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Biosci. Rep. (2013) / 33 / art:e00052 / doi 10.1042/BSR20130051
Proteome adaptations in Ethe1-deficient mice
indicate a role in lipid catabolism and
cytoskeleton organization via post-translational
protein modifications
Tatjana M. HILDEBRANDT*1, Ivano DI MEO†, Massimo ZEVIANI†‡, Carlo VISCOMI† and Hans-Peter BRAUN*
*Institut f¨
ur Pflanzengenetik, Leibniz Universit¨
at Hannover, Herrenh¨
auser Straße 2, 30419 Hannover, Germany, †IRCCS Foundation
Neurological Institute ‘C. Besta’, Via Temolo 4, 20126 Milano, Italy, and ‡Medical Research Council Mitochondrial Biology Unit, Wellcome
Trust/MRC Building, Hills Road, Cambridge CB2 0XY, U.K.
Synopsis
Hydrogen sulfide is a physiologically relevant signalling molecule. However, circulating levels of this highly biologic-
ally active substance have to be maintained within tightly controlled limits in order to avoid toxic side effects. In
patients suffering from EE (ethylmalonic encephalopathy), a block in sulfide oxidation at the level of the SDO (sulfur
dioxygenase) ETHE1 leads to severe dysfunctions in microcirculation and cellular energy metabolism. We used an
Ethe1-deficient mouse model to investigate the effect of increased sulfide and persulfide concentrations on liver,
kidney, muscle and brain proteomes. Major disturbances in post-translational protein modifications indicate that the
mitochondrial sulfide oxidation pathway could have a crucial function during sulfide signalling most probably via
the regulation of cysteine S-modifications. Our results confirm the involvement of sulfide in redox regulation and
cytoskeleton dynamics. In addition, they suggest that sulfide signalling specifically regulates mitochondrial catabol-
ism of FAs (fatty acids) and BCAAs (branched-chain amino acids). These findings are particularly relevant in the context
of EE since they may explain major symptoms of the disease.
Key words: branched-chain amino acid oxidation, ethylmalonic encephalopathy, hydrogen sulfide, mitochondria, redox
regulation, sulfur dioxygenase
Cite this article as: Hildebrandt, T.M., Di Meo, I., Zeviani, M., Viscomi, C. and Braun, H-P. (2013) Proteome adaptations in Ethe1
deficient mice indicate a role in lipid catabolism and cytoskeleton organization via post-translational protein modifications. Biosci.
Rep. 33(4), art:e00052.doi:10.1042/BSR20130051
INTRODUCTION
ETHE1 is a mitochondrial SDO (sulfur dioxygenase) [1], which
takes part in sulfide detoxification by oxidizing persulfides to
sulfite [2]. Animal cells produce H2S during the catabolism of
sulfur-containing amino acids [3], and an additional source is the
large intestine where anaerobic bacteria reduce sulfate to sulf-
ide [4]. Endogenous H2S functions as a gaseous messenger and
has been suggested to be involved in the regulation of several
physiological processes such as vascular tone, insulin secretion
and inflammation, whereas elevated sulfide concentrations are
............................................................................................................................................................................................................................................................................................................
Abbreviations used: BCAA, branched-chain amino acid; BCKDH, branched-chain α-keto acid dehydrogenase; COX, cytochrome coxidase; DTT, dithiothreitol; EE, ethylmalonic
encephalopathy; ETF, electron transfer flavoprotein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IEF, isoelectric focusing; IPG, immobilized pH gradient; KGDH, 3-oxoglutarate
dehydrogenase; PDH, pyruvate dehydrogenase; pI, isoelectric point; PTM, post-translational modification; SCAD, short/branched-chain acyl-CoA dehydrogenase; SDO, sulfur
dioxygenase; QPCR, quantitative PCR.
1To whom correspondence should be addressed (email hildebrandt@genetik.uni-hannover.de).
highly toxic. The exact mechanism of sulfide signalling is largely
unknown and thought to be based on the activation of transcrip-
tion factors as well as direct cysteine S-sulfhydration of target
proteins [5,6].
Mutations that lead to a loss of function in ETHE1 dis-
rupt mitochondrial sulfide oxidation and thereby cause the fatal
metabolic disorder, EE (ethylmalonic encephalopathy; OMIM
#602473) [7,8]. In patients the vascular endothelium is severely
damaged by toxic sulfide concentrations in the bloodstream lead-
ing to the main symptoms of EE: rapidly progressive neurological
failure due to multiple necrotic and haemorrhagic brain lesions,
chronic haemorrhagic diarrhoea, vascular petechial purpura and
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T. M. Hildebrandt and others
orthostatic acrocyanosis [9]. The biochemical profile, an increase
in urinary and plasmatic lactate, acylcarnitine, acylglycine and
ethylmalonic acid levels, indicates disturbances in mitochondrial
energy metabolism. This can at least partially be explained by
sulfide toxicity. H2S is a well-known direct inhibitor of COX
(cytochrome coxidase) [10], and in addition chronic exposure
destabilizes subunits of the enzyme leading to a severe COX
deficiency in muscle, brain and colonic mucosa of EE patients
[11]. The accumulation of ethylmalonic acid, which is a derivat-
ive of butyrate, as well as elevated C4 and C5 acylcarnitines and
acylglycines, reflect a block in oxidative metabolism of BCAAs
(branched-chain amino acids). Indeed H2S has been shown to in-
hibit one enzyme of this pathway, SCAD (short-chain acyl CoA
dehydrogenase), in vitro [1].
