An almost final solution to theEthylmalonic acid syndrome
Valeria TirantiERNDIM Meeting
October 22-23, 2009Basel
CNS•early-onset hypotonia. developmental delay•later spasticity, then global failure
Vascular system•acrocyanosis•petechiae, microematuria, internal bleedings
Gastrointestinal system•chronic diarrhoea
Biochemistry•COX deficiency (only in muscle)•lactic acidosis•ethylmalonic aciduria•elevated C4-C5 acylcarnitine
0123456789
Abundance
urea
ethylmalonate
I.S.
2-ketoglutarate
I.S.isobutyrylglycineisovalerylglycine
Time
EthylmalonateButyryl-CoA CO2 ->CH3-CH2-CH2-CO-SCoA + CH3-CH2-CH-COOH
COOH
CoASH
Ethylmalonic Encephalopathy (EE)
EX1-7del
440del11
11 22 33 44 55 66 77
3G>T
113A>G 131delAG222insA
406A>G
488G>A
487C>T 604insG
559insC375+5G>A
505+1G>A
505+1G>T
554T>G
EX4del
GCCCCdel 376-1G>T187C>T
482G>A230delA34C>T
ETHE1: the gene50 EEpatients28 non-EEEMA patients
HeLa
ETHE1 Mitotrackermerge
Ethe1: the protein
It works as an homodimer
It is a mitochondrial matrix protein
Fe++
It binds iron
Tiranti et al 2004-2006, Mineri et al 2008
days
Surv
ival
pro
babi
lity
(%) +/+
-/-
ETHE1-/- mouse phenotype
Metabolites in body fluids
C4 acylcarnitine
CTR KO CTR EE-patientsmouse human
02468
1012141618
µM
/L
C5 acylcarnitine
CTR KO CTR EE-patients00,5
11,5
22,5
33,5
44,5
µM
/L
mouse human
Ethylmalonic acid
0
50
100
150
200
250
300
350
400
µg/
mg
crea
tinine
CTR KO CTR EE-patientsmouse human
gram
s
weeks02468101214
1 2 3 4
+/+
-/-
Brain
-/- -/-+/+ +/+CI/CS CIV/CS
0
50
100
150
200 Liver
-/- -/-+/+ +/+CI/CS CIV/CS
0
50
100
150
200 Muscle
-/- -/-+/+ +/+CI/CS CIV/CS
Kidney
-/- -/-+/+ +/+CI/CS CIV/CS
-/- -/-+/+ +/+CI/CS CIV/CS
Heart
0
50
100
150
200
** **
**
** p<0.0004
liver
brain
muscle
colon
jejunum-/- +/+
Respiratory chain complex activities in mouse tissues
Western-blot analysis of COX assembly inETHE1 KO mice
+/+ -/- +/+ -/- +/+ -/-
COI
SDH
BRAIN LIVER MUSCLE
+/+
-/-
I dimension
II dimension on Brain
BRAIN
COI
S1 COIHeme AHeme A3 Cox10p
Cu B Sco1pSco2p
Cox17p
COIV
S2 COII Sco1pSco2p
Cox17pCu ACOIII
COVa, Vb, VIc, VIIa, VIII
COVIbCOVIIc
S3 COVIIbCOVIa
S4
Cox11pCox14pCox15p
Pet100pPet117pPet191pSurf1
Oxa1p
LonAfg3Rca1
?
?
?
?
Bioinformatic searchETHE1-like proteins are present in operons containing Rhodanese-likeproteins or as Chimeric proteins ETHE1/Rhodanese
Bradyrhizobium japonicum 27376118 9Legionella pneumophila 54293763 9Legionella pneumophila 52841017 9Danio rerio 47086181 9Caenorhabditis elegans 17538952 9Apis mellifera 48140497 9Drosophila melanogaster 24652740 9Drosophila melanogaster 27819799 9Anopheles gambiae 31210533 9Anopheles gambiae 55240363 9Oryza sativa 56202167 9Oryza sativa 34910930 9Arabidopsis thaliana 12324040 9Arabidopsis thaliana 1644427 9Arabidopsis thaliana 22655048 9Xenopus laevis 27371293 9Pan troglodytes 55649315 9Homo sapiens ETHE1Mus musculus 53236965 9Rattus norvegicus 27676450 9Burkholderia cepacia 46322841 9 FBurkholderia cepacia 46317848 9 FRalstonia eutropha 45515657 9Ralstonia metallidurans 48771425 9Nostoc punctiforme 23128919 9 FSynechococcus elongatus 56750614 9Nostoc sp. 17231396 9Anabaena variabilis 45507470 9Synechocystis sp. 16330445 9Thermosynechococcus elongatus 22299169 9
Polaromonas sp. 54032671 9 F
??
Fusion events Operon??soxY soxA ??soxBsoxZsoxX
??
??
??
