Overexpression of Glutamate-Cysteine Ligase Extends Life Span in Drosophila melanogaster
The hypothesis that overexpression of glutamate-cysteine ligase (GCL), which catalyzes the rate-limiting reaction in de novo glutathione biosynthesis, could extend life span was tested in the fruit fly, Drosophila melanogaster. The GAL4-UAS binary transgenic system was used to generate flies overexpressing either the catalytic (GCLc) or modulatory (GCLm) subunit of this enzyme, in a global or neuronally targeted pattern. The GCL protein content of the central nervous system was elevated dramatically in the presence of either global or neuronal drivers. GCL activity was increased in the whole body or in heads, respectively, of GCLc transgenic flies containing global or neuronal drivers. The glutathione content of fly homogenates was increased by overexpression of GCLc or GCLm, particularly in flies overexpressing either subunit globally, or in the heads of GCLc flies possessing neuronal drivers. Neuronal overexpression of GCLc in a long-lived background extended mean and maximum life spans up to 50%, without affecting the rate of oxygen consumption by the flies. In contrast, global overexpression of GCLm extended the mean life span only up to 24%. These results demonstrate that enhancement of the glutathione biosynthetic capability, particularly in neuronal tissues, can extend the life span of flies, and thus support the oxidative stress hypothesis of aging. The hypothesis that overexpression of glutamate-cysteine ligase (GCL), which catalyzes the rate-limiting reaction in de novo glutathione biosynthesis, could extend life span was tested in the fruit fly, Drosophila melanogaster. The GAL4-UAS binary transgenic system was used to generate flies overexpressing either the catalytic (GCLc) or modulatory (GCLm) subunit of this enzyme, in a global or neuronally targeted pattern. The GCL protein content of the central nervous system was elevated dramatically in the presence of either global or neuronal drivers. GCL activity was increased in the whole body or in heads, respectively, of GCLc transgenic flies containing global or neuronal drivers. The glutathione content of fly homogenates was increased by overexpression of GCLc or GCLm, particularly in flies overexpressing either subunit globally, or in the heads of GCLc flies possessing neuronal drivers. Neuronal overexpression of GCLc in a long-lived background extended mean and maximum life spans up to 50%, without affecting the rate of oxygen consumption by the flies. In contrast, global overexpression of GCLm extended the mean life span only up to 24%. These results demonstrate that enhancement of the glutathione biosynthetic capability, particularly in neuronal tissues, can extend the life span of flies, and thus support the oxidative stress hypothesis of aging. The tripeptide, γ-glutamylcysteinylglycine, i.e. glutathione (GSH), 2The abbreviations used are: GSHreduced glutathioneGCLcglutamate-cysteine ligase catalytic subunitGCLmglutamate-cysteine ligase modulatory subunitHPLChigh-performance liquid chromatographyGSGSH synthaseγ-GCγ-glutamylcysteineSODsuperoxide dismutase is a versatile biological reductant, which is often present in millimolar amounts in tissues (1Meister A. Methods Enzymol. 1995; 251: 3-7Crossref PubMed Scopus (400) Google Scholar, 2Sies H. Free Radic. Biol. Med. 1999; 27: 916-921Crossref PubMed Scopus (1369) Google Scholar). It serves multiple physiological functions, including acting as a substrate in the enzymatic reduction of peroxides, as a conjugant to xenobiotics to facilitate their export from cells, in transport of amino acids, thiolation/dethiolation of proteins, and maintenance of cellular redox state (3Meister A. Anderson M.E. Annu. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5970) Google Scholar, 4Ziegler D.M. Annu. Rev. Biochem. 1985; 54: 305-329Crossref PubMed Scopus (692) Google Scholar, 5Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3653) Google Scholar, 6Dröge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7529) Google Scholar). GSH is synthesized de novo by the consecutive action of two enzymes: glutamate-cysteine ligase (GCL, EC 6.3.2.2), which catalyzes the first and rate-limiting step in de novo synthesis (Reaction 1), and GSH synthase (GS, EC 6.3.2.3), which links glycine to γ-glutamylcysteine (γ-GC) to form GSH (Reaction 2) (4Ziegler D.M. Annu. Rev. Biochem. 