Dithiothreitol causes toxicity in C. elegans by modulating the methionine–homocysteine cycle

Abstract

The redox reagent dithiothreitol (DTT) causes stress in the endoplasmic reticulum (ER) by disrupting its oxidative protein folding environment, which results in the accumulation and misfolding of the newly synthesized proteins. DTT may potentially impact cellular physiology by ER-independent mechanisms; however, such mechanisms remain poorly characterized. Using the nematode model Caenorhabditis elegans, here we show that DTT toxicity is modulated by the bacterial diet. Specifically, the dietary component vitamin B12 alleviates DTT toxicity in a methionine synthase-dependent manner. Using a forward genetic screen, we discover that loss-of-function of R08E5.3, an S-adenosylmethionine (SAM)-dependent methyltransferase, confers DTT resistance. DTT upregulates R08E5.3 expression and modulates the activity of the methionine-homocysteine cycle. Employing genetic and biochemical studies, we establish that DTT toxicity is a result of the depletion of SAM. Finally, we show that a functional IRE-1/XBP-1 unfolded protein response pathway is required to counteract toxicity at high, but not low, DTT concentrations.

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.…”

“…The whole-genome sequence data were analyzed using the web platform Galaxy as described earlier (Gokul and Singh, 2022). Briefly, the forward and reverse FASTQ reads, C. elegans reference genome Fasta file (ce11M.fa), and the gene annotation file (SnpEff4.3 WBcel235.86) were input into the Galaxy workflow.…”

“…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.…”