Dietary-derived vitamin B12 protects Caenorhabditis elegans from thiol-reducing agents

Abstract

Background One-carbon metabolism, which includes the folate and methionine cycles, involves the transfer of methyl groups which are then utilised as a part of multiple physiological processes including redox defence. During the methionine cycle, the vitamin B12-dependent enzyme methionine synthetase converts homocysteine to methionine. The enzyme S-adenosylmethionine (SAM) synthetase then uses methionine in the production of the reactive methyl carrier SAM. SAM-binding methyltransferases then utilise SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids. Results We describe a novel SAM methyltransferase, RIPS-1, which was the single gene identified from forward genetic screens in Caenorhabditis elegans looking for resistance to lethal concentrations of the thiol-reducing agent dithiothreitol (DTT). As well as RIPS-1 mutation, we show that in wild-type worms, DTT toxicity can be overcome by modulating vitamin B12 levels, either by using growth media and/or bacterial food that provide higher levels of vitamin B12 or by vitamin B12 supplementation. We show that active methionine synthetase is required for vitamin B12-mediated DTT resistance in wild types but is not required for resistance resulting from RIPS-1 mutation and that susceptibility to DTT is partially suppressed by methionine supplementation. A targeted RNAi modifier screen identified the mitochondrial enzyme methylmalonyl-CoA epimerase as a strong genetic enhancer of DTT resistance in a RIPS-1 mutant. We show that RIPS-1 is expressed in the intestinal and hypodermal tissues of the nematode and that treating with DTT, β-mercaptoethanol, or hydrogen sulfide induces RIPS-1 expression. We demonstrate that RIPS-1 expression is controlled by the hypoxia-inducible factor pathway and that homologues of RIPS-1 are found in a small subset of eukaryotes and bacteria, many of which can adapt to fluctuations in environmental oxygen levels. Conclusions This work highlights the central importance of dietary vitamin B12 in normal metabolic processes in C. elegans, defines a new role for this vitamin in countering reductive stress, and identifies RIPS-1 as a novel methyltransferase in the methionine cycle.

Thiol reductive stress activates the hypoxia response pathway

“…The thiol antioxidants, DTT and β-mercaptoethanol, cause toxicity in C. elegans by upregulating a SAM-dependent methyltransferase, rips-1 , that results in the modulation of the methionine-homocysteine cycle (Gokul and Singh, 2022; Winter et al, 2022). Mitochondrial dysfunction and the hypoxia-inducible factor might alter rips-1 expression (Winter et al, 2022); however, the full spectrum of physiological changes induced by thiol reductive stress that results in the upregulation of rips-1 , remains to be discovered. To characterize the physiological changes that trigger the upregulation of rips-1 , we designed a forward genetic screen to isolate mutants that had constitutive activation of rips-1 , utilizing a rips-1p::GFP reporter strain (Figure S1).…”

“…Next, we asked whether activation of the hypoxia response pathway conferred any protection against thiol reductive stress. Thiol antioxidants-mediated upregulation of rips-1 modulates the methionine-homocysteine cycle and results in developmental toxicity (Gokul and Singh, 2022; Winter et al, 2022). Therefore, it appeared that the activation of the hypoxia response pathway by thiol antioxidants, which results in the upregulation of rips-1 , might be harmful to the host.…”

“…Recent studies in C. elegans showed that, besides causing ER stress, thiol antioxidants modulate the methionine-homocysteine cycle (Gokul and Singh, 2022;Winter et al, 2022).…”

“…In addition to ER stress, thiol antioxidants can also result in oxidative stress by activating futile oxidative cycles in the ER (Maity et al, 2016). Recent studies in C. elegans showed that, besides causing ER stress, thiol antioxidants modulate the methionine-homocysteine cycle (Gokul and Singh, 2022; Winter et al, 2022). The modulation of the methionine-homocysteine cycle is a consequence of the upregulation of an S-adenosylmethionine (SAM)-dependent methyltransferase, rips-1 , by thiol antioxidants.…”

“…In this study, using C. elegans and human cell lines, we characterized the physiological effects of thiol reductive stress. Because thiols antioxidants result in the increased expression of rips-1 (Gokul and Singh, 2022; Winter et al, 2022), we reasoned that understanding the regulation of rips-1 expression might provide insights into the physiological effects of thiol reductive stress. To this end, we carried out forward genetic screens to understand the regulation of rips-1 expression.…”