Identification of palmitoyl protein thioesterase 1 in human THP-1 monocytes/macrophages and characterization of unique biochemical activities for this enzyme

The profiles of serine hydrolases in human and mouse macrophages are similar yet different. For instance, human macrophages express high levels of carboxylesterase 1 (CES1), whereas mouse macrophages have minimal amounts of the orthologous murine CES1. On the other hand, both species' macrophages exhibit limited expression of the canonical 2-arachidonoylglycerol (2-AG) hydrolytic enzyme, MAGL. Our previous study showed carboxylesterase 1 (CES1) was partly responsible for the hydrolysis of 2-AG (50%) and prostaglandin glyceryl esters (PG-Gs) (80-95%) in human THP1 monocytes/macrophages. However, MAGL and other endocannabinoid hydrolases, FAAH, ABHD6 and ABHD12, did not have a role because of either limited or no expression. Thus, another enzyme was hypothesized to be responsible for the remaining 2-AG hydrolysis activity following chemical inhibition and immunodepletion of CES1 (previous study) or CES1 gene knockdown (this study). Here we identified two candidate serine hydrolases in THP1 cell lysates by activity-based protein profiling (ABPP)–MudPIT and western blotting: cathepsin G and palmitoyl protein thioesterase 1 (PPT1). Both proteins exhibited similar electrophoretic properties to a serine hydrolase in THP1 cells detected by estimated that PPT1 contributed 32-40% of 2-AG hydrolysis activity in the THP1 cell line. In addition, pure recombinant PPT1 catalyzed the hydrolysis of 2-AG, PGE 2 -G and PGF 2 α -G, although the catalytic efficiency of 2-AG hydrolysis by PPT1 was ∼ 10-fold lower than CES1's. PPT1 was also insensitive to several chemical inhibitors that potently inhibit CES1, such as organophosphate poisons and JZL184. This is the first report to document the expression of PPT1 in a human monocyte/macrophage cell line and to show PPT1 can hydrolyze the natural substrates 2-AG and PG-Gs. These findings suggest that PPT1 may participate in endocannabinoid metabolism within specific cellular contexts, and highlights the functional redundancy often exhibited by enzymes involved in lipid metabolism. 5 distilled times. After the a microfuge the supernatant, the captured proteins were on-bead digested trypsin according to standard protocols 23 and the tryptic peptides were desalted and analyzed by LTQ LC-MS/MS. Peptides were separated on a 75-μm i.d. × 15 cm reverse phase C18 column (Thermo) controlled by an Ultimate 3000 nanoflow HPLC (Dionex) and eluted using a 55-min gradient from 2%-55% solvent B (99.9% acetonitrile, 0.1% formic acid) at a flow rate of 0.3 μl/min, and further introduced into an LTQ-OrbiTrap Velos mass spectrometer (Thermo Fisher). The mass spectrometer was operated in LTQ data-dependent mode, automatically switching between MS and MS/MS. Full scan MS spectra (300-2,000 amu) were analyzed in a profile mode by the LTQ analyzer. The seven most intense ions in a scan were selected for collision-induced fragmentation (CID) in the LTQ at normalized collision energy of 35% and activation time of 40 ms. The acquired data were analyzed by Proteome Discoverer 1.4 (Thermo Fisher) using SEQUEST algorithm and a human Uniprot database along with the reversed decoy database. Searches were done using a precursor mass tolerance of 1.8 Da and fragment tolerance of 0.5 Da and the following dynamic modifications were included: oxidation on methionine, N-terminal acetylation, and carbamidomethylation on cysteine. The results were filtered using normalized XCorr values for different charge states 24 and were accepted as valid identifications only if the XCorr values were >1.5, >2.5, >3.75 and >4 for singly, doubly, triply and quadruply charged peptides, respectively. Moreover, the maximum delta Cn was 0.15 for peptides. Proteins hydrolyze the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) and two prostaglandin glyceryl esters, PGE 2 -G and PGF 2 α -G, in vitro . These findings suggest that PPT1 may also have a role in degrading endocannabinoids in certain cellular niches, especially in cell types lacking or having limited expression of MAGL, FAAH, ABHD6, ABHD12 and CES1. Furthermore, we characterized the hydrolytic efficiency of PPT1 using both 2-AG and PG-G substrates and compared the kinetic parameters to CES1, MAGL and FAAH. We also investigated the relative contributions of PPT1 and CES1 to the hydrolysis of 2-AG in human THP1 cells using chemical inhibitors, immunoprecipitation of PPT1, and knockdown of CES1 expression using a lentiviral shRNA system. Collectively, the results suggest that PPT1 has the ability to recognize and hydrolyze lipid glyceryl esters in human THP1 cells, and that PPT1 accounts for ∼ 32% of the 2-AG hydrolytic activity while CES1 contributes ∼ 51%.