Vivo Functions of Drosophila Melanogaster &Alpha
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Insect Biochemistry and Molecular Biology 42 (2012) 220e229 Contents lists available at SciVerse ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb Functional fat body proteomics and gene targeting reveal in vivo functions of Drosophila melanogaster a-Esterase-7 Ruth Birner-Gruenberger c, Iris Bickmeyer a, Julia Lange a,1, Philip Hehlert a,b, Albin Hermetter d, Manfred Kollroser e, Gerald N. Rechberger f, Ronald P. Kühnlein a,b,* a Abteilung Molekulare Entwicklungsbiologie, Max-Planck-Institut für biophysikalische Chemie, Am Fassberg 11, 37077 Göttingen, Germany b Forschungsgruppe Molekulare Physiologie, Max-Planck-Institut für biophysikalische Chemie, Am Fassberg 11, 37077 Göttingen, Germany c Research Group Functional Proteomics, Institute of Pathology, and Proteomics Core Facility, Center of Medical Research, Medical University of Graz, Stiftingtalstrasse 24, 8010 Graz, Austria d Institute of Biochemistry, Graz University of Technology, Petersgasse 12, 8010 Graz, Austria e Institute of Forensic Medicine, Medical University of Graz, Universitätsplatz 4, 8010-Graz, Austria f Institute of Molecular Biosciences, Karl-Franzens-University of Graz, Humboldtstrasse 50, 8010-Graz, Austria article info abstract Article history: Carboxylesterases constitute a large enzyme family in insects, which is involved in diverse functions such Received 18 September 2011 as xenobiotic detoxification, lipid metabolism and reproduction. Phylogenetically, many insect carbox- Received in revised form ylesterases are represented by multienzyme clades, which are encoded by evolutionarily ancient gene 7 December 2011 clusters such as the a-Esterase cluster. Much in contrast to the vital importance attributed to carbox- Accepted 8 December 2011 ylesterases in general, the in vivo function of individual a-Esterase genes is largely unknown. This study employs a functional proteomics approach to identify esterolytic enzymes of the vinegar fly Drosophila Keywords: melanogaster fat body. One of the fat body carboxylesterases, a-Esterase-7, was selected for mutational Drosophila fl a a-Esterase-7 analysis by gene targeting to generate a deletion mutant y. Phenotypic characterization of -Esterase-7 fl a Mutant null mutants and transgenic ies, which overexpress a chimeric -Esterase-7:EGFP gene, reveals Insecticide important functions of a-Esterase-7 in insecticide tolerance, lipid metabolism and lifespan control. The Proteomics presented first deletion mutant of any a-Esterase in the model insect D. melanogaster generated by gene Fat body 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 a-Esterase-7, which is of central importance for naturally occurring insecticide resistance in wild populations of various dipteran insect species. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction family, which promotes organismal diversity during Metazoan evolution (reviewed in Oakeshott et al., 1999). Insect genomes are Carboxylesterases (CEs) play essential roles in lipid and xeno- particularly rich in CE genes and this complexity has been corre- biotic metabolism throughout the animal kingdom. The broad lated with important organismic functions such as the insecticide substrate spectrum and their complex expression profiles reflect resistance in the malaria vector Anopheles gambiae (Ranson et al., functional diversification among members of the CE multigene 2002). Overexpression has been described as one of two CE- mediated strategies for metabolic detoxification of organophos- phorous (OP) insecticides found in insecticide-resistant pop- Abbreviations: CE, carboxylesterase; D.m., Drosophila melanogaster; LD, lipid ulations of dipteran species (reviewed in Bass and Field, 2011; droplet; L.c., Lucilia cuprina; NBD-HE-HP, O-((6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) Oakeshott et al., 2005). This strategy makes use of OP seques- amino)hexanoyl)-aminoethyl-O-(n-hexyl)phosphonic acid p-nitrophenyl ester; OP, organophosphorous; qRT-PCR, quantitative reverse transcription PCR; s.e.m., stan- tering to protect against the neurotoxic effect of the insecticides dard error of the mean. on their target, acetylcholinesterase. Alternatively, CE mutants * Corresponding author. Forschungsgruppe Molekulare Physiologie, Max-Planck- with novel substrate spectrum are employed to detoxify OP Institut für biophysikalische Chemie, Am Fassberg 11, 37077 Göttingen, Germany. insecticides by hydrolytic cleavage (see Section 4). Generally, Tel.: þ49 (0) 551 2011045, fax: þ49 (0) 551 2011755. much in contrast to the vital general importance of CEs, functional E-mail address: [email protected] (R.P. Kühnlein). 