The severe systemic consequences of a dysfunction in the
sulfide oxidation system become obvious in patients suffering
from EE. The aim of this study was to identify the effects
of elevated sulfide concentrations on the protein composition of
different tissues in order to shed light on the role of sulfide in
cellular metabolism. For this purpose, the proteomes of liver, kid-
ney, skeletal muscle and brain were analysed in a recombinant
Ethe1-deficient mouse model, which faithfully recapitulates the
clinical and biochemical features of EE [1].
EXPERIMENTAL
Animals
Recombinant Ethe1 −/−mice were obtained as described [1]. The
mice were maintained on a C57BL6/129Sv mixed background.
Four-week-old Ethe1−/−and Ethe1 +/+female littermates were
considered in this study. Animal studies were in accordance with
the Italian Law D.L. 116/1992 and the EU directive 86/609/CEE.
Mice were maintained in a temperature- and humidity-controlled
animal-care facility, with a 12 h light/dark cycle and free access to
water and food (Standard Diet). Mice were killed by dislocation
of the neck.
Protein extraction and 2D gel electrophoresis
Tissue samples were frozen in liquid nitrogen and ground in
a shaker mill (Retsch). For IEF (isoelectric focusing) 4 mg of
each sample were suspended in 350 μl rehydration buffer (6 M
urea, 2 M thiourea, 50 mM DTT (dithiothreitol), 2 % CHAPS
(w/v), 5 % IPG (immobilized pH gradient) buffer 3–11 nl (v/v),
12 μl/ml DeStreak reagent and a trace of bromphenol blue) and
homogenized by sonication (3×20 s). Samples were centrifuged
at 17000 gfor 10 min and the protein content was adjusted
to 2.14 mg/ml (Quantkit, GE Healthcare). IEF was carried out
on Immobiline DryStrip gels (18 cm, non-linear gradient pH 3–
11) using the Ettan IPGphor 3 system (GE Healthcare). For the
second dimension (SDS/PAGE) IPG strips were equilibrated in
6 M urea, 30 % glycerol (87 %, v/v), 2 % SDS, 50 mM Tris/HCl
pH 8.8, bromphenol blue with (i) 1% DTT (w/v) and (ii) 2.5 %
Iodacetamide (w/v) and transferred horizontally onto 16.5 % Tri-
cine gels. Electrophoresis was carried out for 20 h at 35 mA/mm
gel layer in a Protean IIXL gel system (Biorad) using a broad-
range protein molecular mass marker (10–225 kDa, Promega) as
molecular mass standard.
Gel image analysis
Gels were stained overnight with colloidal CBB (Coomassie Bril-
liant Blue), CBB-250 G (Merck), scanned and analysed with
Delta2D software version 4.2 (Decodon). Six gels were used
for each tissue (three biological replicates) to compare the pro-
tein abundance between Ethe1 knockout mice and wild-type lit-
termates. Only protein spots with significant (Student’s ttest,
P<0.05), at least 1.5-fold differences in the relative spot volume
were assumed to be of different abundance.
Protein identification by MS
Spots were excised from the 2D gels using a manual spot picker
(Genetix), washed with 0.1 M ammonium bicarbonate and de-
hydrated with acetonitrile. For tryptic digestion the gel pieces
were incubated overnight at 37◦C(2μg/ml trypsin in 0.1 M
ammonium bicarbonate, Promega). Tryptic peptides were ex-
tracted by successive incubation with (i) 50% (v/v) acetoni-
trile, 5 % (v/v) formic acid (ii +iii) 50 % (v/v) acetonitrile, 1 %
(v/v) formic acid (iv) 100% acetonitrile for 15 min at 37 ◦C.
Supernatants were pooled and dried by vacuum centrifugation.
Peptides were analysed using an EASY-nLC-system (Proxeon)
coupled to a MicrOTOF-Q-II mass spectrometer (Bruker Dalton-
ics) according to the protocol described in [12]. Proteins were
identified using the MASCOT search algorithm against Swiss-
Prot (www.uniprot.org) and NCBI non-redundant protein data-
base (www.ncbi.nlm.nih.gov/protein).
Data analysis
A co-expression analysis for the Ethe1 gene (Mm.29553,
1417203_at) was performed with Genevestigator (www.
genevestigator.com; Nebion). All 2910 wild-type genetic back-
ground samples available in the mouse 40 k microarray platform
of the manually curated database of Genevestigator were used
for the query. Co-expressed genes were identified by using the
Pearson correlation coefficient as the measure of similarity. The
bioinformatics tool DAVID [13] was used to identify enriched
functional annotation terms within the datasets.