1985; 54: 305-329Crossref PubMed Scopus (692) Google Scholar). The primary determinants of the rate of GSH synthesis are the availability of substrates, activity of GCL, and feedback inhibition of GCL by GSH (7Griffith O.W. Free Radic. Biol. Med. 1999; 27: 922-935Crossref PubMed Scopus (976) Google Scholar). L-Glutamate+L-cysteine+ATP↔L-γ-glutamyl-L-cysteine+ADP+PiREACTION 1 L-γ-Glutamyl-L-cysteine+glycine+ATP↔GSH+ADP+PiREACTION 2 reduced glutathione glutamate-cysteine ligase catalytic subunit glutamate-cysteine ligase modulatory subunit high-performance liquid chromatography GSH synthase γ-glutamylcysteine superoxide dismutase Mammalian GCL is a heterodimeric enzyme, consisting of a catalytic subunit, GCLc, and a regulatory or modulatory subunit, GCLm, which are the products of two distinct genes (8Huang C.S. Anderson M.E. Meister A. J. Biol. Chem. 1993; 268: 20578-20583Abstract Full Text PDF PubMed Google Scholar). The GCLc subunit can catalyze the formation of γ-GC in the absence of GCLm, but its activity is increased substantially by covalent interactions with GCLm. GCL activity is increased under oxidative stress, i.e. an imbalance between antioxidants and prooxidants, by the formation of disulfide bonds between the GCLm and GCLc subunits (7Griffith O.W. Free Radic. Biol. Med. 1999; 27: 922-935Crossref PubMed Scopus (976) Google Scholar, 9Shi M.M. Kugelman A. Iwamoto T. Tian L. Forman H.J. J. Biol. Chem. 1994; 269: 26512-26517Abstract Full Text PDF PubMed Google Scholar). Oxidants can also differentially up-regulate the transcription of GCL genes, thereby providing a protective mechanism against oxidative stress-induced cellular dysfunction. A considerable body of evidence indicates that GSH provides protection against oxidative stress in vivo. For instance, experimental depletion of GSH by l-buthionine-SR-sulfoximine, an inhibitor of GCL activity, results in enhanced vulnerability to oxidative challenges (10Mårtensson J. Jain A. Stole E. Frayer W. Auld P.A.M. Meister A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9360-9364Crossref PubMed Scopus (181) Google Scholar). Transfection of cells with GCLc cDNAs elevates GSH levels as well as resistance to oxidative stress (11Moore W.R. Anderson M.E. Meister A. Murata K. Kimura A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1461-1464Crossref PubMed Scopus (74) Google Scholar). The amount of GSH and the ability to maintain its steady-state level under oxidative stress tend to decrease in some tissues with increasing age, which is hypothesized to be reflective of an impairment of the mechanisms of GSH synthesis (12Bharath S. Hsu M. Kaur D. Rajagopalan S. Andersen J.K. Biochem. Pharmacol. 2002; 64: 1037-1048Crossref PubMed Scopus (344) Google Scholar). Indeed, a recent study of the housefly indicates that GCL has significantly higher affinities for its substrates in young than in old flies, suggesting the occurrence of a catalytic inefficiency of the rate-limiting step of glutathione biosynthesis (13Toroser D. Sohal R.S. Biochem. Biophys. Res. Commun. 2005; 326: 586-593Crossref PubMed Scopus (17) Google Scholar). Several lines of evidence suggest that the level of oxidative stress is enhanced during the aging process (14Sohal R.S. Mockett R.J. Orr W.C. Free Radic. Biol. Med. 2002; 33: 575-586Crossref PubMed Scopus (524) Google Scholar). For instance, rates of mitochondrial reactive oxygen species generation and steady-state amounts of the products of reactions between reactive oxygen speices and macromolecules such as nucleic acids, proteins, and lipids are relatively high in aged animals. Furthermore, aged organisms are more susceptible to induced oxidative damage than the young. In this context, the principal objective of this study was to examine the role of GSH and oxidative stress in the aging process, using Drosophila melanogaster as a model organism. Molecular cloning of both a ∼31-kDa regulatory subunit and a ∼80-kDa catalytic subunit has been accomplished in Drosophila (15Fraser J.A. Saunders R.D.C. McLellan L.I. J. Biol. Chem. 2002; 277: 1158-1165Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Accordingly, the specific hypothesis was that overexpression of Drosophila GCL catalytic and modulatory subunits, using global and tissue-specific promoters, would enhance the ability to synthesize GSH and prolong the life span. Chemicals and Reagents—HPLC calibration standards (γ-GC and GSH) were obtained from Sigma. o-Phosphoric acid was purchased from EMD Science (Gibbstown, NJ). Milli-Q grade water was prepared by reverse osmosis on a Millipore® water-purification system. All chemicals were either analytical grade or of the highest purity commercially available. Construction of pP[UAST]-GCLc and pP[UAST]-GCLm—The EcoRI-BamHI fragment of the GCLc gene was digested from