Zn-hydrolase domain Rhodanese-related sulfurtransferase domain
Domain of unknown functionPredicted permeaseGluthation S-transferase
Sulfur oxidase gene cluster
RHODANESE(TST, Thiosulphate Sulfurtrasferase)
Mitochondrial matrix protein
Unknown biological function (Cyanide
detoxyfication, Iron-sulfur cluster
formation, energetic metabolism)
It is involved in sulfur metabolism
Sulfur metabolism
Szabo, Nat Rev Drug Discov 2007
Thiosulphate in urines
EE-Patients
nmol
/mg
crea
tini
ne
0
500
1000
1500
2000
2500
CTR0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
nmol
/mg
crea
tini
ne
CTR KO mice
kidneybrain liver serummuscle
0
40
80
120
160
200
+/+ +/+ +/+ +/+ +/+-/- -/- -/- -/- -/-
µM
and tissues
LIVER
0,0
500,0
1000,0
1500,0
2000,0
2500,0
3000,0
3500,0
MUSCLE
0,0
500,0
1000,0
1500,0
2000,0
2500,0
CTR KOCTR KO
p<0.0007 p=0.00007
BRAIN
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
90,0
CTR KO
p<0.0016
H2S concentration in tissues ofETHE1 knockout mice
H2S
(nM
)
H2S inhibits cytochrome c oxidase(COX)
Szabo, Nat Rev Drug Discov 2007
H2S inhibits enzymatic activities
0 200 400 600 800 1000 12000
20
40
60
80
100
120
NaSH (µM)
residu
al a
ctivity SCAD in liverCOX/CS
NaSH (µM)
residu
al a
ctivity
120
0 1 2 3 4 5 6 7 8 9 10 11 12 130
20
40
60
80
100
NaCN
MUSCLE
BRAIN
LIVER
0 2 4 6 8 10 12
0
20
40
60
80
100
120
NaSH (µM)
residu
al a
ctivity
CI and CS in muscle
CI/CS
CS
Conclusions 1
•The pathogenetic mechanism of Ethylmalonic Encephalopathy(EE) relies on high levels of H2S, that are toxic for both COXand SCAD
•H2S can act as a vasodilator thus explaining the acrocyanosisand can be toxic for the microvessels thus explaining thepetechiae
Which is the role of ETHE1????
H2S (sulfide) is the first inorganic substrate formitochondrial respiratory chain in mammals
H2SSQR RSSH+H2O+O2 H2SO3
SDO
H2S2O3
TST sulfide quinoneoxidoreductase
sulfur dioxygenase
sulfurtranferase
matrixO2 2H2O
2H2S
intermembrane space CIII CIV
SQR
S S
inner mitochondrialmembrane
H2SO3
H2S2O3
-SSHR
e-
e- e-
Cyt cUQH2
UQ
e-
R
H2O
O2
TST/Rhodanese(Thiosulfate Sulfur Transferase)
SDO(SDO: Sulfur Dioxygenase)
(thiosulfate)
(sulfite)
(hydrogen sulfide) (persulfide)-SSHR(persulfide)
(SQR: Sulfide quinone Oxidoreductase)
SDO activity in liver of ETHE1 -/-homogenates
0
1
2
3
4
5
6
7
8
9
10
liver homogenate
SDO a
ctivity
(nmol/m
g/min)
+/+ -/-
Sulfide
ETHE1
SDH
Liver-/- +/+
matrixO2 2H2O
H2S
intermembrane space CIII CIV
SQR
S S
inner mitochondrialmembrane
H2SO3
H2S2O3
-SSHR
e-
e- e-
Cyt cUQH2
UQ
e-
R
Rhodanese(Thiosulfate Sulfur Transferase)
ETHE1(SDO: Sulfur Dioxygenase)
(sulfite)
(persulfide)-SSHR(persulfide)
(SQR: Sulfide quinone Oxidoreductase)
Conclusions 2ETHE1, encodes a mitochondrial sulfur dioxygenase thattakes part in aerobic energetic exploitation of, anddetoxification from, H2S
EE is the first example of a mitochondrial disorder causedby genetically determined poisoning of the respiratory chain
The creation of organ-specific conditional KO animals, canbe useful to identify the main source of H2S generation inEE, e.g. exogenous absorption from the gut flora vs.endogenous production
COX
KOKO
WTWT
1011-1012 cfu/ml
104-107 cfu/ml
101-103 cfu/ml
H&E
H2S-producing gut flora and mucosalCOX
The toxic mechanism underpinning EthylmalonicEncephalopathy makes effective therapy a realisticgoal: for instance antibiotic control of H2S-producingbacterial flora vs. H2S neutralization using GSH as asulfide acceptor
(1) the diffusion of H2S through the colonicmucosa could be a major, but not the onlycause of disease in Ethe1-/- mice andpatients
(2) the restriction of bacterial production ofH2S and its neutralization by NAC, may betherapeutically beneficial in the mouse, andpossibly human, disease condition.
Final considerations and futureperspectives
A specific treatment against the proliferation ofH2S-producing anaerobic bacterial flora in the largeintestine leads to substantial improvement of theclinical conditions in experimental murine EE
CollaborationsTatjana HildebrandtMike LevittMarco RimoldiAlessandro PrelleGigliola FagiolariCecilia Tiveron
THANK YOU!!Unit of Molecular Neurogenetics
Egill BriemRossana Mineri
Carlo Viscomi
Eleonora Lamantea
Ivano DiMeo
Barbara Garavaglia
All the clinicians world-widewho referred the patients toour Centre
Massimo Zeviani
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