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首页> 外文期刊>Biochemistry >Identification of palmitoyl protein thioesterase 1 in human thp1 monocytes and macrophages and characterization of unique biochemical activities for this enzyme
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Identification of palmitoyl protein thioesterase 1 in human thp1 monocytes and macrophages and characterization of unique biochemical activities for this enzyme

机译：人thp1单核细胞和巨噬细胞中棕榈酰蛋白硫酯酶1的鉴定和该酶独特的生化活性的表征

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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, PGE_2-G, and PGF_(2α)-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.
机译：人和小鼠巨噬细胞中丝氨酸水解酶的谱图相似但不同。例如，人类巨噬细胞表达高水平的羧酸酯酶1（CES1），而小鼠巨噬细胞具有最小量的直系同源小鼠CES1。另一方面，来自两种物种的巨噬细胞均显示出规范的2-花生四烯酰甘油（2-AG）水解酶MAGL的有限表达。我们以前的研究表明CES1负责人THP1单核细胞和巨噬细胞中2-AG（50％）和前列腺素甘油酯（PG-Gs）（80-95％）的水解。但是，MAGL和其他内源性大麻素水解酶FAAH，ABHD6和ABHD12由于表达受限或没有表达而没有作用。因此，假设另一种酶负责化学抑制和CES1（以前的研究）或CES1基因敲低（这项研究）的免疫消耗后剩余的2-AG水解活性。在这里，我们通过基于活性的蛋白谱分析（ABPP）-MUDPIT和Western blotting在THP1细胞裂解物中鉴定了两个候选丝氨酸水解酶：组织蛋白酶G和棕榈酰蛋白硫酯酶1（PPT1）。两种蛋白在31-32 kDa处均显示出与基于凝胶的ABPP检测到的THP1细胞丝氨酸水解酶相似的电泳特性。然而，只有PPT1表现出脂解活性和在体外水解2-AG。有趣的是，PPT1在THP1细胞中强烈表达，但对基于活性的探针氟代磷酸酯生物素的反应性明显低于组织蛋白酶G。在THP1细胞中也鉴定出另一种丝氨酸水解酶KIAA1363，但没有明显的脂解活性。基于化学计量学分析，免疫耗竭研究和化学抑制剂谱，我们估计PPT1在THP1细胞系中贡献了32-40％的2-AG水解活性。另外，尽管PPT1水解2-AG的催化效率比CES1低约10倍，但纯重组PPT1催化2-AG，PGE_2-G和PGF_（2α）-G的水解。 PPT1对几种有效抑制CES1的化学抑制剂也不敏感，例如有机磷酸盐毒物和JZL184。这是第一个报道PPT1在人单核细胞和巨噬细胞细胞系中表达并显示PPT1可以水解天然底物2-AG和PG-Gs的报告。这些发现表明，PPT1可能参与特定细胞环境中的内源性大麻素代谢，并突显了参与脂质代谢的酶通常表现出的功能冗余。

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