Western blotting
Mouse tissues were homogenized in 10×(v/w)10 mM potassium
phosphate buffer, pH 7.4 and centrifuged at 800 gfor 10 min
in the presence of protease inhibitors. For the analysis of mito-
chondrial proteins, an additional high-speed centrifugation step
was used to collect the mitochondria. Samples were frozen and
thawed twice in liquid nitrogen. Approximately 60 μg of proteins
was used for each sample in denaturing SDS/PAGE. Western blot
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which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.
Proteome changes in Ethe1 deficiency
Figure 1 Changes in protein abundance in Ethe1-deficient mouse tissues
Total proteins (0.75 mg) from liver (A), kidney (B), brain (C) and muscle (D) of three knockout mice and three wild-type
littermates were separated by IEF/SDS/PAGE (pH 3–11, 16% acr ylamide). The Coomassie Brilliant Blue stained gels were
analysed with Delta2D, and significantly different spots (P<0.05) with at least 1.5-fold changes in volume are numbered
in the fused images. Proteins were identified via nanoLC-MS/MS (see Table 1, Supplementary Table S1).
analysis was performed with α-ETHE1 [1], α-ETF (α-electron
transfer flavoprotein) [14], α-SCAD (abcam), α-SDH-A (Invitro-
gen), α-GAPDH (α-glyceraldehyde 3-phosphate dehydrogenase)
(Millipore), α-Actin (Millipore) and α-ACTA1 (Dako) antibod-
ies, using the ECL (enhanced chemiluminescence) (Amersham),
as described elsewhere [11].
PCR
Tissue-derived RNA was isolated with TRIzol reagent (Invitro-
gen). Two micrograms of total RNA was treated with RNase-free
DNase and retro-transcribed by using the Cloned AMV First-
strand cDNA Synthesis kit and protocol (Invitrogen). Approx-
imately 2–5 ng of cDNA was used for SYBR-GREEN based
real-time PCR using primers specific for the amplification of
the genes of interest (oligo sequences available on request) ac-
cording to the ABI-Primer Express software. Standard transcript
HPRT (hypoxanthine-guanine phosphoribosyltransferase) was
co-amplified using suitable primers. Real-time QPCR (quantitat-
ive PCR) was carried out using an ABI PRISM 7000 Sequence
Detection System. The amplification profile was according to
a two-step protocol: one cycle at 50◦C for 2 min, one cycle at
95 ◦C for 10 min and then 40 cycles of 95◦C for 15 s and 60 ◦C
for 1 min. A final dissociation step (95 ◦C for 15 s, 60 ◦C for 20 s,
95◦for 15 s) was added to assess for unspecific primer–dimer
amplifications.
RESULTS
Effects of Ethe1 deficiency on mouse liver, kidney,
brain and muscle proteomes
Total protein extracts from liver, kidney, brain and skeletal muscle
were separated by 2D IEF/SDS/PAGE resulting in 70 spots with
significantly different volumes in Ethe1-deficient mice com-
pared with wild-type littermates (Figure 1) that contained 81
proteins (Tab l e 1 , Supplementary Table S1 available at http://
www.bioscirep.org/bsr/033/bsr033e052add.htm). These proteins
identified in spots with a changed volume will be described by the
shorter term ‘changed/affected proteins’ throughout the paper. In
parallel, an unbiased co-expression analysis based on microar-
ray data available in public repositories was carried out using
the Genevestigator software. The 81 top scoring genes for co-
expression with Ethe1 in wild-type mouse tissues were identified
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577
T. M. Hildebrandt and others
Table 1 Proteins with potentially changed abundance in liver,
kidney, brain or muscle of Ethe1-deficient mice
Proteins were separated by IEF/SDS/PAGE (Figure 1) and spots
with at least 1.5-fold changes in volume (k.o./wt) were analysed by
nanoLC-MS/MS. For additional information see Supplementary Table
S1 (available at http://www.bioscirep.org/bsr/033/bsr033e052add.
htm).
