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Ethylmalonic encephalopathy (EE) is a rare autosomal recessive inborn error of metabolism. To study the molecular effects of ETHE1 p. D165H mutation, we employed mass spectrometry-based mitochondrial proteome and phosphoproteome profiling in the human skeletal muscle. Eighty-six differentially altered proteins were identified, of which thirty-seven mitochondrial proteins were differentially expressed, and most of the proteins (37%) were down-regulated in the OXPHOS complex-IV. Also, nine phosphopeptides that correspond to eight mitochondrial proteins were significantly affected in EE patient. These altered proteins recognized are involved in several pathways and molecular functions, predominantly in oxidoreductase activity. This is the first study that has integrated proteome and phosphoproteome of skeletal muscle and identified multiple proteins associated in the pathogenesis of EE.

We report here the effects of hydrogen sulfide (sulfide), that accumulates in ETHE1 deficiency, in rat cerebellum. Sulfide impaired electron transfer and oxidative phosphorylation. Sulfide also induced mitochondrial swelling, and decreased ΔΨm and calcium retention capacity in cerebellum mitochondria, which were prevented by cyclosporine A (CsA) plus ADP, and ruthenium red, suggesting mitochondrial permeability transition (mPT) induction. Melatonin (MEL) and N-ethylmaleimide also prevented sulfide-induced alterations. Prevention of sulfide-induced decrease of ΔΨm and viability by CsA and MEL was further verified in cerebellum neurons. The data suggest that sulfide induces mPT pore opening via thiol modification and ROS generation.

The mitochondrial enzyme ETHE1 is a persulfide dioxygenase essential for cellular sulfide detoxification, and its deficiency causes the severe and complex inherited metabolic disorder ethylmalonic encephalopathy (EE). In spite of well-described clinical symptoms of the disease, detailed cellular and molecular characterization is still ambiguous. Cellular redox regulation has been described to be influenced in ETHE1 deficient cells, and to clarify this further we applied image cytometry and detected decreased levels of reduced glutathione (GSH) in cultivated EE patient fibroblast cells. Cell growth initiation of the EE patient cells was impaired, whereas cell cycle regulation was not. Furthermore, Seahorse metabolic analyzes revealed decreased extracellular acidification, i. e. decreased lactate formation from glycolysis, in the EE patient cells. TMT-based large-scale proteomics was subsequently performed to broadly elucidate cellular consequences of the ETHE1 deficiency. More than 130 proteins were differentially regulated, of which the majority were non-mitochondrial. The proteomics data revealed a link between ETHE1-deficiency and down-regulation of several ribosomal proteins and LIM domain proteins important for cellular maintenance, and up-regulation of cell surface glycoproteins. Furthermore, several proteins of endoplasmic reticulum (ER) were perturbed including proteins influencing disulfide bond formation (e.g. protein disulfide isomerases and peroxiredoxin 4) and calcium-regulated proteins. The results indicate that decreased level of reduced GSH and alterations in proteins of ribosomes, ER and of cell adhesion lie behind the disrupted cell growth of the EE patient cells.

Ethylmalonic encephalopathy (EME) is a rare autosomal recessive inherited metabolic disease characterized by developmental delay, ecchymotic patches, and acrocyanosis with chronic diarrhea. We report a first case of EME from India who primarily presented with chronic diarrhea since early infancy and developmental delay. Mutation analysis of ETHE1 showed presence of a previously reported homozygous mutation in exon 4 confirming the diagnosis. She was started on riboflavin, CoQ, carnitine, metronidazole and N-acetyl cysteine with improvement in diarrhea but neurological features continue to progress. Prenatal diagnosis was performed in the next pregnancy. Though, EME is a devastating inherited metabolic disorder with mortality usually in early infancy, here we have reported a case presented as late as at 4 years. A high index of suspicion followed by specific molecular diagnosis not only ends the diagnostic odyssey but also ensures prenatal diagnosis in the future pregnancies.

Ethylmalonic encephalopathy (EE) is a rare metabolic disorder caused by dysfunction of ETHE1 protein, a mitochondrial dioxygenase involved in hydrogen sulfide (H2S) detoxification. EE is usually a fatal disease with a severe clinical course mainly associated with developmental delay and regression, recurrent petechiae, orthostatic acrocyanosis, and chronic diarrhoea. Treatment includes antioxidants, antibiotics that lower H2S levels and antispastic medications, which are not curative. The mutations causing absence of the ETHE1 protein, as is the case for the described patient, usually entail a severe fatal phenotype. Although there are rare reported cases with mild clinical findings, the mechanism leading to these milder cases is also unclear. Here, we describe an 11-year-old boy with an ETHE1 gene mutation who has no neurocognitive impairment but chronic diarrhoea, which is controlled by oral medical treatment, and progressive spastic paraparesis that responded to Achilles tendon lengthening.

Sulfhydryl groups on protein Cys residues undergo an array of oxidative reactions and modifications, giving rise to a virtual redox zip code with physiological and pathophysiological relevance for modulation of protein structure and functions. While over two decades of studies have established NO-dependent S-nitrosylation as ubiquitous and fundamental for the regulation of diverse protein activities, proteomic methods for studying H2S-dependent S-sulfhydration have only recently been described and now suggest that this is also an abundant modification with potential for global physiological importance. Notably, protein S-sulfhydration and S-nitrosylation bear striking similarities in terms of their chemical and biological determinants, as well as reversal of these modifications via group-transfer to glutathione, followed by the removal from glutathione by enzymes that have apparently evolved to selectively catalyze denitrosylation and desulfhydration. Here we review determinants of protein and low-molecular-weight thiol S-sulfhydration/desulfhydration, similarities with S-nitrosylation/denitrosylation, and methods that are being employed to investigate and quantify these gasotransmitter-mediated cell signaling systems.

Hydrogen sulfide (H(2)S) is a recently described endogenously produced gaseous signaling molecule that influences various cellular processes in the central nervous system, cardiovascular system, and gastrointestinal tract. The biogenesis of H(2)S involves the cytoplasmic transsulfuration enzymes, cystathionine β-synthase and γ-cystathionase, whereas its catabolism occurs in the mitochondrion and couples to the energy-yielding electron transfer chain. Low steady-state levels of H(2)S appear to be controlled primarily by efficient oxygen-dependent catabolism via sulfide quinone oxidoreductase, persulfide dioxygenase (ETHE1), rhodanese, and sulfite oxidase. Mutations in the persulfide dioxgenase, i.e. ETHE1, result in ethylmalonic encephalopathy, an inborn error of metabolism. In this study, we report the biochemical characterization and kinetic properties of human persulfide dioxygenase and describe the biochemical penalties associated with two patient mutations, T152I and D196N. Steady-state kinetic analysis reveals that the T152I mutation results in a 3-fold lower activity, which is correlated with a 3-fold lower iron content compared with the wild-type enzyme. The D196N mutation results in a 2-fold higher K(m) for the substrate, glutathione persulfide.

ETHE1 (ethylmalonic encephalopathy protein 1) is a beta-lactamase fold-containing protein that is essential for the survival of a range of organisms. In spite of the apparent importance of this enzyme, very little is known about its function or biochemical properties. In this study Arabidopsis ETHE1 was over-expressed and purified and shown to bind tightly to 1.2+/-0.2 equivalents of iron. (1)H NMR and EPR studies demonstrate that the predominant oxidation state of Fe in ETHE1 is Fe(II), and NMR studies confirm that two histidines are bound to Fe(II). EPR studies show that there is no antiferromagnetically coupled Fe(III)Fe(II) center in ETHE1. Gel filtration studies reveal that ETHE1 is a dimer in solution, which is consistent with previous crystallographic studies. Although very similar in terms of amino acid sequence to glyoxalase II, ETHE1 exhibits no thioester hydrolase activity, and activity screening assays reveal that ETHE1 exhibits low level esterase activity. Taken together, ETHE1 is a novel, mononuclear Fe(II)-containing member of the beta-lactamase fold superfamily.

In patients suffering from ethylmalonic encephalopathy hydrogen sulfide accumulates in the bloodstream due to a block in mitochondrial sulfide oxidation at the level of the sulfur dioxygenase ETHE1. The chronic exposure to elevated sulfide concentrations causes fatal dysfunctions in various organ systems. In particular, the vascular endothelium is severely damaged leading to multiple necrotic and haemorrhagic brain lesions, chronic haemorrhagic diarrhea, vascular petechial purpura and orthostatic acrocyanosis. The biochemical profile, an increase in urinary and plasmatic lactate, acylcarnitine, acylglycine, and ethylmalonic acid levels, furthermore indicates disturbances in mitochondrial energy metabolism. While the effects of sulfide on cytochrome c oxidase have been thoroughly investigated, and shown to be due to an increased degradation of specific subunits, the additional systemic consequences are not completely understood. We used an Ethe1 deficient mouse model, which faithfully recapitulates the clinical and biochemical features of ethylmalonic encephalopathy, to investigate the effect of increased sulfide and persulfide concentrations on liver, kidney, muscle and brain proteomes. Our results provide evidence for a crucial role of the mitochondrial sulfide oxidation pathway in signaling. The mechanism of this regulatory function is most likely based on cysteine S-modifications. Increased sulfide concentrations promote cysteine S-sulfhydrations and thereby shift the balance between different forms of post-translational modifications. In addition, ETHE1 as a sulfur dioxygenase could directly take part in the removal of sulfhydrations by oxidizing the cysteine derived persulfide group. Notably, the main pathways affected by elevated sulfide concentrations on proteomic level are the mitochondrial oxidation of branched-chain amino acids and fatty acids as well as cytoskeleton organization. Enzymes involved in lipid catabolism are also most clearly coexpressed with Ethe1. These findings clarify the original clinical description of ethylmalonic encephalopathy that was thought to be a defect in the beta-oxidation pathway and indicate a close functional connection between sulfide oxidation and mitochondrial acyl-CoA metabolism. In addition, sulfide must be considered an important factor in the control of cytoskeleton dynamics. The increase in sulfide and persulfide concentrations elicits changes in structural proteins as well as in enzymes involved in the organization of the cytoskeleton specifically in brain and muscle of Ethe1 deficient mice. These results are consistent with the severe defects in motor activity and rapidly progressive neurological failure observed in patients and the mouse model. Thus, the present study provides a new perspective on the pathogenesis of ethylmalonic encephalopathy and also reveals new aspects about the metabolic function of mitochondrial sulfide oxidation.

Hydrogen sulfide (H 2 S) is an endogenously produced gaseous signaling molecule with beneficial effects in various cellular processes in the central nervous system, cardiovascular system and gastrointestinal tract. The biogenesis of H 2 S involves the enzymes of the transsulfuration pathway cystathionine b-synthase and g-cystathionase. H 2 S oxidation and removal occurs in the mitochondria and the electrons from this oxidation are used in electron transfer chain to generate energy. Steady-state levels of H 2 S are controlled by its biogenesis in the transsulfuration pathway and its efficient oxygen-dependent catabolism in the mitochondria by a 3-enzyme pathway including sulfide quinone oxidoreductase, persulfide dioxygenase (ETHE1) and a sulfur transferase. Mutations in the persulfide dioxgenase, ETHE1, result in ethylmalonic encephalopathy, an inborn error of metabolism. In this study, we report the biochemical characterization and kinetic properties of human persulfide dioxygenase and describe the biochemical penalties associated with two patient mutations, T152I and D196N. Steady-state kinetic analysis reveals that the T152I mutation results in a 3-fold lower activity, which is correlated with a 3-fold lower iron content compared to the wild-type enzyme. The D196N mutation results in a 2-fold higher K M for the substrate, glutathione persulfide (GSSH).

Background Hydrogen sulfide (H 2 S) is a biologically active metabolite, which influences many physiological processes. While H 2 S can be produced and degraded in different mammalian tissues, the kinetics of its turnover, which determine its steady state levels, have not been well studied. We have assessed the rates of H 2 S production in murine liver, kidney, and brain homogenates at physiologically relevant conditions. We have also studied the kinetics of H 2 S clearance by liver, kidney, and brain homogenates under aerobic and anaerobic conditions. Methods Organs were obtained from 2 month old BALB/C males. Kinetics of H 2 S production and clearance were studied in tissue homogenates diluted 1:9 with 100 mM HEPES buffer, pH 7.4. Production of H 2 S was studied under anaerobic conditions at 37 °C and cysteine concentration from 0 to 5.0 mM. Kinetics of H 2 S clearance was studied at 25 °C under both anaerobic and aerobic conditions with initial H 2 S concentration of 20 ppm. Concentration of H 2 S was determined using gas chromatography with DB-1 column (30 m, 0.32 mm, 1.0 μm) and 355 Sulfur Chemiluminescence Detector (Agilent). Results We found that the rate of H 2 S production by tissue homogenates is considerably higher than background rates observed in the absence of exogenous substrates. At physiological cysteine concentration of 100 μM our estimations for the rate of H 2 S production (mean ± SD) in mouse liver and brain are 484 ± 271 μmol/h kg ( n = 7) and 29 ± 7 μmol/h kg ( n = 4) respectively. In kidney the rate of H 2 S production of 104 ± 44 μmol/h kg ( n = 3) was obtained at 0.5 mM cysteine. In the absence of cysteine H 2 S added to homogenate decays exponentially with time and, as expected, the decay is significantly faster under aerobic conditions. The half-life for H 2 S under aerobic conditions is 2.0, 2.8, and 10.0 min with liver, kidney and brain homogenate, respectively. Western-blot analysis of the sulfur dioxygenase, ETHE1, involved in H 2 S catabolism, demonstrates higher protein levels in liver and kidney versus brain. At pH 5.8 the rate of H 2 S production decreases dramatically and becomes negligible at physiologically relevant cysteine levels, while the rate of H 2 S clearance decreases only slightly. By combining experimental and simulation approaches, we demonstrate high rates of tissue H 2 S turnover and provide estimates of in vivo steady-state H 2 S levels which are 29, 8.7, and 18 nmoles per kg tissue in mouse liver, kidney, and brain respectively. Conclusion Our study reveals that mammalian tissues maintain a high metabolic flux of sulfur through H 2 S, providing the possibility for rapid regulation of H 2 S levels.