AT05811 cDNA clone (BDGP) plasmid DNA and subcloned into corresponding sites of the pBluescript SKII(–) vector. The 5′-EcoRI site was derived from the polylinker of the POT7 vector of the AT05811 clone, whereas the BamHI site was located in the 3′-untranslated region of the GCLc mRNA. The resulting construct contained 42 bp of 5′-untranslated sequence of the GCLc gene, the entire coding region (2157 bp) and 374 bp downstream from the GCLc gene stop codon. This 2573-bp GCLc fragment was subsequently excised from the EcoRI and XbaI sites, which flank the sequence, and introduced into corresponding sites in the polylinker of the pP[UAST] vector. For the construction of pP[UAST]-GCLm, the EcoRI-XhoI fragment of the GCLm gene was extracted from GH01757 cDNA clone (BDGP) and subcloned into the corresponding EcoRI-XhoI sites of the polylinker of pP[UAST] vector. The 5′-EcoRI and the 3′-XhoI sites were both provided by the polylinker of pOT2A vector of the GH01757 clone. The resulting construct contained 77 bp of 5′-untranslated sequence of the GCLm mRNA, the entire coding domain (857 bp) and 202 bp downstream from the stop codon. Drosophila Strains, P-mediated Transformation, and Life Span Studies—The y w strain used in these studies contains the X chromosome markers yellow (y) and white (w) and has been maintained in this laboratory for >12 years. The Tub-GAL4, elav-GAL4, Appl-GAL4, and D42-GAL4 driver lines were kindly supplied by Blanka Rogina (University of Connecticut Health Science Center). DNA preparations of pP[UAST]-GCLc and pP[UAST]-GCLm constructs were injected along with pTurbo plasmid (16Tomlinson A. Kimmel B.E. Rubin G.M. Cell. 1988; 55: 771-784Abstract Full Text PDF PubMed Scopus (129) Google Scholar) carrying transposase into Drosophila embryos of the y w recipient strain. Southern analysis and chromosome mapping were used to confirm the presence of single individual transgenes at distinct loci after microinjection. The collection of transgenic lines was further expanded using transient remobilization by the Δ2–3 element (17Robertson H.M. Preston C.R. Phillis R.W. Johnson-Schlitz D.M. Benz W.K. Engels W.R. Genetics. 1988; 118: 461-470Crossref PubMed Google Scholar) following crosses with CyO/Sp; Sb Δ2–3/TM6, Ubx flies. All transgenic lines, including the driver lines, were back-crossed a minimum of six times to the same yw isoline, to ensure that the genetic backgrounds were equivalent. Experimental flies were then obtained from crosses between virgin females containing the GAL4 driver and males containing the UAS-GCLc or UAS-GCLm responder transgene. Control flies containing the GCL transgene alone or the GAL4 driver alone were obtained by substituting the parental yw strain for either the driver or responder strain in parallel crosses. Life-span studies were performed as described previously (18Mockett R.J. Orr W.C. Sohal R.S. Methods Enzymol. 2002; 349: 213-220Crossref PubMed Scopus (12) Google Scholar). Immunoblot Analysis—Antibodies were raised against recombinant GCLc and GCLm proteins. Fragments of GCLc and GCLm genes comprising the whole-length coding sequences were inserted in pProEx vector (Invitrogen) in-frame with histidine-6x sequence. Recombinant proteins were expressed in Escherichia coli strain DH5-α and purified by nickel-agarose chromatography. Purified proteins were sent to Covance Research Products (Denver, PA) for the preparation of rabbit antisera. Immunoblot analysis was performed as described previously (19Radyuk S.N. Klichko V.I. Orr W.C. Arch. Insect Biochem. Physiol. 2000; 45: 79-93Crossref PubMed Scopus (27) Google Scholar). Primary antibodies (∼0.2–0.5 mg/ml) were diluted 1:5,000 (GCLc) or 1:20,000 (GCLm). Anti-actin antibodies used as a loading control were purchased from MP Biomedicals (Aurora, OH). The secondary antibodies were horseradish peroxidase anti-rabbit and anti-mouse conjugates (Invitrogen and Amersham Biosciences, respectively). HPLC-based Assays of GCL Enzyme Activity and Glutathione Content in Fly Homogenates—GCL enzyme activity was measured as described previously for houseflies (13Toroser D. Sohal R.S. Biochem. Biophys. Res. Commun. 