Spot Accession Name k.o./wt
Liver
1 P60710 β-Actin 1 0.43
2 Q64374 Regucalcin 0.53
3 Q91Y97 Fructose-bisphosphate aldolase B 0.54
Q8VCH0 3-ketoacyl-CoA thiolase B 0.54
4 P97823 Acyl-protein thioesterase 1 0.57
5 Q8R086 Sulfite oxidase 0.57
Q63836 Selenium-binding protein 2 0.57
6 P29758 Ornithine aminotransferase 0.59
7 Intermediate filament protein 0.62
O08709 Peroxiredoxin 6 0.62
8 Q80W21 Glutathione S-transferase Mu 7 0.62
9 P30416 Peptidyl-prolyl cis-trans isomerase 0.66
10 Q03265 ATP synthase subunit alpha 1.57
11 P10649 GST Mu 1 1.58
12 P63038 HSP 60 1.66
13 P55264 Adenosine kinase 1.68
14 Q8BH95 Enoyl-CoA hydratase 2.15
15 Q9DCW4 Electron transfer flavoprotein 2.62
16 Q9QXF8 Glycine N-methyltransferase 2.85
17 P35700 Peroxiredoxin 1 3.01
Kidney
18 Q60866 Phosphotriesterase 0.34
19 P28825 Meprin A subunit alpha 0.37
20 Q9JHW2 Omega-amidase NIT2 0.41
21 Q9QUM9 Proteasome subunit alpha 0.42
22 Q9DBJ1 Phosphoglycerate mutase 1 0.42
23 Q9QYR9 Acyl-coenzyme A thioesterase 0.47
24 Q8CAQ8 Mitofilin 0.51
25 Q99J99 3-mercaptopyruvate sulfurtransferase 0.52
26 Q9WU78 PCD 6-interacting protein 0.53
27 Q60866 Phosphotriesterase 0.53
28 P11352 Glutathione peroxidase 1 0.57
29 P27773 Protein disulfide-isomerase A3 0.58
30 P28825 Meprin A 0.58
31 P47738 Aldehyde DH 0.59
32 P28825 Meprin A 0.61
33 P09103 Protein disulfide-isomerase A1 0.61
34 Q9D964 Glycine amidinotransferase 0.66
35 Q9JK42 PDH kinase 1.64
36 Q9DBL1 SCAD 1.64
37 Q62433 Protein NDRG1 1.67
38 Q6NZD1 Sulfotransferase 1.70
39 Q91VR2 ATP synthase subunit gamma 1.74
Q64669 NAD(P)H DH 1.74
Brain
40 P09103 Protein disulfide-isomerase A1 0.44
Table 1 Continue
Spot Accession Name k.o./wt
41 Q03265 ATP synthase subunit alpha 0.47
42 P14206 40S ribosomal protein SA 0.56
Q61937 Nucleophosmin 0.56
43 O55131 Septin-7 0.59
44 Q64332 Synapsin-2b 0.59
P06745 Glucose-6-phosphate isomerase 0.59
45 P52480 Pyr uvate kinase 0.64
46 P17751 Triosephosphate isomerase 1.54
47 Q03265 ATP synthase subunit alpha 1.58
48 Q61792 LIM and SH3 domain protein 1 1.59
49 P38647 Stress-70 protein 1.61
50 P35505 Fumarylacetoacetase 1.65
51 P80317 T-complex protein 1 1.87
52 P27773 Protein disulfide-isomerase A3 1.88
53 Q91YT0 NADH DH flavoprotein 1 1.94
P10126 Elongation factor 1-alpha 1 1.94
O55131 Septin-7 1.94
54 P16858 GAPDH 2.08
55 Q60932 VDAC protein 1 2.14
P00920 Carbonic anhydrase 2 2.14
56 P17742 Peptidyl-prolyl cis-trans isomerase A 2.19
57 P63101 14-3-3 protein ζ/δ3.06
58 Q9D2G2 2-oxoglutarate DH E2 3.45
Muscle
59 Q9JKS4 LIM domain-binding protein 3 0.28
60 Q9JIF9 Myotilin 0.34
61 Q9D0F9 Phosphoglucomutase-1 0.41
62 Q62167 RNA helicase DDX3X 0.53
63 P19157 GST P1 0.61
64 Q00898 α-1-antitrypsin 1-5 0.65
P62737 Actin, aortic smooth muscle 0.65
65 P62737 Actin, aor tic smooth muscle 1.50
P07758 α-1-antitrypsin 1-1 1.50
66 Q9D6R2 Isocitrate DH 1.58
Q9JHR4 myosin heavy chain IIB 1.58
67 Q99JY0 3-ketoacyl-CoA-thiolase 1.61
68 P14602 HSPβ1 1.61
69 P97457 Myosin regulator y light chain 2 1.67
70 P04117 FA-binding protein 2.25
(Supplementary Table S2 available at http://www.bioscirep.org/
bsr/033/bsr033e052add.htm) and will be called ‘co-expressed
genes/proteins’.
In order to obtain a general overview on which pathways or
functional categories are associated with Ethe1, we carried out en-
richment analysis with the software tool DAVID on the two com-
plete data sets generated by either proteomics or co-expression
analysis. The majority of differentially abundant proteins were
those regulated by PTMs (post-translational modifications, acet-
ylation and phosphorylation) or with specific subcellular local-
ization (cytosolic and mitochondrial) (Tab l e 2 ). While the latter
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Proteome changes in Ethe1 deficiency
Table 2 Functional annotation analysis
Proteins with different abundance in Ethe1 deficient compared with wild-type mouse liver, kidney, brain and muscle (proteomics) and of genes that
are co-expressed with Ethe1 in wild-type mouse samples (co-expression) were analysed with DAVID.
Proteomics Co-expression
Functional annotation Number of proteins Fold enrichment Number of proteins Fold enrichment
Acetylation 48 5.0 21 2.0
Phosphorylation 48 1.8 Not enriched Not enriched
Cytosol 37 2.9 Not enriched Not enriched
Mitochondrion 30 4.9 38 5.7
Figure 2 Biological processes affected by Ethe1 deficiency
Proteins with different abundance in Ethe1 deficient compared with
wild-type mouse liver (black bars), kidney (white bars), brain (light grey
bars) or muscle (dark grey bars) were assigned to pathways according to
the information provided by UniProt. Coex., number of proteins from the
categories listed that are co-expressed with Ethe1 in wild-type mouse
samples.
result was somewhat expected, given the mitochondrial localiz-
ation of Ethe1, the former suggests a role for Ethe1 in PTMs.
