Overexpression of Glutamate-Cysteine Ligase Extends Life Span in Drosophila melanogaster

William C Orr,Svetlana N Radyuk,Leena Prabhudesai,Dikran Toroser,Judith J Benes,James M Luchak,Robin J Mockett,Igor Rebrin,J Gregory Hubbard,Rajindar S Sohal

doi:10.1074/jbc.m508272200

Abstract

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.

Highlights

Mammalian glutamate-cysteine ligase (GCL) is a heterodimeric enzyme, consisting of a catalytic subunit, generate flies overexpressing either the catalytic (GCLc), and a regulatory or modulatory subunit, GCLm, which are the products of two distinct genes [8]
Expression of GCLc and GCLm driven by Tub-GAL4 was increased by 4- to 6-fold and 60 –100%, respectively, in comparison with controls, as revealed by immunoblot analysis and the GCL enzyme activity assay (Fig. 1)
When neuronal drivers were used, only slight increases in GCL activity were noted in whole body homogenates (Fig. 1A); the same was true when GCL protein levels were determined by Western analysis

Summary

Introduction

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 [8]. Several lines of evidence suggest that the level of oxidative stress is enhanced during the aging process [14]. 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. 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 ϳ31-kDa regulatory subunit and a ϳ80-kDa catalytic subunit has been accomplished in Drosophila [15]. 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

Objectives

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Conclusion