Abstract

Reactive oxygen species (ROS) are signalling molecules whose study in intact organisms has been hampered by their potential toxicity. This has prevented a full understanding of their role in organismal processes such as development, aging and disease. In Caenorhabditis elegans, the development of the vulva is regulated by a signalling cascade that includes LET-60ras (homologue of mammalian Ras), MPK-1 (ERK1/2) and LIN-1 (an ETS transcription factor). We show that both mitochondrial and cytoplasmic ROS act on a gain-of-function (gf) mutant of the LET-60ras protein through a redox-sensitive cysteine (C118) previously identified in mammals. We show that the prooxidant paraquat as well as isp-1, nuo-6 and sod-2 mutants, which increase mitochondrial ROS, inhibit the activity of LET-60rasgf on vulval development. In contrast, the antioxidant NAC and loss of sod-1, both of which decrease cytoplasmic H202, enhance the activity of LET-60rasgf. CRISPR replacement of C118 with a non-oxidizable serine (C118S) stimulates LET-60rasgf activity, whereas replacement of C118 with aspartate (C118D), which mimics a strongly oxidised cysteine, inhibits LET-60rasgf. These data strongly suggest that C118 is oxidized by cytoplasmic H202 generated from dismutation of mitochondrial and/or cytoplasmic superoxide, and that this oxidation inhibits LET-60ras. This contrasts with results in cultured mammalian cells where it is mostly nitric oxide, which is not found in worms, that oxidizes C118 and activates Ras. Interestingly, PQ, NAC and the C118S mutation do not act on the phosphorylation of MPK-1, suggesting that oxidation of LET-60ras acts on an as yet uncharacterized MPK-1-independent pathway. We also show that elevated cytoplasmic superoxide promotes vulva formation independently of C118 of LET-60ras and downstream of LIN-1. Finally, we uncover a role for the NADPH oxidases (BLI-3 and DUOX-2) and their redox-sensitive activator CED-10rac in stimulating vulva development. Thus, there are at least three genetically separable pathways by which ROS regulates vulval development.

Highlights

  • Reactive oxygen species (ROS) are signalling molecules that participate in regulating many cellular processes [1,2,3], including in C. elegans [4,5,6,7,8]

  • In contrast to what is observed in cultured mammalian cells, we find that C118 is oxidized by hydrogen peroxide, rather than by superoxide or nitric oxide, and that its oxidation inhibits rather than activates the pathway

  • We find that the regulation of LET-60ras by oxidation does not act through MPK-1 to affect vulva development

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Summary

Introduction

Reactive oxygen species (ROS) are signalling molecules that participate in regulating many cellular processes [1,2,3], including in C. elegans [4,5,6,7,8]. Hydrogen peroxide (H2O2) is produced by the action of superoxide dismutases, which convert superoxide to hydrogen peroxide [13], as well as directly by enzymes like xanthine oxidase [14] and monoamine oxidase [15]. It is produced by the Dual Oxidases (DUOXs), a sub-class of NOX proteins that possess a peroxidase domain, and can convert superoxide to hydrogen peroxide [16]. There are two NOXs of the Duox sub-class, with BLI-3 required for proper development of the cuticle via tyrosine cross-linking [21,22,23], and the very similar DUOX-2 to which no function has yet be assigned [24, 25]

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