ROS regulation of RAS and vulva development in Caenorhabditis elegans

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 H 2 0 2 , 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 H 2 0 2 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.

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“…Using CRISPR/Cas9, we mutated the redox-sensitive Cys366 to serine (Ser), a non-oxidizable Cys mimetic 30 and found no overt phenotypes (Fig. 6 , Supplementary Fig.…”

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“…To avoid damage from H 2 O 2 , C. elegans rely on conserved cellular defenses, including H 2 O 2 -degrading catalases (Chavez et al, 2007; Schiffer et al, 2020). However, inducing those defenses at inappropriate times can cause undesirable side effects, including developmental defects (Doonan et al, 2008; Kramer-Drauberg et al, 2020), because H 2 O 2 modulates the function of proteins involved in a wide variety of cellular processes, including signal transduction and differentiation (Hourihan et al, 2016; Kramer-Drauberg et al ., 2020; Meng et al, 2021; Veal et al, 2007). We recently found that ten classes of sensory neurons in the nematode’s brain manage the challenge of deciding when the nematode’s tissues induce H 2 O 2 defenses (Schiffer et al ., 2020).…”

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