Dataset Information

DAGL-Beta Functions as a PUFA-Specific Triacylglycerol Lipase in Macrophages.

ABSTRACT: Here, we apply quantitative chemical proteomics and untargeted lipidomics to assign a polyunsaturated fatty acid (PUFA)-specific triacylglycerol (TAG) lipase activity for diacylglycerol lipase-beta (DAGLβ) in macrophages. We demonstrate that DAGLβ but not DAGLα is expressed and active in bone marrow-derived macrophages (BMDMs) as determined by activity-based protein profiling analysis of SILAC BMDMs. Genetic disruption of DAGLβ resulted in accumulation of cellular TAGs composed of PUFA but not saturated/low unsaturated fatty acid counterparts, which is recapitulated in wild-type macrophages treated with a DAGLβ-selective inhibitor. Biochemical assays with synthetic substrates confirm PUFA-TAGs as authentic DAGLβ substrates. In summary, our findings identify DAGLβ as a PUFA-specific TAG lipase in primary macrophages.

SUBMITTER: Shin M

PROVIDER: S-EPMC7093258 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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DAGL-Beta Functions as a PUFA-Specific Triacylglycerol Lipase in Macrophages.

Shin Myungsun M Ware Timothy B TB Hsu Ku-Lung KL

Cell chemical biology 20200127 3

Here, we apply quantitative chemical proteomics and untargeted lipidomics to assign a polyunsaturated fatty acid (PUFA)-specific triacylglycerol (TAG) lipase activity for diacylglycerol lipase-beta (DAGLβ) in macrophages. We demonstrate that DAGLβ but not DAGLα is expressed and active in bone marrow-derived macrophages (BMDMs) as determined by activity-based protein profiling analysis of SILAC BMDMs. Genetic disruption of DAGLβ resulted in accumulation of cellular TAGs composed of PUFA but not s ...[more]

PMID: 31991095

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Project description:BackgroundLipolytic enzymes of hyperthermophilic archaea generally prefer small carbon chain fatty acid esters (C2-C12) and are categorized as esterases. However, a few have shown activity with long-chain fatty acid esters, but none of them have been classified as a true lipase except a lipolytic enzyme AFL from Archaeglobus fulgidus. Thus, our main objective is to engineer an archaeal esterase into a true thermostable lipase for industrial applications. Lipases which hydrolyze long-chain fatty acid esters display an interfacial activation mediated by the lid domain which lies over active site and switches to open conformation at the oil-water interface. Lid domains modulate enzyme activities, substrate specificities, and stabilities which have been shown by protein engineering and mutational analyses. Here, we report engineering of an uncharacterized monoacylglycerol lipase (TON-LPL) from an archaeon Thermococcus onnurineus (strain NA1) into a triacylglycerol lipase (rc-TGL) by replacing its 61 N-terminus amino acid residues with 118 residues carrying lid domain of a thermophilic fungal lipase-Thermomyces lanuginosus (TLIP).ResultsTON-LPL and rc-TGL were cloned and overexpressed in E. coli, and the proteins were purified by Ni-NTA affinity chromatography for biochemical studies. Both enzymes were capable of hydrolyzing various monoglycerides and shared the same optimum pH of 7.0. However, rc-TGL showed a significant decrease of 10 °C in its optimum temperature (Topt). The far UV-CD spectrums were consistent with a well-folded α/β-hydrolase fold for both proteins, but gel filtration chromatography revealed a change in quaternary structure from trimer (TON-LPL) to monomer (rc-TGL). Seemingly, the difference in the oligomeric state of rc-TGL may be linked to a decrease in temperature optimum. Nonetheless, rc-TGL hydrolyzed triglycerides and castor oil, while TON-LPL was not active with these substrates.ConclusionsHere, we have confirmed the predicted esterase activity of TON-LPL and also performed the lid engineering on TON-LPL which effectively expanded its substrate specificity from monoglycerides to triglycerides. This approach provides a way to engineer other hyperthermophilic esterases into industrially suitable lipases by employing N-terminal domain replacement. The immobilized preparation of rc-TGL has shown significant activity with castor oil and has a potential application in castor oil biorefinery to obtain value-added chemicals.

Dataset Information

DAGL-Beta Functions as a PUFA-Specific Triacylglycerol Lipase in Macrophages.

Publications

DAGL-Beta Functions as a PUFA-Specific Triacylglycerol Lipase in Macrophages.

Similar Datasets

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