Cooperative enzymatic control of N-acyl amino acids by PM20D1 and FAAH
- Department of Pathology, Stanford University School of Medicine, United States
- Stanford ChEM-H, Stanford University, United States
- Department of Biology, Stanford University, United States
Figures
Figure 1
Detection of a residual N-acyl amino acid hydrolase activity in PM20D1-KO tissues.
(a) Schematic of the enzymatic assay that monitors conversion of C20:4-Phe or C20:4-Gly into arachidonic acid. (b, c) C20:4-Phe (b) and C20:4-Gly (c) hydrolysis activities across the indicated wild-type (blue) or PM20D1-KO (orange) tissues. For (b) and (c), activity assays were conducted with 100 µM substrates and 100 µg tissue lysate in phosphate-buffered saline (PBS) for 1 hr at 37°C. Data are shown as means ± SEM, N = 3/group. All experiments were performed once, with N corresponding to biological replicates. *, p<0.05; **, p<0.01, ***, p<0.001 for the indicated comparison.
Figure 2
Identification of fatty acid amide hydrolase (FAAH) as the enzyme responsible for the PM20D1-independent N-acyl amino acid hydrolase activity.
(a, b) C20:4-Gly hydrolysis activity of cell lysates transfected with the indicated mammalian M20 peptidase (a) or of the indicated liver homogenate fraction from PM20D1-KO animals (b). (c, d) Effect on the C20:4-Gly hydrolysis activity from PM20D1-KO liver membranes of the indicated inhibitors. Activity assays were conducted with 100 µM substrates and 100 µg tissue lysate in PBS for 1 hr at 37°C. For panel (b), membrane and soluble fractions of liver lysate were separated by centrifugation at 100,000 x g for 1 hr. For panel (c), inhibitors were pre-incubated at 1 mM for EDTA and 10 µM for all other compounds for 10 min before the start of the assay. Data are shown as means ± SEM, N = 3/group. All experiments were performed once, with N biological replicates. *, p<0.05; ***, p<0.001 for the comparison versus DMSO or GFP control.
Figure 3 with 1 supplement
N-acyl amino acid hydrolase and synthase substrate scope in vitro for FAAH and PM20D1.
(a) Phylogenetic alignment of the five murine M20 peptidases with mouse FAAH and a FAAH-related enzyme, QRSL1. Orange, M20 peptidases; gray, FAAH-related sequences. (b) Anti-flag western blot for cell lysates (left) and conditioned media (right) transfected with the indicated plasmids. (c–f) N-acyl amino acid hydrolysis and synthase activities of FAAH- and mock-transfected cell lysates (b, c) or PM20D1-transfected and mock-transfected conditioned media (d, e). Activity assays were conducted with 100 µM substrates and 100 µg protein in PBS for 1 hr at 37°C. Data are shown as means ± SEM, N = 3/group. All experiments were performed once, with N biological replicates. *, p<0.05; **, p<0.01; ***, p<0.001 for the indicated comparison.
Figure 3—figure supplement 1
Additional characterization of FAAH and PM20D1 enzyme activities in vitro.
(a) Hydrolysis activities using the indicated substrate from transfected cell lysates (FAAH) or transfected conditioned media (PM20D1). (b) C20:4-Gly hydrolysis activity with purified PM20D1 in the presence or absence of C20:4-NAT competitor (100 µM). Activity assays were conducted with 100 µM substrates and 100 µg protein in PBS for 1 hr at 37°C. Data are shown as means ± SEM, N = 3/group. All experiments were performed once, with N biological replicates. ***, p<0.001 for the indicated comparison.
Figure 4 with 1 supplement
Changes in N-acyl amino acids upon selective blockade of FAAH in vivo.
(a, b) Fold change of the indicated N-acyl amino acids compared to the control for each of the indicated comparisons from liver (a) or blood (b). For drug treatment, PF-3845 was administered intraperitoneally at 10 mg/kg once (acute) or for three consecutive days (chronic). Tissues were harvested 3 hr after the final dose. No bars are shown for N-acyl amino acids that were below the limit of detection. Data are shown as means ± SEM, N = 4–5 mice/group for each of the indicated comparisons. All experiments were performed once, with N biological replicates. *, p<0.05; **, p<0.01; ***, p<0.001 by ANOVA with Dunnett’s multiple comparisons test versus control animals.
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Figure 4—source data 1
Absolute quantitation of N-acyl amino acids in liver and plasma following FAAH blockade.
- https://cdn.elifesciences.org/articles/55211/elife-55211-fig4-data1-v1.xlsx
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Figure 4—source data 2
Absolute quantitation of N-acyl ethanolamines and N-acyl taurines in wild-type mouse liver.
- https://cdn.elifesciences.org/articles/55211/elife-55211-fig4-data2-v1.xlsx
Figure 4—figure supplement 1
PM20D1 activity in FAAH-KO plasma.