In particular, proteins regulated by acetylation were five times
enriched in our sample compared with the whole proteome. In-
terestingly, similar results were obtained by clustering the genes
from co-expression analysis, confirming a close functional con-
nection between Ethe1 and PTMs.
Next, we sorted all proteins into biological processes accord-
ing to the information available in the Uniprot database (www.
uniprot.org) (Figure 2). Among proteins significantly changed,
only four were enzymes directly related to sulfur metabol-
ism, namely sulfite oxidase, glycine N-methyltransferase, 3-
mercaptopyruvate sulfurtransferase and a sulfotransferase. The
largest group of proteins, including 17 enzymes from the proteo-
mic experiment and 14 from co-expression analysis, catalyse re-
actions during the post-translational processing of proteins, some
taking part in general protein handling such as folding (several
chaperones, peptidyl-prolyl cis–trans isomerase) and degradation
(proteasome subunits, proteases), other being involved in specific
PTMs (ubiquitinylation, glycosylation, methylation, sialylation,
glutathionylation and acylation).
In brain and muscle, we found evidence for an influence on
proteins related to cell structure. Actin and myosin were iden-
tified in four spots with a changed volume in Ethe1-less mouse
muscle. In addition, several proteins that take part in the organiz-
ation of the cytoskeleton, such as chaperones [T-complex protein
1andHSPβ1 (heat-shock protein β1)], LIM domain containing
or binding proteins, a septin and myotilin, were affected, sug-
gesting that alterations of the cytoskeleton may be involved in
the pathogenesis of EE.
Our analysis shows that Ethe1 has a key role in energy meta-
bolism affecting central processes as different as the glycolysis,
the TCA (tricarboxylic acid) cycle and the OXPHOS system.
Interestingly, the correlation to lipid metabolism was most pro-
nounced. Six enzymes, all catalysing catabolic reactions, were
affected by Ethe1 deficiency. Co-expression analysis resulted in
19 genes related to lipid metabolism, including 14 enzymes of
FA (fatty acid) or BCAA oxidation, two proteins involved in the
regulation of lipid catabolism, two in FA binding and transport
and only one enzyme of FA synthesis (Supplementary Table S2).
These findings prompted us to investigate further the potential
role of Ethe1 in the oxidative catabolism of FAs and BCAAs.
Effect of Ethe1 deficiency on FA/BCAA catabolic
enzymes
The BCAAs valine, leucine and isoleucine are oxidized in the
mitochondria, and some of the reactions overlap with FA β
oxidation (Figure 3)[15]. Enzymes catalysing these common
steps were most clearly correlated to Ethe1. As an example, the
tissue distribution of Ethe1, SCAD and the committed step in
BCAA oxidation, BCKDH (branched-chain keto acid dehydro-
genase), on expression level is shown in Figure 4. Four enzymes
of FA/BCAA oxidation, SCAD, ETF, enoyl-CoA hydratase and
3-ketoacyl-CoA thiolase, are co-expressed with Ethe1 and also
present in spots with an increased volume (1.6- to 2.6-fold) in
Ethe1-deficient mice (Figure 3). Separation on the basis of the
pI (isoelectric point) detects not only differences in total protein
abundance but also some PTMs that modify the charge of the
protein. We therefore tested whether these enzymes were actu-
ally up-regulated by measuring transcript expression and protein
amount (Figure 5). No differences were detected in either ex-
pression level or total abundance of SCAD, ETF or enoyl-CoA
hydratase indicating an influence on PTMs. In contrast, BCKDH
subunits E2 and E3, but not E1, were significantly less expressed
in the Ethe1-less mice. Similar to PDH (pyruvate dehydrogenase)
and KGDH (3-oxoglutarate dehydrogenase), BCKDH is a large
protein complex consisting of three subunits present in multiple
copies, subunit E3 being common to all three dehydrogenases.
However, the expression of PDH and KGDH E2 subunits was
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T. M. Hildebrandt and others
Figure 3 Correlation of Ethe1 and FA/BCAA
The enzymes catalysing common reactions in BCAA and FA oxidative metabolism are affected by Ethe1 deficiency (arrows
mark increased abundance in Ethe1-deficient mouse liver, kidney, brain or muscle) and also co-expressed with Ethe1
(asterisks). Valine oxidation is shown in solid lines, leucine oxidation in dashed lines and isoleucine oxidation in dotted
lines. BCKDH, branched-chain α-keto acid dehydrogenase; SCAD, short/branched-chain acyl-CoA dehydrogenase; ETF,
electron transfer flavoprotein.