2005; 326: 586-593Crossref PubMed Scopus (17) Google Scholar). GSH present in the fly homogenates at the time of sacrifice was also quantified by HPLC, using a Shimadzu Class VP solvent delivery system and CoulArray 6500 electrochemical detector, as described previously (20Rebrin I. Bayne A.C.V. Mockett R.J. Orr W.C. Sohal R.S. Biochem. J. 2004; 382: 131-136Crossref PubMed Scopus (67) Google Scholar). Oxygen Consumption—Respiration rates were determined for three to five groups of 25 male flies per genotype, at ages 13–20 days, using an Oxzilla Dual Absolute and Differential Oxygen Analyzer (Sable Systems International, Las Vegas, NV). Flies in each group were placed in a respiration chamber with nearly the same dimensions as standard vials used for life-span studies. Air was passed through the chamber and baselining system in alternating 10-min intervals, with a flow rate of 50 ml·min–1 and sampling interval of 2 s. The calculated rate of oxygen consumption for each vial of flies was the average of five to six consecutive chamber minus baseline estimations, after subtraction from oxygen concentration in a parallel reference channel. Statistical Analysis—Enzyme activities (overexpressor versus control) were compared by unpaired Student's t tests using Microsoft Excel software. Rates of oxygen consumption were compared by analysis of variance, with on The of between was determined for each experimental and its corresponding and using tests the mean life spans and times of were compared by analysis of variance, with between GCLc and the corresponding and of GCLc and of the recombinant element carrying UAS-GCLc and carrying UAS-GCLm Southern analysis was performed to fly lines carrying which were to specific by For each three to transgenic lines with a single were and back-crossed a minimum of six times into a reference yw background to ensure genetic The responder carrying the transgenes were to the following driver Tub-GAL4, which and Appl-GAL4, and which distinct of neuronal of GCLc and GCLm by was increased by to and respectively, in with as by analysis and the GCL enzyme activity neuronal were only in GCL activity were in whole body homogenates the same was GCL protein levels were determined by in GCL content were in proteins extracted from heads, using either global or neuronal of GCLc overexpression GCL enzyme activity was increased significantly in heads but in the and of flies analysis of GCL proteins were from heads of GCLc transgenic flies, and of each was using primary antibodies raised against Drosophila GCLc or as a loading control was a in the GCL protein content of flies containing the or driver in with the GCLc responder of the obtained for against loading activities in heads and of flies. are expressed as the per of protein and are mean of three to in activity and t for each experimental group are on with the corresponding Neuronal of GCLc Life of life spans between experimental and control groups were using male flies per Experimental flies contained of distinct UAS-GCLc transgenes and either the Tub-GAL4, Appl-GAL4, or for each transgene the UAS-GCLc transgene without the driver and the driver without its transgene The experimental groups overexpressing GCLc with the in life in with their In the presence of and D42-GAL4 neuronal was a and in on tests or of mean life were also with the but these were only for the mean and maximum life spans were increased by up to and the on maximum life span were only for two of for the and D42-GAL4 drivers. the of on life span was D42-GAL4 life spans of GCLc transgenic in a life spans of GCLc transgenic flies in w w in a In to the results obtained for GCLc, neuronal overexpression of the regulatory subunit of glutamate-cysteine GCLm, on the driver was a but of life span was to for mean life GSH or overexpression of GCLc or GCLm in an increased content of from a of the transgenic flies were by was a in GSH content in experimental flies containing the with the global Flies with the transgene but in GSH levels the that neuronal only a of the body with this GSH content was increased in the heads but only in the of flies In GCLm containing the global the in GSH content from content in heads and in of flies. are expressed per of fly and are mean of three were calculated from the of heads, and heads of fly determined by of of in GSH content and t for each experimental group are on with the corresponding The GSH content of whole body homogenates of control flies was to the amount measured in whereas between control and experimental groups were between for heads and for to for three Oxygen and to Experimental of oxygen consumption were compared between flies containing both the or driver and the responder transgene in and containing either the driver or responder transgene was between experimental and control groups for either driver suggesting that the of life span was an resulting from respiration rates in the long-lived flies. were on stress resistance for of the at of age, and flies increased resistance to and and of flies to for each of experimental versus