Life span and stress resistance of Caenorhabditis elegans are differentially affected by glutathione transferases metabolizing 4-hydroxynon-2-enal

doi:10.1016/j.mad.2006.11.025

Mechanisms of Ageing and Development

Volume 128, Issue 2, February 2007, Pages 196-205

https://doi.org/10.1016/j.mad.2006.11.025 Get rights and content

Abstract

The lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) forms as a consequence of oxidative stress, and acts as a signaling molecule or, at superphysiological levels, as a toxicant. The steady-state concentration of the compound reflects the balance between its generation and its metabolism, primarily through glutathione conjugation. Using an RNAi-based screen, we identified in Caenorhabditis elegans five glutathione transferases (GSTs) capable of catalyzing 4-HNE conjugation. RNAi knock-down of these GSTs (products of the gst-5, gst-6, gst-8, gst-10, and gst-24 genes) sensitized the nematode to electrophilic stress elicited by exposure to 4-HNE. However, interference with the expression of only two of these genes (gst-5 and gst-10) significantly shortened the life span of the organism. RNAi knock-down of the other GSTs resulted in at least as much 4-HNE adducts, suggesting tissue specificity of effects on longevity. Our results are consistent with the oxidative stress theory of organismal aging, broadened by considering electrophilic stress as a contributing factor. According to this extended hypothesis, peroxidation of lipids leads to the formation of 4-HNE in a chain reaction which amplifies the original damage. 4-HNE then acts as an “aging effector” via the formation of 4-HNE-protein adducts, and a resulting change in protein function.

Introduction

A role for the accumulation of oxidative and free radical damage in aging was proposed many decades ago (Pearl, 1928, Harman, 1956). This theory went through numerous modifications and refinements, but has withstood the test of time remarkably well, and remains a major paradigm of research into the aging process. Recent work suggests that other types of molecular damage, brought about by reactive xenobiotics and/or products of endogenous metabolism, also play a key role in organismal senescence (McElwee et al., 2004, Gems and McElwee, 2005). Integrating the two lines of reasoning, we proposed an extension to the oxidative damage theory of aging. This hypothesis (Ayyadevara et al., 2005a, Ayyadevara et al., 2005b, McEwen et al., 2005) postulates that the lipid peroxidation chain reaction, initiated by a reaction of reactive oxygen species (ROS) with lipids, amplifies an original oxidative insult. The end products of lipid peroxidation, in particular electrophilic aldehydes exemplified by 4-hydroxynon-2-enal (4-HNE) are the effectors which act in parallel with ROS to cause molecular damage, and ultimately aging. We have previously provided evidence for the above hypothesis by experimentally modulating the capacity of Caenorhabditis elegans to metabolize 4-HNE. To this end, we transgenically overexpressed glutathione transferases (GSTs) with high catalytic activity for 4-HNE, including the murine enzyme mGSTA4-4, and the endogenous C. elegans CeGSTP2-2 (the gst-10 gene product), or knocked down the latter using RNAi, and observed the predicted effects on life span of the nematode (Ayyadevara et al., 2005a, Ayyadevara et al., 2005b).

Forty-four annotated gst genes, and a number of additional GST-like proteins, are listed in release 156 of WormBase (www.wormbase.org). A bioinformatics analysis of the C. elegans genome identified 57 genes encoding proteins that match the C-terminal portion of GSTs (Supplemental Table S4 in Holt et al., 2002). Therefore, we posed the question whether C. elegans GSTs other than the already characterized gst-10 gene product have the ability to modulate life span by affecting 4-HNE levels in critical cells of the organism. To answer this question, in the present work we carried out an RNAi-based screen of GSTs. The results indicate that, in addition to the gst-10 gene product, at least four other C. elegans GSTs have the ability to metabolize 4-HNE to a significant extent. Each of these five enzymes protects the organism against electrophilic stress caused by exposure to 4-HNE, but only two of these significantly affect life span.

Section snippets

C. elegans culture conditions

C. elegans strain Bristol-N2 was used in all experiments except for the life span reported in Fig. 9 which was carried out on strain NL2099, obtained from the Caenorhabditis Genetics Center (St. Paul, MN). The animals were cultured at 20 °C in nematode growth medium (NGM: 25 mM potassium phosphate, pH 6.0, 50 mM NaCl, 0.25%, w/v, peptone, 0.5%, w/v, cholesterol, 1 mM MgCl₂, 1 mM CaCl₂) and fed with Escherichia coli strain OP50 (2 × 10⁹ cells/ml) grown in 3XD medium (10.5 g/l of Na₂HPO₄, 4.5 g/l of KH₂PO₄

A subset of C. elegans GSTs is capable of conjugating 4-HNE

RNA interference targeted to the subset of 26 gst genes represented in the Ahringer RNAi library (Kamath and Ahringer, 2003, Kamath et al., 2003) was carried out in wild-type C. elegans (strain Bristol-N2). In addition, gst-10 was targeted as described previously (Ayyadevara et al., 2005a). Assays of worm homogenates demonstrated that knock-down of CeGSTP2-2 (gst-10 gene product) reduced 4-HNE-conjugating activity, as previously reported (Ayyadevara et al., 2005b). Furthermore, RNAi targeted to

Discussion

We have previously reported that 4-HNE, and/or a chemically related product of lipid peroxidation, affects life span of C. elegans (Ayyadevara et al., 2005a, Ayyadevara et al., 2005b). A part of the experimental evidence supporting this conclusion was the finding that overexpression of CeGSTP2-2, the gst-10 gene product, extends life span of C. elegans, while RNAi knock-down of gst-10 has the opposite effect. However, both immunodepletion of CeGSTP2-2 and the yield of its biochemical

Acknowledgments

This work was supported in part by National Institutes of Health grants R01 AG18845 and R01 ES07804 (to P.Z.), and Program Project grant P01 AG20641 (to R.J.S.R.). P.Z. and R.J.S.R. are recipients of VA Research Career Scientist Awards. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). We thank Dr. Dennis R. Petersen, University of Colorado, Denver, for the generous gift of an

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¹: These authors contributed equally to this work.

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Life span and stress resistance of Caenorhabditis elegans are differentially affected by glutathione transferases metabolizing 4-hydroxynon-2-enal

Abstract

Introduction

Section snippets

C. elegans culture conditions

A subset of C. elegans GSTs is capable of conjugating 4-HNE

Discussion

Acknowledgments

Neuron

FEBS Lett.

J. Biol. Chem.

Free Radic. Biol. Med.

Mech. Ageing Dev.

Tetrahedron Lett.

Methods

Curr. Opin. Neurobiol.

Free Radic. Biol. Med.

J. Biol. Chem.

Mech. Ageing Dev.

Ageing Res. Rev.

Anal. Biochem.

J. Chromatogr.

Curr. Biol.

J. Chromatogr. B

J. Lipid Mediators Cell Signal.

Mech. Ageing Dev.

Arch. Biochem. Biophys.

Prog. Lipid Res.

Free Radic. Biol. Med.