Figure 4 Tissue distribution of Ethe1 and BCAA catabolic
enzymes
Similar expression pattern of BCKDH (E1 α-subunit), SCAD and ETHE1:
gene expression in wild-type mouse skeletal muscle, brain, liver and
kidney was analysed using Genevestigator.
unaffected (results not shown). Notably, these findings clarify
the original clinical description of the disease that was thought to
be a defect in the β-oxidation pathway.
Effect of Ethe1 deficiency on PTMs
GAPDH and actin are both known to be regulated by diverse
forms of PTMs. Among all proteins affected by Ethe1 deficiency,
these two have the highest number of annotations in Uniprot,
with GAPDH being subject to ADP-ribosylation, acetylation,
methylation, phosphorylation, S-nitrosylation and ubiquitin con-
jugation, and actin being annotated for acetylation, methylation,
nitration, oxidation, phosphorylation and ubiquitin conjugation
(Supplementary Table S1). In addition, both proteins are reg-
ulated by different forms of cysteine S-modifications such as
glutathionylation, palmitoylation and most interestingly sulfhy-
dration, which is not yet included in the annotations [16]. The
IEF/SDS results indicate an influence of elevated sulfide and per-
sulfide concentrations on these PTMs. GAPDH was present in
a 2.1-fold larger spot in Ethe1-deficient mouse brain that was
located at a more acidic pH than the calculated pI of the protein.
Such a shift can be caused by different modifications that either
introduce an additional negative charge, such as phosphorylation,
or mask a positive charge, as in lysine acetylation. A pI shift was
also detectable for αactin in muscle with a 1.5-fold decrease in
spot 64 and a matching increase in spot 65 at a slightly lower
pH (Figure 1). Expression levels of GAPDH and actin were con-
sistently elevated in Ethe1-deficient mice, whereas an increase in
total protein abundance was only detectable for muscle GAPDH
(Figure 6).
DISCUSSION
The pathogenic mechanism of EE is related to the accumulation
of high levels of hydrogen sulfide in tissues and body fluids of
the patients and of the animal model of the disease. While the
effects of sulfide on COX have been thoroughly investigated, and
shown to be because of an increased degradation of COX subunits
[11], the additional systemic consequences are not completely
understood.
Sulfide in protein regulation
Our results suggest that Ethe1 and the sulfide oxidation pathway
have a major role in post-translational protein modifications. Pro-
teins that are regulated by such modifications were enriched in
both lists of proteins, those affected by Ethe1 deficiency as well as
those co-expressed with Ethe1. The data sets also contained many
enzymes involved in the catalysis of reactions during the post-
translational processing of proteins. Two examples of changed
PTM patterns were directly detectable on our 2D gels via acidic
spot shifts (actin and septin).
Apart from direct inhibition, cysteine S-sulfhydration is the
only known mechanism of enzyme regulation by hydrogen
sulfide. Cysteine thiol groups can be converted to hydropersulf-
ides (-SSH), which mostly leads to increased protein activity
[16]. Since Ethe1 oxidizes persulfide groups, it could play a role
as a negative regulator of S-sulfhydration. The activity as well
as stability of enzymes can be regulated by an interaction of
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which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.
Proteome changes in Ethe1 deficiency
Figure 5 Effect of Ethe1 deficiency on the expression and protein content of enzymes involved in BCAA and FA oxidation
in mouse liver
(A) Gene expression was analysed by QPCR and expression in Ethe−/−samples (grey bars) is shown in relation to the
corresponding wild-type samples (black bars). (B) Protein abundance was estimated by Western blot. Numbers indicate the
relative densities of the bands (percentage of wild-type). E1, E2, E3, subunits of the BCKDH; SCAD, short/branched-chain
acyl-CoA dehydrogenase; ETF, electron transfer flavoprotein; ECH, enoyl-CoA hydratase; SDH-A, subunit a of succinate
dehydrogenase complex. *Significantly different from wild-type (P<0.05).
Figure 6 Effect of Ethe1 deficiency on the expression and protein content of GAPDH and actin
(A,C) Gene expression was analysed by QPCR and expression in Ethe1−/−samples (grey bars) is shown in relation to the
corresponding wild-type samples (black bars). (B,D) Protein abundance was estimated by Western blot. Numbers indicate
the relative densities of the bands (percentage of wild-type). (A,B) mouse liver, (C,D) mouse skeletal muscle. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; ACTA1, actin α1; SDH-A, subunit a of succinate dehydrogenase complex.
*Significantly different from wild-type (P<0.05).
different cysteine S-modifications such as S-nitrosylation, S-
acylation and S-glutathionylation. We found that acyl-protein
thioesterase, which hydrolyses regulatory FAs bound to cysteine
thiols, as well as several GSTs (glutathione transferases), which
remove cysteine S-glutathionylations, were affected by the
knock-out of Ethe1. Thus, our results indicate a general disturb-
ance in the modification of regulatory cysteines, as a consequence
of a block in sulfide and persulfide oxidation.
