Dataset Information

Identification of palmitoyl protein thioesterase 1 in human THP1 monocytes and macrophages and characterization of unique biochemical activities for this enzyme.

ABSTRACT: 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, macrophages from both species exhibit limited expression of the canonical 2-arachidonoylglycerol (2-AG) hydrolytic enzyme, MAGL. Our previous study showed CES1 was partly responsible for the hydrolysis of 2-AG (50%) and prostaglandin glyceryl esters (PG-Gs) (80-95%) in human THP1 monocytes and macrophages. However, MAGL and other endocannabinoid hydrolases, FAAH, ABHD6, and ABHD12, did not have a role because of limited expression 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 electrophoretic properties similar to those of a serine hydrolase in THP1 cells detected by gel-based ABPP at 31-32 kDa; however, only PPT1 exhibited lipolytic activity and hydrolyzed 2-AG in vitro. Interestingly, PPT1 was strongly expressed in THP1 cells but was significantly less reactive than cathepsin G toward the activity-based probe, fluorophosphonate-biotin. KIAA1363, another serine hydrolase, was also identified in THP1 cells but did not have significant lipolytic activity. On the basis of chemoproteomic profiling, immunodepletion studies, and chemical inhibitor profiles, we 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, PGE2-G, and PGF2?-G, although the catalytic efficiency of hydrolysis of 2-AG by PPT1 was ~10-fold lower than that of CES1. 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 and 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.

SUBMITTER: Wang R

PROVIDER: S-EPMC4102699 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Identification of palmitoyl protein thioesterase 1 in human THP1 monocytes and macrophages and characterization of unique biochemical activities for this enzyme.

Wang Ran R Borazjani Abdolsamad A Matthews Anberitha T AT Mangum Lee C LC Edelmann Mariola J MJ Ross Matthew K MK

Biochemistry 20131018 43

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, macrophages from both species exhibit limited expression of the canonical 2-arachidonoylglycerol (2-AG) hydrolytic enzyme, MAGL. Our previous study showed CES1 was partly responsible for the hydrolysis of 2-AG (50%) and prostagl ...[more]

PMID: 24083319

Similar Datasets

Project description:Two major isoforms of human carboxylesterases (CEs) are found in metabolically active tissues, CES1 and CES2. These hydrolytic enzymes are involved in xenobiotic and endobiotic metabolism. CES1 is abundantly expressed in human liver and monocytes/macrophages, including the THP1 cell line; CES2 is expressed in liver but not in monocytes/macrophages. The cholesteryl ester hydrolysis activity in human macrophages has been attributed to CES1. Here, we report the direct inhibitory effects of several endogenous oxysterols and fatty acids on the CE activity of THP1 monocytes/macrophages and recombinant human CES1 and CES2. Using THP1 whole-cell lysates we found: (1) 27-hydroxycholesterol (27-HC) is a potent inhibitor of carboxylesterase activity (IC50=33 nM); (2) 24(S),25-epoxycholesterol had moderate inhibitory activity (IC(50)=8.1 microM); and (3) cholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 25-hydroxycholesterol each had little inhibitory activity. 27-HC was a partially noncompetitive inhibitor of recombinant CES1 (K(iapp)=10 nM) and impaired intracellular CES1 activity following treatment of intact THP1 cells. In contrast, recombinant CES2 activity was not inhibited by 27-HC, suggesting isoform-selective inhibition by 27-HC. Furthermore, unsaturated fatty acids were better inhibitors of CES1 activity than saturated fatty acids, while CES2 activity was unaffected by any fatty acid. Arachidonic acid (AA) was the most potent fatty acid inhibitor of recombinant CES1 and acted by a noncompetitive mechanism (K(iapp)=1.7 microM); when not complexed to albumin, exogenous AA penetrated intact THP1 cells and inhibited CES1. Inhibition results are discussed in light of recent structural models for CES1 that describe ligand binding sites separate from the active site. In addition, oxysterol-mediated inhibition of CES1 activity was demonstrated by pretreatment of human liver homogenates or intact THP1 cells with exogenous 27-HC, which resulted in significantly reduced hydrolysis of the pyrethroid insecticide bioresmethrin, a CES1-specific xenobiotic substrate. Collectively, these findings suggest that CE activity of recombinant CES1, cell lysates, and intact cells can be impaired by naturally occurring lipids, which may compromise the ability of CES1 to both detoxify environmental pollutants and metabolize endogenous compounds in vivo.

