Functional fat body proteomics and gene targeting reveal in vivo functions of Drosophila melanogaster α-Esterase-7

Abstract

Carboxylesterases constitute a large enzyme family in insects, which is involved in diverse functions such as xenobiotic detoxification, lipid metabolism and reproduction. Phylogenetically, many insect carboxylesterases are represented by multienzyme clades, which are encoded by evolutionarily ancient gene clusters such as the α-Esterase cluster. Much in contrast to the vital importance attributed to carboxylesterases in general, the in vivo function of individual α-Esterase genes is largely unknown. This study employs a functional proteomics approach to identify esterolytic enzymes of the vinegar fly Drosophila melanogaster fat body. One of the fat body carboxylesterases, α-Esterase-7, was selected for mutational analysis by gene targeting to generate a deletion mutant fly. Phenotypic characterization of α-Esterase-7 null mutants and transgenic flies, which overexpress a chimeric α-Esterase-7:EGFP gene, reveals important functions of α-Esterase-7 in insecticide tolerance, lipid metabolism and lifespan control. The presented first deletion mutant of any α-Esterase in the model insect D. melanogaster generated by gene targeting not only provides experimental evidence for the endogenous functions of this gene family. It also offers an entry point for in vivo structure-function analyses of α-Esterase-7, which is of central importance for naturally occurring insecticide resistance in wild populations of various dipteran insect species.

Structure and function of an insect α-carboxylesterase (αEsterase7) associated with insecticide resistance

“…Although the physiological substrates of insect α-CBEs are unknown, recent MS results have shown that D. melanogaster αE7 is expressed in the fat body lipid droplet proteome (11), implying a role in lipid or cholesterol metabolism. This work has been extended to show that D. melanogaster αE7-null mutants exhibit reduced lifespan, lowered insecticide tolerance, and reduced lipid storage capacity (6).…”

“…However, α-CBEs, such as αE7 in L. cuprina (LcαE7), confer a significant protective effect on insects owing to their ability to bind and slowly hydrolyze OPs (6). It has also been shown that this CBE-mediated OP resistance can be increased through G137D and W251L point mutations (4, 7).Insect α-CBEs, including LcαE7, are thought to play important physiological roles in lipid and xenobiotic metabolism (6,8). They are one of the most abundant protein families in insects, with between 20 and 40 active CBEs predicted to be expressed by the fruit fly Drosphila melanogaster (9, 10).…”

“…Insect α-CBEs, including LcαE7, are thought to play important physiological roles in lipid and xenobiotic metabolism (6,8). They are one of the most abundant protein families in insects, with between 20 and 40 active CBEs predicted to be expressed by the fruit fly Drosphila melanogaster (9, 10).…”

“…Soaking of LcαE7 crystals in OPs led to the capture of a crystallographic snapshot of LcαE7 in its phosphorylated state, which allowed comparison with acetylcholinesterase and rationalization of its ability to protect insects against the effects of OPs. Finally, inspection of the active site of LcαE7 reveals an asymmetric and hydrophobic substrate binding cavity that is well-suited to fatty acid methyl esters, which are hydrolyzed by the enzyme with specificity constants (∼10 6 M −1 s −1 ) indicative of a natural substrate.…”

“…OPs inhibit acetylcholinesterase (AChE) at cholinergic synapses in the central and peripheral nervous systems, which leads to interminable nerve signal transduction and death (5). However, α-CBEs, such as αE7 in L. cuprina (LcαE7), confer a significant protective effect on insects owing to their ability to bind and slowly hydrolyze OPs (6). It has also been shown that this CBE-mediated OP resistance can be increased through G137D and W251L point mutations (4,7).…”