1 Present address: ICON Clinical Research, Heinrich-Hertz-Straße 26, 63225 information about the in vivo role of particular CE family members Langen, Germany. is limited. 0965-1748/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2011.12.004 R. Birner-Gruenberger et al. / Insect Biochemistry and Molecular Biology 42 (2012) 220e229 221 Electrophoretic zymograms of adult Drosophila melanogaster determined (Bio-Rad Protein Assay #500-0006) and adjusted to flies identified 22 soluble esterase isozymes, among them ten CEs 0.5 mg/ml. Homogenates were incubated with 20 mM the fluorescent (Healy et al., 1991). On the other hand computational annotation of activity-based probe O-((6-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) the fly genome lists 35 genes, which encode enzymes with exper- amino)hexanoyl)aminoethyl-O-(n-hexyl)phosphonic acid p-nitro- imentally supported or predicted carboxylesterase activity (GO phenyl ester (NBD-HE-HP) (synthesized as published in Oskolkova term 0004091; Tweedie et al., 2009; www.flybase.org). Among et al., 2003; Schmidinger et al., 2005) and 1 mM Triton X-100 for 2 h them are the ten genes (a-Est1-10) of the a-Esterase cluster located at 28 C with mild shaking (550 rpm), quick-frozen in liquid on the right arm of chromosome 3. Recently, the first two a-Est nitrogen and kept at À20 C until further use. Unlabeled controls genes were assigned to esterase isozyme activities, i.e. a-Est5 to were also tested to exclude autofluorescent proteins. Proteins were EST9 and a-Est7 to EST23 by peptide fingerprinting combined with precipitated with 10% trichloroacetic acid (w/v) and subjected to recombinant protein expression (Campbell et al., 2003). This SDS-PAGE in Tris/glycine buffer (5% stacking gel, 10% resolving gel) approach started to link the wealth of biochemical information on or 2D-gel electrophoresis (pH 3e10 NL IPG strips; 10% SDS-PAGE) EST isozymes to the molecular identity of the corresponding genes. for detection of fluorescent NBD-HE-HP-labeled protein bands/ However, the in vivo roles of individual a-Est cluster genes remains spots by laser scanning as described (Birner-Gruenberger et al., obscure as none of them has been functionally characterized by 2005). Relative fluorescence intensity spot volumes were deter- mutant analysis. mined using Delta2D 4.0.8 software for spot detection and quan- Correlational analyses support diverse functions for a-Est genes. titation after normalization on the total fluorescence intensity of all For example, a-Est1, a-Est2 and a-Est8 have a pronounced expres- detected spots. sion in adult fly heads (Campbell et al., 2003; Chintapalli et al., Protein spots were excised from gels and tryptically digested 2007). In heads of adult males a-Est1 and a-Est8 are transcrip- according to the method by Shevchenko et al. (1996). Peptide tionally up-regulated in fly lines selected for aggressive behavior extracts were dissolved in 0.1% formic acid, separated by nano- (Dierick and Greenspan, 2006) and a-Est2 behaves as a mating- HPLC and analyzed by LC-MS/MS with a Thermo-Finnigan LTQ responsive gene (Ellis and Carney, 2010). Moreover, the a-EST2 linear iontrap mass spectrometer as described (Birner-Gruenberger and a-EST7 proteins are members of the fat body lipid droplet et al., 2008). Proteins were identified by searching the D. mela- proteome (Beller et al., 2006) suggesting a role in lipid metabolism. nogaster NCBI non-redundant public database with SpectrumMill Notably, seven of the ten a-Esterase cluster genes are at least Rev. 03.03.078 (Agilent). Acceptance parameters were two or more moderately expressed in the Drosophila fat body (Chintapalli et al., identified distinct peptides according to the parameters defined by 2007). Multiple a-Est gene expression in the fat body highlights its Carr et al. (2004) except for the single high confident peptide of a- central role of this organ as a metabolic hub in insects. Besides its EST2 (see Fig. S1). most prominent role as major energy storage organ, the fat body is involved in vital organismal functions such as immunity, growth 2.2. Esterase activity assay control and detoxification. The role of particular esterase activities in these processes awaits clarification. Fat body cell homogenates were essentially prepared as This study employs a functional proteomics approach to described in Section 2.1. Specific total esterase activity was deter- molecularly identify esterases of the Drosophila fat body. Tissue mined from the linear increase of absorbance at 405 nm of p- profiling for esterase activities with subsequent enzyme identifi- nitrophenolate released from 100 mM p-nitrophenyl butyrate in cation via mass spectrometry has been successfully applied to 20 mM TriseHCl, pH8, 150 mM NaCl and 0.01%Triton X-100 (Shirai characterize