Identification of palmitoyl protein thioesterase 1 in human THP1 monocytes and macrophages and characterization of unique biochemical activities for this enzyme.
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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DOI：
10.1021/bi401138s
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发表时间：
2013-10-29
期刊：
影响因子：
2.9
通讯作者：
Ross, Matthew K.
中科院分区：
文献类型：
--
作者：
Wang, Ran;Borazjani, Abdolsamad;Matthews, Anberitha T.;Mangum, Lee C.;Edelmann, Mariola J.;Ross, Matthew K.
人和小鼠巨噬细胞中的丝氨酸水解酶谱相似但又不同。例如，人巨噬细胞表达高水平的羧酸酯酶1（CES 1），而小鼠巨噬细胞具有最少量的正向鼠CES 1。另一方面，两种物种的巨噬细胞均表现出典型的2-花生四烯酸甘油（2-AG）水解酶MAGL的有限表达。我们以前的研究表明，羧酸酯酶1（CES 1）在人THP 1单核/巨噬细胞中部分负责2-AG（50%）和前列腺素甘油酯（PG-Gs）（80-95%）的水解。然而，MAGL和其他内源性大麻素水解酶，FAAH，ABHD 6和ABHD 12，没有作用，因为有限或没有表达。因此，假设另一种酶负责化学抑制和免疫耗竭CES 1（先前研究）或CES 1基因敲除（本研究）后剩余的2-AG水解活性。在这里，我们确定了两个候选人丝氨酸水解酶THP 1细胞裂解物的活性为基础的蛋白质谱（ABPP）-MudPIT和蛋白质印迹：组织蛋白酶G和棕榈酰蛋白硫酯酶1（PPT 1）。这两种蛋白质表现出类似的电泳特性，丝氨酸水解酶在THP 1细胞检测基于凝胶的ABPP在31-32 kDa的，但是，只有PPT 1表现出脂解活性和水解2-AG在体外。有趣的是，PPT 1在THP 1细胞中高度表达，但对基于活性的探针氟磷酸盐-生物素的反应性显著低于组织蛋白酶G。KIAA 1363是另一种丝氨酸水解酶，也在THP 1细胞中鉴定，但没有显著的脂解活性。在化学蛋白质组学分析、免疫耗竭研究和化学抑制剂谱的基础上，我们估计PPT 1在THP 1细胞系中贡献了32-40%的2-AG水解活性。此外，纯的重组PPT 1催化2-AG、PGE 2-G和PGF 2 α-G的水解，尽管PPT 1对2-AG的催化效率比CES 1低约10倍。PPT 1也对几种有效抑制CES 1的化学抑制剂不敏感，如有机磷毒物和JZL 184。这是第一个报告，文件中的人单核细胞/巨噬细胞系的表达，并显示PPT 1可以水解天然底物2-AG和PG-Gs。这些发现表明，PPT 1可能参与内源性大麻素代谢的特定细胞环境中，并强调了功能冗余往往表现出参与脂质代谢的酶。
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 gel-based ABPP at 31-32 kDa; however, only PPT1 exhibited lipolytic activity and hydrolyzed 2-AG in vitro. Interestingly, PPT1 was highly 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 PGF2α-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.
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影响因子：
3.8
作者：
Lu JY;Hu J;Hofmann SL
通讯作者：
Hofmann SL
DOI：
10.1016/s0165-3806(99)00115-7
发表时间：
1999-12-10
期刊：
DEVELOPMENTAL BRAIN RESEARCH
影响因子：
--
作者：
Isosomppi, J;Heinonen, O;Peltonen, L
通讯作者：
Peltonen, L
影响因子：
3.5
作者：
Lehtovirta, M;Kyttälä, A;Jalanko, A
通讯作者：
Jalanko, A
DOI：
10.1073/pnas.251485198
发表时间：
2001-11-20
影响因子：
11.1
作者：
Gupta, P;Soyombo, AA;Hofmann, SL
通讯作者：
Hofmann, SL
影响因子：
--
作者：
Blankman, Jacqueline L.;Simon, Gabriel M.;Cravatt, Benjamin F.
通讯作者：
Cravatt, Benjamin F.