C20:4-Gly hydrolysis activities in from WT, FAAH-KO, or PM20D1-KO plasma. Activity assays were conducted with 100 µM substrates and 100 µg plasma in PBS for 1 hr at 37°C. Data are shown as means ± SEM, N = 3/group. All experiments were performed once, with N biological replicates. ***, p<0.001 versus WT.
Figure 5
Cooperative interactions between PM20D1 and FAAH regulate endogenous N-acyl amino acid levels.
(a) C20:4-Gly hydrolysis activity in livers from PM20D1-WT, PM20D1-KO, or PM20D1-KO treated with PF-3845. (b, c) Relative fold change of the indicated N-acyl amino acids in PM20D1-KO mice or in PM20D1-KO mice treated with PF-3845 versus wild-type mice in liver (b) or blood (c). For drug treatment, PF-3845 was administered intraperitoneally at 10 mg/kg for three consecutive days and tissues were harvested 3 hr after the final dose. No bars are shown for N-acyl amino acids that were below the limit of detection. Data are shown as means ± SEM, N = 4–5 mice/group for each of the indicated comparisons. All experiments were performed once, with N biological replicates. *, p<0.05; **, p<0.01; ***, p<0.001 in color are versus PM20D1-WT levels, whereas those in black are for the indicated comparison by ANOVA with Tukey’s multiple comparison test.
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Figure 5—source data 1
Absolute quantitation of N-acyl amino acids in liver and plasma following PM20D1 or dual PM20D1/FAAH blockade.
- https://cdn.elifesciences.org/articles/55211/elife-55211-fig5-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Mouse line (Mus musculus) | PM20D1-KO | Long et al., 2018 (PMID:29967167) | ||
| Mouse line (M. musculus) | C57BL/6J | Jackson Labs | 000664 | |
| Transfected construct (M. musculus) | PM20D1-flag | Addgene | 84566 | |
| Transfected construct (M. musculus) | FAAH-flag | Origene | MR209084 | |
| Transfected construct (M. musculus) | ACY1-flag | Origene | MR206415 | |
| Transfected construct (M. musculus) | CNDP1-flag | Origene | MR219018 | |
| Transfected construct (M. musculus) | CNDP2-flag | Origene | MR207616 | |
| Transfected construct (M. musculus) | PM20D2-flag | Origene | MR222068 | |
| Cell line (Homo sapiens) | HEK293T | ATCC | CRL-3216 | |
| Antibody | Anti-flag M2, mouse monoclonal | Sigma | F1804 | (1:1000) |
| Antibody | Anti-tubulin, rabbit polyclonal | Abcam | Ab6046 | (1:1000) |
| Chemical compound | PF-3845 | Selleckchem | S2666 | |
| Chemical compound | C20:4-Gly | Cayman | 90051 | |
| Chemical compound | C20:4-Ser | Cayman | 10005455 | |
| Chemical compound | C20:4-Phe | Abcam | Ab141612 | |
| Chemical compound | Arachidonic acid | Sigma-Aldrich | 10931 | |
| Chemical compound | WWL70 | Sigma-Aldrich | SML1641 | |
| Chemical compound | Talabostat | R and D | 3719 | |
| Chemical compound | MAFP | Fisher Scientific | 14-21-5 | |
| Chemical compound | C20:4-NAT | Cayman | 10005537 | |
| Chemical compound | Anandamide | Sigma-Aldrich | A0580 | |
| Chemical compound | C16:0-Phe | Lin et al., 2018 (PMID:29533650) | ||
| Chemical compound | C18:0-Phe | Lin et al., 2018 (PMID:29533650) | ||
| Chemical compound | C20:0-Phe | Lin et al., 2018 (PMID:29533650) | ||
| Chemical compound | C22:6-Phe | Lin et al., 2018 (PMID:29533650) | ||
| Chemical compound | C18:1-Asn | Lin et al., 2018 (PMID:29533650) | ||
| Chemical compound | C18:1-Gly | Cayman | 90269 | |
| Chemical compound | C18:1-Lys | Lin et al., 2018 (PMID:29533650) | ||
| Chemical compound | C18:1-Met | Lin et al., 2018 (PMID:29533650) | ||
| Chemical compound | C18:1-Ser | Cayman | 13058 | |
| Chemical compound | C18:1-Trp | Lin et al., 2018 (PMID:29533650) | ||
| Chemical compound | C18:1-Tyr | Lin et al., 2018 (PMID:29533650) | ||
| Chemical compound | C18:1-Gln | Lin et al., 2018 (PMID:29533650) |
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Cooperative enzymatic control of N-acyl amino acids by PM20D1 and FAAH
eLife 9:e55211.
https://doi.org/10.7554/eLife.55211