GAPDH is a well-studied example of enzyme regulation by
different forms of cysteine S-modifications. It is inactivated by
binding of GSH, NO (nitric oxide) or palmitic acid to specific
cysteine residues, whereas the activity is 7-fold increased after
sulfhydration of Cys150 [16–19]. The acidic shift of GAPDH on
our 2D gels indicates differences in the PTM pattern. GAPDH
expression was increased in liver and muscle of Ethe1-less mice
with no concomitant increase in total protein abundance in liver.
While in muscle glycolysis is probably up-regulated by the
block in mitochondrial respiration, dysregulation might lead to
decreased stability of GAPDH in the liver. Similar discrepan-
cies between expression level, total protein abundance and 2D
spot size, were also detected for actin and several enzymes of
BCAA/FA oxidation, which will be discussed in subsequent para-
graphs.
Sulfide in BCAA/FA catabolism
The metabolite profile in blood and urine of EE patients and
Ethe1-deficient mice, characterized by the accumulation of ethyl-
malonic acid and C4–C5 acylcarnitines, indicates major disturb-
ances in the oxidation of BCAAs and FAs. A similar profile is
indeed also present in defects of acyl-CoA dehydrogenases such
as SCAD, isobutyryl-CoA dehydrogenase and 2-methylbutyryl-
CoA dehydrogenase [20–22]. Thus a simple and elegant explan-
ation for the effect of Ethe1 deficiency on lipid metabolism is the
inhibition of these enzymes by accumulating sulfide or persulf-
ides. SCAD is directly inhibited by sulfide [1] and, in addition,
CoA persulfide might play a role, as a powerful inhibitor of acyl-
CoA dehydrogenases [23,24].
The results presented here reveal an additional level of interac-
tion between Ethe1 and the metabolism of FAs and BCAAs that
may explain the original clinical description of the disease as a β-
oxidation defect. Lipid catabolism is most clearly co-expressed
with Ethe1 and several related protein abundances change as a
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581
T. M. Hildebrandt and others
result of the knock-out of this gene. Interestingly, the prevalence
of Ethe1 in liver and muscle parallels the tissue distribution of
FA/BCAA oxidizing enzymes [25]. In addition, there is evidence
for an influence on the regulatory level. Gene expression for two
of the three subunits of the BCKDH complex was decreased in
Ethe1−/−liver. As the flux-generating step in the catabolism of
BCAAs BCKDH is tightly regulated [26]. In the knockout mice
either sulfide or, more likely, the accumulating reaction products
of BCKDH down-regulate E2 and E3 subunits. The expression
of enzymes catalysing subsequent steps in FA/BCAA oxidation
was unchanged so that in this case an effect of sulfide or other
metabolites on transcription factors can be excluded. Also sulfide
does not interfere with the stability of ETF subunits or SCAD as
the block in sulfide oxidation had no influence on total protein
abundance. Since direct enzyme inhibition would not be visible
on the gels, the differences detected in the 2D proteome are most
probably caused by an altered pattern of PTMs. Mitochondrial FA
βoxidation has been shown to be regulated by lysine acetylation
[27]. Also the enzymes catalysing common steps in FA/BCAA
oxidation are subject to different kinds of cysteine S modific-
ations [28,29]. Accumulating sulfide or persulfide probably in-
terferes with this regulatory system. However, the mechanism
of this interference will have to be further elucidated in order
to fully understand the physiological role of the SDO in lipid
metabolism.
A general connection between sulfide and lipid metabolism
has previously been reported. Sulfide concentrations were de-
creased in the plasma of overweight compared with lean men and
low sulfide levels are associated with the development of insulin
resistance in Type 2 diabetes [30]. Baiges et al. [31] found that
Ethe1 and sulfide quinone oxidoreductase, which catalyses the
first step in the mitochondrial sulfide oxidation pathway, were
decreased by more than 50 % in rats fed with a high-fat diet.
Thus there seems to be a reciprocal influence of FA and sulfide
metabolism with a block of lipid catabolism by high sulfide con-
centrations and a down-regulation of sulfide metabolism by high
concentrations of FAs.
Sulfide in cell structure
EE patients and Ethe1-deficient mice show severe defects in mo-
tor activity and rapidly progressive encephalopathy, both of which
are not fully understood yet. Our results show that the increase in
sulfide and persulfide concentrations elicits changes in structural
proteins as well as in enzymes involved in the organization of the
cytoskeleton in brain and muscle, indicating an organ-specific
role similar to the isolated COX deficiency, which is also preval-
ent in these tissues. Accordingly, co-expression analysis provided
no hints for a general correlation of genes associated with the cell
structure and Ethe1. The reasons for this tissue-specificity are
currently unknown. However, it should be mentioned that neither
brain – nor muscle – restricted Ethe1 knockout mice, show a
clinical phenotype, in spite of tissue-specific COX deficiency,
suggesting that the pathogenesis of the disease relies on the ab-
sence of Ethe1 in other tissues, particularly in the colon [11].
In fact, the combined administration of NAC (N-acetylcysteine),
a glutathione precursor and metronidazole, a bactericidal agent
against the anaerobic bacteria of the colon, ameliorates the clin-
ical conditions of both mice and patients [32]. Di Meo et al.
recently demonstrated that a gene therapy approach based on
adeno-associated viral vectors specifically targeting the liver in
the very same mouse model used in this study is highly effect-
ive in prolonging the lifespan, likely by scavenging most of the
circulating sulfide [33].
