Mitochondrial Sulfide Quinone Oxidoreductase Prevents Activation of the Unfolded Protein Response in Hydrogen Sulfide

Abstract

Hydrogen sulfide (H2S) is an endogenously produced gaseous molecule with important roles in cellular signaling. In mammals, exogenous H2S improves survival of ischemia/reperfusion. We have previously shown that exposure to H2S increases the lifespan and thermotolerance in Caenorhabditis elegans, and improves protein homeostasis in low oxygen. The mitochondrial SQRD-1 (sulfide quinone oxidoreductase) protein is a highly conserved enzyme involved in H2S metabolism. SQRD-1 is generally considered important to detoxify H2S. Here, we show that SQRD-1 is also required to maintain protein translation in H2S. In sqrd-1 mutant animals, exposure to H2S leads to phosphorylation of eIF2α and inhibition of protein synthesis. In contrast, global protein translation is not altered in wild-type animals exposed to lethally high H2S or in hif-1(ia04) mutants that die when exposed to low H2S. We demonstrate that both gcn-2 and pek-1 kinases are involved in the H2S-induced phosphorylation of eIF2α. Both ER and mitochondrial stress responses are activated in sqrd-1 mutant animals exposed to H2S, but not in wild-type animals. We speculate that SQRD-1 activity in H2S may coordinate proteostasis responses in multiple cellular compartments.

Keywords: Caenorhabditis elegans (C. elegans); eukaryotic initiation factor 2 (eIF2); hydrogen sulfide; proteostasis; translation initiation.

FIGURE 1.

SQRD-1 is required for optimal protein translation in H₂S. A, SQRD catalyzes the oxidation of H₂S at the mitochondria. H₂S is oxidized, resulting in the sulfur atom from H₂S (red) forming a persulfite intermediate on SQRD. Electrons from H₂S are fed into the quinone pool of the electron transport chain. The SQRD persulfite intermediate is resolved by oxidation with another cellular sulfur moiety to form the final -R-S-S-H species. R can include a variety of species, including sulfhydryl residues of cellular proteins (8, 9). B, experimental strategy. Worms were fed [³⁵S]methionine labeled OP50 in liquid culture for 4 h to label cellular amino acid precursor pools and then transferred to solid NGM plates seeded with unlabeled OP50 for exposure to either H₂S or room air. C, mutants lacking SQRD-1 do not efficiently incorporate [³⁵S]methionine into protein when exposed to H₂S. Incorporation of [³⁵S]methionine was measured by autoradiograms from three independent experiments. All samples were normalized to room air exposed wild-type animals (N2). Plot shows average ± standard deviation. D, representative autoradiogram of proteins from animals exposed to H₂S. Proteins were extracted from wild-type (N2) and sqrd-1(tm3378) mutant animals 3 h after transfer to NGM plates, and separated by SDS-PAGE. Coomassie-stained gels (left) show total protein and autoradiogram (right) shows proteins with incorporated [³⁵S]methionine.

FIGURE 2.

Decrease in translation in H₂S is associated with sqrd-1 deficiency. A, polysome profile of wild-type (N2) animals exposed to H₂S (solid red line) compared with controls that remained in room air (black dotted line). Arrows point to peaks containing free 40S and 60S ribosome subunits. The 80S monosome peak is marked, and polysome fractions are bracketed. B, polysome profile of sqrd-1(tm3378) mutant animals exposed to H₂S (solid red line) compared with controls that remained in room air (black dotted line). Annotations as in A. C, quantification of change in percent of ribosomes actively translating after exposure to H₂S. In addition to exposure to 50 ppm H₂S (first three bars), the change in translation was also measured for wild-type (N2) animals exposed to 150 ppm H₂S or hypoxia (far right). ΔTranslation = (% active H₂S) − (%active room air). Number of independent replicates: N2, n = 5; sqrd-1, n = 3; hif-1, n = 7; N2 in 150 ppm H₂S, n = 3. N2 in hypoxia n = 3

FIGURE 3.

SQRD-1 prevents ER and mitochondrial stress in H₂S. A, phosphorylation of eIF2α is stimulated in sqrd-1(tm3378) mutant animals exposed to H₂S. Western blots to detect phosphorylated eIF2α. All strains except wild-type (N2) have the sqrd-1(tm3378) allele. In top blot, phosph-eIF2α is indicated by arrow, the * is a nonspecific band present in all samples. Bottom blot shows total eIF2α staining as a loading control. B, relative quantification of phospho-eIF2α staining from replicate Western blot experiments. Data shown are average of five independent biological replicates (error bars show S.D.) for each genotype. C, change in transcript abundance of gene products measured by qRT-PCR after exposure to H₂S. Avg fold change calculated from ΔΔC_t (ΔC_t^H₂S - ΔC_t^RA), error bars show S.D. N2, n = 4; sqrd-1 n = 5 independent experiments. D, fold-change of stress response genes, measured by qRT-PCR of wild-type (N2) animals exposed to 150 ppm H₂S for 3 h (n = 3 independent biological replicates). For comparison, data for N2 in 50 ppm is same as in panel C.