Project description:Oxidative stress in cells and tissues leads to the formation of an assortment of lipid electrophiles, such as the quantitatively important 4-hydroxy-2-trans-nonenal (HNE). Although this cytotoxic aldehyde is atherogenic the mechanisms involved are unclear. We hypothesize that elevated HNE levels can directly inactivate esterase and lipase activities in macrophages via protein adduction, thus generating a biochemical lesion that accelerates foam cell formation and subsequent atherosclerosis. In the present study we examined the effects of HNE treatment on esterase and lipase activities in human THP1 monocytes/macrophages at various physiological scales (i.e., pure recombinant enzymes, cell lysate, and intact living cells). The hydrolytic activities of bacterial and human carboxylesterase enzymes (pnbCE and CES1, respectively) were inactivated by HNE in vitro in a time- and concentration-dependent manner. In addition, so were the hydrolytic activities of THP1 cell lysates and intact THP1 monocytes and macrophages. A single lysine residue (Lys105) in recombinant CES1 was modified by HNE via a Michael addition reaction, whereas the lone reduced cysteine residue (Cys389) was found unmodified. The lipolytic activity of cell lysates and intact cells was more sensitive to the inhibitory effects of HNE than the esterolytic activity. Moreover, immunoblotting analysis using HNE antibodies confirmed that several cellular proteins were adducted by HNE following treatment of intact THP1 monocytes, albeit at relatively high HNE concentrations (>50μM). Unexpectedly, in contrast to CES1, the treatment of a recombinant human CES2 with HNE enhanced its enzymatic activity ∼3-fold compared to untreated enzyme. In addition, THP1 monocytes/macrophages can efficiently metabolize HNE, and glutathione conjugation of HNE is responsible for ∼43% of its catabolism. The functional importance of HNE-mediated inactivation of cellular hydrolytic enzymes with respect to atherogenesis remains obscure, although this study has taken a first step toward addressing this important issue by examining the potential of HNE to inhibit this biochemical activity in a human monocyte/macrophage cell line.

Project description:Carboxylesterases (CES) have important roles in pesticide and drug metabolism and contribute to the clearance of ester-containing xenobiotics in mammals. Tissues with the highest levels of CES expression are the liver and small intestine. In addition to xenobiotics, CES also harness their broad substrate specificity to hydrolyze endobiotics, such as cholesteryl esters and triacylglycerols. Here, we determined if two human CES isoforms, CES1 and CES2, hydrolyze the endocannabinoids 2-arachidonoylglycerol (2AG) and anandamide (AEA), and two prostaglandin glyceryl esters (PG-Gs), which are formed by COX-mediated oxygenation of 2AG. We show that recombinant CES1 and CES2 efficiently hydrolyze 2AG to arachidonic acid (AA) but not amide-containing AEA. Steady-state kinetic parameters for CES1- and CES2-mediated 2AG hydrolysis were, respectively, kcat, 59 and 43 min(-1); Km, 49 and 46 μM; and kcat/Km, 1.2 and 0.93 μM(-1) min(-1). kcat/Km values are comparable to published values for rat monoacylglycerol lipase (MAGL)-catalyzed 2AG hydrolysis. Furthermore, we show that CES1 and CES2 also efficiently hydrolyze PGE2-G and PGF2α-G. In addition, when cultured human THP1 macrophages were treated with exogenous 2AG or PG-G (10 μM, 1 h), significant quantities of AA or PGs were detected in the culture medium; however, the ability of macrophages to metabolize these compounds was inhibited (60-80%) following treatment with paraoxon, the toxic metabolite of the insecticide parathion. Incubation of THP1 cell lysates with small-molecule inhibitors targeting CES1 (thieno[3,2-e][1]benzothiophene-4,5-dione or JZL184) significantly reduced lipid glyceryl ester hydrolase activities (40-50% for 2AG and 80-95% for PG-Gs). Immunodepletion of CES1 also markedly reduced 2AG and PG-G hydrolase activities. These results suggested that CES1 is in part responsible for the hydrolysis of 2AG and PG-Gs in THP1 cells, although it did not rule out a role for other hydrolases, especially with regard to 2AG metabolism since a substantial portion of its hydrolysis was not inactivated by the inhibitors. An enzyme (Mr 31-32 kDa) of unknown function was detected by serine hydrolase activity profiling of THP1 cells and may be a candidate. Finally, the amounts of in situ generated 2AG and PG-Gs in macrophages were enhanced by treating the cells with bioactive metabolites of OP insecticides. Collectively, the results suggest that in addition to MAGL and fatty-acid amide hydrolase (FAAH), which have both been documented to terminate endocannabinoid signaling, CES may also have a role. Furthermore, since PG-Gs have been shown to possess biological activities in their own right, CES may represent an important enzyme class that regulates their in vivo levels.

Dataset Information

Identification of palmitoyl protein thioesterase 1 in human THP1 monocytes and macrophages and characterization of unique biochemical activities for this enzyme.

Publications

Identification of palmitoyl protein thioesterase 1 in human THP1 monocytes and macrophages and characterization of unique biochemical activities for this enzyme.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets