Proteomics Reveals that Redox Regulation Is Disrupted in Patients

Mar 16, 2011 - Samples from six patients were analyzed and revealed seven differentially regulated proteins compared with healthy controls. Two protei...
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Proteomics Reveals that Redox Regulation Is Disrupted in Patients with Ethylmalonic Encephalopathy Johan Palmfeldt,*,† Søren Vang,†,‡ Vibeke Stenbroen,† Evangelos Pavlou,§ Mila Baycheva,|| Gebhard Buchal,^ Ardeshir Ahmad Monavari,# Persephone Augoustides-Savvopoulou,z Hanna Mandel,b and Niels Gregersen† †

Research Unit for Molecular Medicine, Aarhus University Hospital, Skejby, Denmark Department of Molecular Medicine, Aarhus University Hospital, Skejby, Denmark § Second Department of Pediatrics, Aristotle University of Thessaloniki, Thessaloniki, Greece Clinic of Gastroenterology and Hepatology, University Pediatric Hospital, Sofia, Bulgaria ^ Department of Pediatrics, Hospital of Kirchen, Germany # The National Centre of Inherited Metabolic Diseases, Children's University Hospital, Dublin, Ireland z First Department of Pediatrics, Aristotle University of Thessaloniki, Thessaloniki, Greece b Meyer Children's Hospital, Rambam Medical Center, Technion-Israel Institute of Technology, Haifa, Israel

)

‡

bS Supporting Information ABSTRACT: Deficiency of the sulfide metabolizing protein ETHE1 is the cause of ethylmalonic encephalopathy (EE), an inherited and severe metabolic disorder. To study the molecular effects of EE, we performed a proteomics study on mitochondria from cultured patient fibroblast cells. Samples from six patients were analyzed and revealed seven differentially regulated proteins compared with healthy controls. Two proteins involved in pathways of detoxification and oxidative/reductive stress were underrepresented in EE patient samples: mitochondrial superoxide dismutase (SOD2) and aldehyde dehydrogenase X (ALDH1B). Sulfide:quinone oxidoreductase (SQRDL), which takes part in the same sulfide pathway as ETHE1, was also underrepresented in EE patients. The other differentially regulated proteins were apoptosis inducing factor (AIFM1), lactate dehydrogenase (LDHB), chloride intracellular channel (CLIC4) and dimethylarginine dimethylaminohydrolase 1 (DDAH1). These proteins have been reported to be involved in encephalopathy, energy metabolism, ion transport, and nitric oxide regulation, respectively. Interestingly, oxidoreductase activity was overrepresented among the regulated proteins indicating that redox perturbation plays an important role in the molecular mechanism of EE. This observation may explain the wide range of symptoms associated with the disease, and highlights the potency of the novel gaseous mediator sulfide. KEYWORDS: ethylmalonic encephalopathy, quantitative proteomics, HSCO, oxidative stress, H2S

’ INTRODUCTION Ethylmalonic encephalopathy (EE) is a severe disease caused by homozygous or compound heterozygous gene variations in the ethylmalonic encephalopathy gene (ETHE1). The disease manifests a wide range of symptoms including lactic acidosis, chronic diarrhea, skin discoloration, and encephalopathy and often leads to death within the first few years of life.1 A decade after the initial phenotypic description of the disease, the causative gene deficiency was characterized;2 however, the gene function was only recently described.3 ETHE1, a mitochondrial protein functioning in sulfide (H2S) detoxification, is deficient in patients with EE.3 Sulfide toxicity has been studied extensively in relation to sulfide leaking from volcanic sources, causing acute toxicity elicited mainly by inhibition of cytochrome c oxidase.4 However, over the past few years sulfide has been described as a potent endogenous gaseous mediator, in analogy with gaseous r 2011 American Chemical Society

carbon monoxide and nitric oxide,5,6 and is involved in important pathways including vasorelaxation7 and cardioprotection.8 The elevated sulfide levels in EE patients does not cause immediate lethality, since patients are able to live from a few months to several years; however, the wide range of symptoms suggests that there are pleiotropic effects on biological functions. A very recent study showed that disease progression in mice and patients could be slowed down by a combination treatment of an antibiotic, which diminished sulfide generated by bacteria in the intestine, and N-acetylcysteine which putatively acts as a sulfide scavenger.9 The identification of ETHE1 as a sulfide-metabolizing enzyme resulted in a marked increase in our understanding of EE and has allowed for administration of life-extending therapy that reduces the sulfide concentrations. However, further resolution of the Received: December 6, 2010 Published: March 16, 2011 2389

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Journal of Proteome Research molecular etiology of EE is instrumental for a better understanding and treatment of the various symptoms of EE. Large scale proteomics is an invaluable tool for gaining insight into the molecular effects behind this severe disease with multiple symptoms. Mass spectrometry (MS)-based proteomics using relative quantification, such as iTRAQ (Isobaric Tag for Relative and Absolute Quantitation) allows mapping of mitochondrial proteins from several patients and has previously been performed on cultivated human skin fibroblasts.10 Fibroblast cultures are advantageous since they can be established from a small patient skin biopsy. Although some tissue-specific phenomena are missed in fibroblasts they can serve as good models for cellular physiology and biochemistry related to disease, especially since the fibroblasts are of human origin and can be cultured to get sufficient material for detailed studies. In this study, mitochondrial proteomics was performed on cultivated skin fibroblasts isolated from six EE patients as well as three healthy controls and several pathways of metabolism and stress response were mapped. Our data suggest that EE is linked to alterations in levels of several redox-active proteins, and we have found that mitochondrial superoxide dismutase 2 (SOD2) and apoptosis-inducing factor 1 (AIFM1) are two proteins altered in the EE patients.

’ MATERIALS AND METHODS Cell Cultivation and Mitochondrial Enrichment

Primary human dermal fibroblasts were isolated from newborn males, from three healthy individuals (Cambrex #CC-2509, ATCC #CRL-2429, and ATCC #CRL-2450), and from six patients diagnosed with EE caused by ETHE1 deficiency. The studies were approved by The Danish Ethical Committee (ref # M-20090216). The fibroblast cells were cultivated in 150 cm2 flasks with RPMI 1640 medium (BioWhittaker, Cambrex Bioscience, Walkersville, MD) supplemented with 10% (v/v) fetal calf serum (BioWhittaker) and 2 g/L glucose. Cells for experiments were used at a relatively low passage number, between passage 9 and 13, to minimize risk for phenotypic differentiation. After preculturing, the cells were harvested at subconfluence from four 150 cm2 flasks. The cells were lysed by 30 strokes in Dounce homogenizer, and the mitochondria were subsequently isolated by differential centrifugation from the 60010 000 g fraction, as previously described.10 The entire experiment was repeated twice for each fibroblast cell line. The mitochondrial samples from the three healthy individuals were pooled and used as a control sample. Molecular Genetic Analysis of ETHE1

Sequence analyses of the promoter (400 nucleotides), all seven exons and flanking introns were performed on DNA isolated from patient samples. The primers used and the protocol for PCR amplification of the seven ETHE1 exons were performed as described by Tiranti et al.2 All primers were tagged with the M13 nucleotide sequence to facilitate subsequent sequencing reactions. Nucleotide sequence data were collected on an ABI 3100 Avant Genetic Analyzer (Applied Biosystems, Foster City, CA), and analyzed using Sequencher v3.1.1 (Gene Codes Corp., Ann Arbor, MI). All references to nucleotides or amino acids in the text are based on the cDNA sequence of ETHE1 (NM_014297). The initiating ATG codon is numbered as bp 13, and the initiator methionine is numbered as amino acid 1.

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MS Detection of ETHE1 Protein in SDS Gel

To assess the quantity of ETHE1 protein (30 μg) from enriched mitochondria was separated by SDS-PAGE, followed by in-gel trypsin cleavage and LCMS/MS analysis, essentially as previously described.11 ETHE1 protein and the reference protein VDAC1 were quantitated by extracted ion chromatograms (XIC) from proteotypic peptides identified with expectation values below 5  105. Both proteins were detected in the same SDS gel piece, containing proteins smaller than 35 kDa. Quantification was performed on two peptides from ETHE1, EAVLIDPVLETAPR (m/z 761.9279) and LSGAQADLHIEDGDSIR (m/z 599.6311), and three from VDAC1, LTFDSSFSPNTGK (m/z 700.8388), YQIDPDACFSAK (m/z 707.8196), and VNNSSLIGLGYTQTLKPGIK (m/z 1052.0946). Labeling, Fractionation and LCMS/MS

The proteomics procedures were performed as previously described.10 Labeling with iTRAQ (Applied Biosystems) was applied to 100 μg of protein (50 μg from each replicate cultivation). See Figure 2 for an overview. Each patient sample and healthy control was labeled with a randomly chosen iTRAQ label, to minimize any nonspecific effects due to the different labels. The controls in the two studies had different iTRAQ labels, 117 and 116, in study A and B, respectively. The labeled peptides were mixed and separated by isoelectric focusing (IEF) on an Immobiline Drystrip Gradient (IPG) gel (pH 3.54.5) (GE Healthcare, Uppsala, Sweden). The gel strip was cut into thirteen pieces of equal size and peptides were then extracted in two steps, of one hour each, with 100 μL 5% AcN, 0.5% trifluoracetic acid (TFA), purified on PepClean C-18 Spin Columns (Pierce, Rockford, IL) according to manufacturer’s protocol. Peptide mixtures were analyzed by nanoliquid chromatography (Easy nLC from Proxeon, Denmark) coupled to mass spectrometry (MS) (LTQ-Orbitrap, Thermo Fisher), with separation on a reverse phase column (75 μm, 100 mm and 3.5 μm C18 particles) at a flow rate of 300 nL/min using a 100 min gradient (5  35%) of acetonitrile in 0.4% acetic acid. The MS detection constituted a full scan (m/z 4002000) in Orbitrap ( T

Exon 4 Exon 4/Intron 4

Not translated2 Splicing error. Missing protein.2

1

3G > T

Exon 1

Met 1 Ile. Missing protein.2

1

494A > G

Exon 4

Asp 165 Gly

have previously, by Western blot analysis, been shown to lack the ETHE1 protein.16 We confirmed those data by an MS based approach, and in addition showed that the patient sample with the variation Asp 165 Gly contains the ETHE1 protein at a level similar to the healthy controls (Figure 1). EE gives rise to multiple symptoms, and while certain symptoms are not manifested in some patients, the most common symptoms are observed in almost all patients (Figure 2). ETHE1 is a mitochondrial protein and therefore we sought to characterize alterations in the mitochondrial proteome in EE patients by applying an iTRAQ-based proteomics analysis to fibroblast mitochondria. To reduce inherent variation from the cultivation and the enrichment of mitochondria, each of the biological samples was isolated from cells cultivated in two different experiments (Figure 3). To assess the effects of biological and analytical variation we compared with data from a previous study,10 in which one fibroblast was cultured in three independent experiments, and analyzed with the same analytical setting as here. In that study the median standard error of the mean (SEM), of all quantitated proteins, was below 6%. However, the corresponding value in the present study, including variation from samples with different biological background, was above 8% for both control and ETHE1 deficient fibroblasts, indicating that the biological variation, as expected, adds an additional layer of variation. More than thousand proteins were detected in each study and approximately 60% of the detected proteins had robust iTRAQ data. We focused the data treatment on proteins with known presence in mitochondria and obtained stable quantitative data, in all samples, from almost 150 mitochondrial proteins. They were sorted into the following ten functional pathways: fatty acid oxidation, citric acid cycle þ pyruvate dehydrogenase, respiratory chain, amino acid metabolism, translation, protein quality control system, antioxidants, mitochondrial morphology, and apoptosis (Supplementary Table 1, Supporting Information). Seven proteins were found to be differentially expressed, 4 decreased, and 3 increased in the EE patient cells compared to healthy controls (Figure 4). The protein with the most pronounced alteration in expression levels was mitochondrial superoxide dismutase (SOD2), which had approximately a 2-fold reduction. SOD2 is an important member of the oxidative stress response family. When we quantified the expression level of several other members of this family, we found that the expression level of other oxidative stress proteins did not seem to be altered (Figure 5). To validate the altered levels of SOD2 we again cultured the fibroblasts and performed Western blots with an antibody specific to SOD2, which confirmed the proteomics data (Figure 6). The additional six proteins with altered levels in EE patients appeared to represent a wide range of biological functions as assessed by Gene Ontology (GO) (Table 2). Interestingly, while 2391

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Figure 1. Analysis of the presence of ETHE1 protein in the six patient samples (numbered 16) compared with two control samples. Sample six is the sample with the Asp 165 Gly variant. ETHE1 protein and the reference protein VDAC1 were quantitated by extracted ion chromatograms (XIC) from proteotypic peptides identified with expectation values below 5  105. Both proteins were measured in the same SDS gel piece, containing proteins smaller than 35 kDa. XIC of the ETHE1 peptide EAVLIDPVLETAPR (m/z 761.9279) (peak to the right) is compared with the peptide peak of LTFDSSFSPNTGK (m/z 700.8388) of VDAC1.

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Figure 3. Overview of the method used to study the mitochondrial proteome of cultivated human skin fibroblasts isolated from patients compared with healthy controls. In each iTRAQ experiment, three patient samples were compared with a pool of three healthy controls. The gel used for isoelectric focusing was cut into 13 pieces, from which peptides were extracted, purified, and subsequently analyzed by nanoLCMS/MS. IEF, isoelectric focusing.

function was p < 0.02 (calculated as 0.376  (1  0.37)  7 = 0.011), which indicate that this function was enriched among the seven differentially expressed proteins due to EE. To investigate the phenotype of the EE fibroblast cells and to test if they were sensitive to superoxide, assays of mitochondrial potential and cellular viability were performed after exposure to the superoxide inducing agent Antimycin A. No difference in mitochondrial potential was seen between EE and control cells (data not shown) and the EE cells did not have lower viability than the control cells (Supplementary Figure 1, Supporting Information). Figure 2. Overview of EE symptoms reported for the six patients. The different shades of gray indicate the six different patients. Some symptoms, for example, the biochemical markers, have a fluctuating magnitude, whereas encephalopathy develops gradually and is sometimes diagnosed later in the disease progress. EMA, ethylmalonic acid.

observing the molecular function of these proteins by the GO terms, 6 out of the 7 of them (approximately 86%) appeared to possess “oxidoreductase activity”. In the complete list of proteins detected in the mitochondria, 37% possessed “oxidoreductase activity”. Therefore, the probability to have 6 out of 7 with the

’ DISCUSSION In this study, we successfully detected proteins linked to the systemic effects of severe EE by performing mitochondrial proteomics of cultivated skin fibroblasts. EE, a disease whose etiology is challenging to understand, is caused by deficiency of ETHE1 protein,2 participating in mitochondrial sulfide metabolism. ETHE1 serves as a dioxygenase that has been described to have activity downstream of sulfide:quinone oxidoreductase (SQRDL) and upstream of a rhodanese.3 In the present study, the expression of seven proteins was significantly and consistently 2392

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Figure 6. Western blot depicting protein expression of SOD2 in three randomly selected patient samples and three different healthy controls. The mitochondrial protein VDAC1 is used as reference protein and loading control.

Figure 4. Proteins altered in EE patient cells compared with healthy controls. Values depicted are an average of three patient values divided by value from healthy controls. Blue and red bars correspond to data from three different patients. Error bars represent standard error of the mean. All shown data passed a threshold test of at least 26% alteration (based on three times the global standard error). “*” indicates p < 0.05 in the student’s t-test.

Figure 5. Proteomics data for oxidative stress proteins. Relative protein level between patient and healthy controls. Values are an average of three patient values divided by value of healthy controls. Blue and red bars correspond to data from three different patients. Error bars represent standard error of the mean. None of the proteins had statistically significant alterations.

altered in cultivated fibroblasts isolated from the EE patients. Interestingly, SQRDL was found to be decreased by a factor of approximately two, confirming that perturbation in the sulfide metabolism pathway occurs at protein level in the patient fibroblasts. Decreased levels of SQRDL suggests that the disruption of ETHE1 perturbs the SQRDL level. In this context, it is interesting to note that ETHE1 and SQRDL have been shown to be coregulated in rats during metabolic perturbation.12 It is

becoming increasingly clear that sulfide is the main causative factor in EE, however, the underlying mechanisms are not well understood, probably since perturbed sulfide metabolism can result in multiple pleiotropic effects. Our data indicate that proteins with oxidoreductase activities are overrepresented among the proteins with significant changes in ETHE1 deficient fibroblasts. This observation, together with the decreased levels of SOD2, indicates that redox balance and antioxidant activity were affected in fibroblasts isolated from EE patients. SOD2 has also been found to be down-regulated in cells from patients with variations in short chain acyl coenzyme A dehydrogenase (ACADS), and these patients have urinary excretion of ethylmalonic acid and certain symptoms in common with EE patients.17 Although, SOD2 was down-regulated in the present study no sensitivity to superoxide could be detected. Actually, previous studies have shown that presence of air alleviates some biochemical traits of EE cells. Inhibition of COX and ACADS, for example, have been shown to be fully reverted in vitro in the presence of air.3 Furthermore, these two enzymatic activities are normal in cultivated EE patient fibroblasts, but not in tissue samples.18,19 We attempted to study the degree of sulfide exposure in the cultivated cells by measuring the presence of its metabolic product thiosulfate in the cultivation media. Unfortunately, all the measurements were below the detection level of the applied assay20(approximately 0.1 μmol/mL) (data not shown). Altogether, it seems like the cultivated fibroblasts have a quite mild cellular phenotype, compared with other tissues. Future studies may better elucidate the role of oxygen in this context, and the detailed relationship between oxidoreductase activities and sulfide toxicity. Sulfide is known to react with redox-regulated cysteine residues and to regulate protein activities.5,21 Therefore sulfide toxicity may influence the activity of many different proteins. Very recently, the symptoms of EE were shown to be relieved by administration of N-acetylcysteine (NAC),9 which favors the synthesis of glutathione, and may improve glutathionedependent sulfide clearance and protection of protein thiols. Additionally, cells isolated from EE patients had altered levels of aldehyde dehydrogenase X (ALDH1B1), whose activity has recently been characterized.22 This protein has catalytic activity toward acetaldehyde, short- and medium-chain aliphatic aldehydes, as well as the products of lipid peroxidation, 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). Although the main physiological function of ALDH1B1 is unclear, the accumulation of the above aldehyde substrates may be toxic in cells, and the low levels of the ALDH1B1 enzyme observed in EE patient cells may worsen the conditions caused by aldehyde accumulation. Another dehydrogenase, L-lactate dehydrogenase B (LDH B), also was increased in patient samples, which often results in an increase in lactate production and causes lactic 2393

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Table 2. Gene Ontology (GO) Molecular Function of the Seven Proteins with Altered Level in EE Patients gene name; gene symbol Aldehyde dehydrogenase X,

GO molecular function oxidoreductase activity

GO biological process carbohydrate metabolic process

mitochondrial; ALDH1B1

nucleobase, nucleoside, nucleotide and nucleic acid metabolic process cellular amino acid and derivative metabolic process

N(G),N(G)-Dimethylarginine

hydrolase activity

mesoderm development

oxidoreductase activity

tricarboxylic acid cycle

oxidoreductase activity

carbohydrate metabolic process sulfur metabolic process

oxidoreductase activity

immune system process

dimethylaminohydrolase 1; DDAH1 L-Lactate

dehydrogenase B chain; LDHB

Sulfide:quinone oxidoreductase,

angiogenesis

mitochondrial; SQRDL Apoptosis-inducing factor 1, mitochondrial; AIFM1

respiratory electron transport chain apoptosis ferredoxin metabolic process oxygen and reactive oxygen species metabolic process

Chloride intracellular channel protein 4; CLIC4

oxidoreductase activity; transferase activity;

immune system process anion transport

racemase and epimerase activity;

intracellular signaling cascade

structural constituent of cytoskeleton;

oxygen and reactive oxygen species metabolic process

anion channel activity;

protein metabolic process

translation factor activity;

signal transduction;response to toxin

nucleic acid binding; receptor binding; Superoxide dismutase [Mn], mitochondrial; SOD2

translation elongation factor activity oxidoreductase activity

immune system process oxygen and reactive oxygen species metabolic process

acidosis that is associated with EE. Lactate production is induced by poor cellular energy status, which in the case of EE is caused by sulfide-mediated inhibition of cytochrome c oxidase.23,24 LDH predominantly functions as a cytosolic protein, however it also has activity inside mitochondria related to usage of lactate as an aerobic energy source.25 Furthermore, the other subunit of LDH, LDH A, also appeared to be overrepresented (30%) in our studies, although the increases were not statistically significant. Dimethylarginine dimethylaminohydrolase (DDAH1), was up-regulated in patient cells. This enzyme is an effector in the synthesis of nitric oxide (NO) and has been implicated in certain circulatory diseases.26,27 Both NO and sulfide (H2S) are gaseous mediators and the two compounds have been found to have partially overlapping functions. Sulfide is able to lower the level of NO through a variety of mechanisms, including a direct reaction to form nitrosothiol,28 or alternatively through affecting NO synthetase activity directly29 or linked through bicarbonate exchange.5 Therefore, sulfide can potentially suppress NO and the altered levels of DDAH1 observed in the present study may counteract this suppression. The chloride intracellular channel 4 (CLIC4), is a protein with little known function and the GO terms suggest that it functions in several different pathways (Table 2). The channel is localized to several cellular different compartments and may function in apoptotic pathways.30 CLIC4 is structurally related to glutathione-S-transferase and has a cysteine residue in its putative active site.31 This redox active cysteine may be why CLIC4 is influenced by sulfide accumulation caused by ETHE1 deficiency.

The apoptosis-inducing factor 1 (AIFM1), is underrepresented in EE patient mitochondria, which is possibly due to its release from mitochondria resulting in a subsequent induction of caspase-independent apoptosis through nuclear signaling.32 Interestingly, genetic variation in the AIFM1 gene has been found to be associated with mitochondrial encephalopathy33 and AIFM1 might therefore serve as the causative step leading to encephalopathy in EE patients. When localized to mitochondria, AIFM1 also serves as a redox active enzyme, a FAD containing NAD(P)H oxidase generating superoxide anions, an activity that is functionally separated from its role in apoptosis.34 However there may be a casual relationship between the two activities of AIFM1, that is, perturbation of redox balance (e.g., by sulfide) could putatively mediate cell death-related disease symptoms, including encephalopathy. Furthermore, we confirmed previous studies describing that ETHE1 protein in many cases are absent in the patients.2,16 However, in one of our patient samples, with variant Asp 165 Gly, the ETHE1 protein was indeed present. The proteomic data from this patient sample did not deviate from the other samples indicating that the ETHE1 function is lacking despite presence of protein. This supports previous, similar data suggesting position 161 and 163 to be close to catalytic site.16 In conclusion, the cultivated patient fibroblasts had high viability, and thus seem to have only mild phenotypic effects caused by the ETHE1 deficiency, a deficiency that is fatal for patients. By performing detailed proteomic studies, however, we managed to locate clear changes in the mitochondrial proteome and show that various biochemical pathways and oxidoreductases 2394

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Journal of Proteome Research are affected. The affected proteins include enzymes that detoxify aldehydes and superoxides, as well as apoptosis inducing factor 1 (AIFM1). These proteins are all involved in redox stress and might play important roles in the pathogenesis of the disease.

’ ASSOCIATED CONTENT

bS

Supporting Information Supplementary Table 1. Quantitative data of mitochondrial proteins. The ratios, patient value divided by control value, were calculated from at least four iTRAQ values. The proteins were sorted into mitochondrial pathways as previously described.10 Supplementary Table 2. Mass spectrometry data on peptide and protein identification, and relative quantification by iTRAQ, for study A. Supplementary Table 3. Mass spectrometry data on peptide and protein identification, and relative quantification by iTRAQ, for study B. Supplementary Figure 1. Viability of cultivated fibroblast cells measured by Nucleocounter technology based on fluorescence assay. The cells were treated in three different ways: no treatment (reference), or exposure to 100 μM of the superoxide inducing agent antimycin A for 15 min or 2 h, respectively. Blue bars depict fibroblast cells from healthy controls (n = 3) and red bars cells from EE patient fibroblasts (n = 3). Error bars are standard deviations of three independent experiments. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Fax: þ45 8949 6018. Phone: þ45 8949 5150.

’ ACKNOWLEDGMENT We acknowledge Kristian Knudsen for technical assistance as well as the Institute of Clinical Medicine at Aarhus University, Denmark and The John and Birthe Meyer Foundation for financial support. ’ REFERENCES (1) Burlina, A. B.; Dionisi-Vici, C.; Bennett, M. J.; Gibson, K. M.; Servidei, S.; Bertini, E.; Hale, D. E.; Schmidt-Sommerfeld, E.; Sabetta, G.; Zacchello, F.; et al. A new syndrome with ethylmalonic aciduria and normal fatty acid oxidation in fibroblasts. J. Pediatr. 1994, 124 (1), 79–86. (2) Tiranti, V.; D’Adamo, P.; Briem, E.; Ferrari, G.; Mineri, R.; Lamantea, E.; Mandel, H.; Balestri, P.; Garcia-Silva, M. T.; Vollmer, B.; Rinaldo, P.; Hahn, S. H.; Leonard, J.; Rahman, S.; Dionisi-Vici, C.; Garavaglia, B.; Gasparini, P.; Zeviani, M. Ethylmalonic encephalopathy is caused by mutations in ETHE1, a gene encoding a mitochondrial matrix protein. Am. J. Hum. Genet. 2004, 74 (2), 239–52. (3) Tiranti, V.; Viscomi, C.; Hildebrandt, T.; Di Meo, I.; Mineri, R.; Tiveron, C.; Levitt, M. D.; Prelle, A.; Fagiolari, G.; Rimoldi, M.; Zeviani, M. Loss of ETHE1, a mitochondrial dioxygenase, causes fatal sulfide toxicity in ethylmalonic encephalopathy. Nat. Med. 2009, 15 (2), 200–5. (4) Smith, R. P.; Abbanat, R. A. Protective effect of oxidized glutathione in acute sulfide poisoning. Toxicol. Appl. Pharmacol. 1966, 9 (2), 209–17. (5) Gadalla, M. M.; Snyder, S. H. Hydrogen sulfide as a gasotransmitter. J. Neurochem. 2010, 113 (1), 14–26. (6) Wang, R. Hydrogen sulfide: the third gasotransmitter in biology and medicine. Antioxid. Redox Signal. 2010, 12 (9), 1061–4.

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dx.doi.org/10.1021/pr101218d |J. Proteome Res. 2011, 10, 2389–2396