control on flies per results were obtained with and transgenes in with each driver of flies to for flies per results were obtained with the and The of this study was that overexpression of the catalytic subunit of glutamate-cysteine GCLc, in tissues of the extended the mean and maximum life spans of D. melanogaster by up to 50%, whereas global overexpression on In contrast, GCLm overexpression in the on life whereas global overexpression in a in The raised by these is overexpression of GCLc, in the the life span of flies. The of aging Drosophila is to a of including amounts of neuronal and and a of which be products of mitochondrial M.M. J. M. PubMed Scopus Google Scholar). the level of oxidative stress in flies, by rates of mitochondrial disulfide and amounts of oxidative with (14Sohal R.S. Mockett R.J. Orr W.C. Free Radic. Biol. Med. 2002; 33: 575-586Crossref PubMed Scopus (524) Google Scholar, I. Bayne A.C.V. Mockett R.J. Orr W.C. Sohal R.S. Biochem. J. 2004; 382: 131-136Crossref PubMed Scopus (67) Google Scholar). In the present GCLc overexpression was to enhance the amount of which has multiple in cells, acting as a or a for the of reactive It can with a of which after reactions a substrate of superoxide dismutase Free Radic. Biol. Med. 1993; PubMed Scopus Google Scholar). Glutathione and acting in are hypothesized to a mechanism for the of reactive oxygen with acting as an Free Radic. Biol. Med. 1993; PubMed Scopus Google Scholar). The of such a could the results for GCL are to obtained by overexpressing in the nervous which also the life span of Drosophila (14Sohal R.S. Mockett R.J. Orr W.C. Free Radic. Biol. Med. 2002; 33: 575-586Crossref PubMed Scopus (524) Google Scholar, D. PubMed Scopus Google Scholar). The of D. melanogaster be relatively to in oxidative stress, has a content of in with tissues V.I. S.N. Orr W.C. 1999; PubMed Scopus Google Scholar). The absence of life resulting from global of GSH content in GCLc transgenic flies the that in tissues are and that of overexpression the the in the the of GCL overexpression be with in activity oxidative stress, with or protein In support of this the global in GCL activity and GSH content were in than in flies. that flies to GCLc in the nervous is with the that GCL protein content was in the heads of GCLc transgenic flies possessing global versus neuronal drivers. the of life span to be by overexpressing GCL at than high and by the overexpression of GCL to the The of GCLc and GCLm overexpression be to in their in GSH biosynthesis, and in substrate The Drosophila GCLc catalytic activity (15Fraser J.A. Saunders R.D.C. McLellan L.I. J. Biol. Chem. 2002; 277: 1158-1165Abstract Full Text Full Text PDF PubMed Scopus (48) Google but the formation of disulfide with GCLm the for from to and feedback inhibition by This in could for the of life span by global overexpression of GCLm, the concentration of in the Drosophila is A. H. 1993; Google that the GCLc subunit in GSH synthesis the than the heterodimeric with the hypothesis that in GCL activity the in for could also the of global overexpression of both GCLc and GCLm during the absence of life resulting from neuronal overexpression of GCLm be by tissue-specific in the of or in the of GCLc GCLc is by during oxidative stress J. PubMed Scopus (12) Google A of the of such as is the of an between and rate (14Sohal R.S. Mockett R.J. Orr W.C. Free Radic. Biol. Med. 2002; 33: 575-586Crossref PubMed Scopus (524) Google Scholar). Experimental that the rate of oxygen consumption extend the life span. in the present was to such an mechanism for life-span by the overexpression of oxygen consumption was measured in vials with dimensions to used for life-span to ensure levels of activity during the life span and GCLc overexpression by or on the rate of oxygen This a as the of the in Furthermore, the of life span was in long-lived genetic backgrounds life spans were for both the neuronal driver and UAS-GCLc control which the that an increased GSH biosynthetic in the has a on aging. The present that enhancement of the for de novo GSH synthesis the life span of flies by up to 50%, has for the oxidative stress hypothesis of aging. the of this hypothesis is the between levels and an is that levels would of this study that overexpression of GCLc in a long-lived background can extend the life span and resistance to oxidative stress, without affecting the such was only tissues of the central nervous system an increased glutathione biosynthetic This tissue-specific the of the nervous system to oxidative stress and the that the overexpression of antioxidants could the aging