Our results indicate a role of PTMs in the regulation of cyto-
skeleton dynamics by sulfide. An intermediate filament protein
and actin were present in smaller spots in Ethe1-less mouse liver,
but no changes were detected in total actin. Similarly, the actin
content of knockout mouse muscle tissue was unchanged al-
though gene expression was increased, and the IEF/SDS results
show a higher ratio of modified α-actin. Mustafa et al. identified
actin as one of the three most abundant sulfhydrated proteins in
sulfide treated mouse liver and estimated that about 12% of the
actin molecules are sulfhydrated under basic physiological con-
ditions [16]. Sulfhydration enhances actin polymerization in vitro
and incubation with 100 μM NaHS leads to rearrangements of the
actin cytoskeleton in HEK-293 (human embryonic kidney 293).
Sulfhydration itself would not be visible on IEF/SDS gels as the
charge of the protein is not changed. However, there could be
direct or indirect effects on other forms of modifications such as
phosphorylation or acetylation that possibly influence its stabil-
ity. Sulfide has been shown to induce a reorganization of the
actin cytoskeleton in human vascular endothelial cells via
the small GTPase Rac1 [34]. Interestingly, we also found a pH
shift of the cytoskeletal GTPase Septin 7 in the brain of Ethe1-
deficient mice (Figure 1,Tab l e 1 , spots 43 and 53) indicating
again dysregulation. Additional hints confirming that accumulat-
ing sulfide interferes with the organization of the cell structure
are changes in abundance of structure specific chaperones and
several regulatory cytoskeletal elements. Therefore, the proteo-
mic results show that sulfide signalling must be considered an
important factor in the control of cytoskeleton dynamics.
Additional aspects
Surprisingly, effects of Ethe1 deficiency on sulfur metabolic en-
zymes were limited. Sulfite oxidase and β-mercaptopyruvate
sulfurtransferase were both detected in spots with a decreased
volume. A down-regulation of these enzymes would indeed make
sense since sulfite concentrations are decreased in the knockout
mice and the sulfurtransferase produces additional sulfide. It has
been suggested that probably alternative pathways for sulfide de-
toxification exist at least in liver or kidney, which are currently
unknown [1]. However, there is no hint so far about a possible
involvement of one of the enzymes found in increased spots in
these tissues, glycine-N-methyltransferase or sulfotransferase in
this process.
H2S acts as a cytoprotective agent by activating Nrf2 (nuclear
factor-erythroid 2 p45 subunit-related factor 2), which induces the
expression of several antioxidant enzymes [6]. In addition, the
sulfide molecule itself has antioxidant properties. Our results
show that increased sulfide concentrations due to disruption of
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which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.
Proteome changes in Ethe1 deficiency
the sulfide oxidation pathway have several effects on the antiox-
idant system (e.g. glutathione peroxidase, peroxiredoxin), thus
confirming the findings of Palmfeldt et al., who detected alter-
ations in the redox mitochondrial proteome from cultured skin
fibroblasts of six EE patients [35].
Conclusions
Our results provide evidence for a crucial role of the mitochon-
drial sulfide oxidation pathway in sulfide signalling. Increased
sulfide concentrations, due to a block in this pathway at the level
of the SDO, lead to major disturbances in post-translational pro-
tein modifications, which can explain the pleiotropic devastating
effects of EE. This study confirms the involvement of sulfide in
redox regulation and cytoskeleton dynamics. In addition, we sug-
gest that sulfide signalling also specifically regulates mitochon-
drial BCAA/FA catabolism and thus takes part in the coordina-
tion of the energy source used by the organism. 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 PTMs. In addition, ETHE1 as an SDO could take part in
the removal of sulfhydrations by oxidizing the cysteine-derived
persulfide group.
AUTHOR CONTRIBUTION
Tatjana Hildebrandt designed the study, performed the proteomics
experiments and co-expression analysis, analysed and interpreted
the data, and wrote the paper. Ivano Di Meo performed the qPCR
and Western blot analysis. Carlo Viscomi provided the mouse tis-
sues, analysed and interpreted the data, and wrote the paper.
Massimo Zeviani and Hans-Peter Braun supervised the work and
revised the paper.
FUNDING
We acknowledge support by Deutsche Forschungsgemeinschaft
and Open Access Publishing Fund of Leibniz Universit¨
at Hannover.
This work was supported by the Deutsche Forschungsgemeinschaft
[grant number HI 1471/1-1 (to T.H.)]; Telethon grant [grant numbers
GPP10005 and AFM15927 (to M.Z.)]; and the Pierfranco and Luisa
Mariani Foundation Italy.
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Received 15 May 2013; accepted 29 May 2013
Published as Immediate Publication 26 June 2013, doi 10.1042/BSR20130051
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584 c
2013 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC-BY) (http://creativecommons.org/licenses/by/3.0)
which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.
