Abstract
Hydrolysis of triacylglycerols and cholesteryl esters is a key event in energy homeostasis of animals. However, many lipolytic activities still await their molecular identification. Here we report on a novel tool for concomitant analysis of lipases in complex proteomes. Fluorescent activity tags mimicking lipid substrates were used to label the proteome of mouse adipose tissue. Analysis by two-dimensional gel electrophoresis and LC-MS/MS led to the identification of all known intracellular lipases as well as a number of novel candidates. One of them was recently shown to be involved in triacylglycerol mobilization in adipocytes and therefore named adipose triglyceride lipase. Functional characterization of expressed enzymes demonstrated that lipolytic and esterolytic activities could be well discriminated. Thus our results show the first map of the lipolytic proteome of mouse adipose tissue and demonstrate the general applicability of our method for rapid profiling and identification of lipolytic activities in complex biological samples.
Highlights
Hydrolysis of triacylglycerols and cholesteryl esters is a key event in energy homeostasis of animals
We have developed a set of activity-based probes to target lipolytic enzymes and applied it for mapping the lipolytic proteome of mouse adipose tissue
Over other published affinity-based probes for functional proteomics, which contain bulky and charged fluorophores for visualization, the small and uncharged NBD fluorophore has the advantage to not interfere with the isoelectric focusing step of 2D gel electrophoresis. It appears to be beneficial for the recognition of the probes by lipolytic enzymes, which are specific for hydrophobic substrates
Summary
Animals—HSL-deficient (HSL-ko) mice were generated by targeted homologous recombination [26]. Lipase and Esterase Activity Assays—p-Nitrophenyl laurate was used as artificial substrate to measure lipolytic activity of both mouse adipose tissue and cytoplasmic extracts of transfected COS-7 cells as described previously [34]. P-Nitrophenyl acetate was used as a model substrate to measure esterase activity in cytoplasmic extracts of transfected COS-7 cells as described previously [35]. Radioactive assays for measurement of neutral triacylglycerol lipase and cholesteryl esterase activity in cytosolic extracts of transfected COS-7 cells contained 100 nmol of triolein/assay with [9,10-3H]triolein (12,000 cpm/nmol, PerkinElmer Life Sciences) as radioactive tracer or 10 nmol of cholesteryl oleate and the corresponding tracer cholesteryl [9,10-3H]oleate (50,000 cpm/nmol), respectively, and were performed as described by Holm et al [36]
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