydrolysis (gray bar) of 100 nm [¹⁴C]AEA by HeLa cells transiently transfected with rFAAH-FLAG(low), FAAH2(N)-rΔTMFAAH-FLAG, FAAH2-FLAG, or vector controls following 1- or 5-min incubations. Statistical significance was determined between transfected cells and rFAAH-FLAG(low)-transfected controls. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (n = 3). F, intracellular hydrolysis of [¹⁴C]AEA in transfected HeLa cells following uptake at 3 s. **, p < 0.01; ***, p < 0.001 (n = 3).