An almost final solution to the Ethylmalonic acid syndrome Valeria Tiranti ERNDIM Meeting October 22-23, 2009 Basel Ethylmalonic Encephalopathy (EE) urea 9 e 8 c n ethylmalonate a 7 d 2-ketoglutarate n u 6 b A 5 4 isobutyrylglycine 3 I.S. isovalerylglycine I.S. 2 1 Time 0 COOH CH3-CH2-CH2-CO-SCoA + CO2 -> CH3-CH2-CH-COOH Butyryl-CoA Ethylmalonate CNS CoASH •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 440del11 ETHE1: the gene 505+1G>A 406A>G 487C>T 3G>T 187C>T 505+1G>T 604insG GCCCCdel 376-1G>T 488G>A 113A>G 131delAG 559insC 50 EE 375+5G>A 222insA 482G>A 554T>G 34C>T 230delA patients 1 2 3 4 5 6 7 7 28 non-EE EX4del EMA patients EX1-7del Ethe1: the protein HeLa It is a mitochondrial matrix protein ETHE1 merge Mitotracker It works as an homodimer Fe++ It binds iron Tiranti et al 2004-2006, Mineri et al 2008 ETHE1-/- mouse phenotype +/+ %) 14 ( +/+ 12 10 8 -/- grams 6 4 2 Survival probability -/- 0 1 2 3 4 weeks days Metabolites in body fluids 400 C4 acylcarnitine C5 acylcarnitine 350 Ethylmalonic acid 300 4,5 250 18 4 16 3,5 200 14 3 /L 12 /L 150 M 2,5 M µ 10 µ 2 8 /mg creatinine 100 1,5 g 6 µ 4 1 50 2 0,5 0 0 0 CTR KO CTR EE-patients CTR KO CTR EE-patients CTR KO CTR EE-patients mouse human mouse human mouse human Respiratory chain complex activities in mouse tissues 200 Liver Kidney 150 liver 100 50 0 +/+ -/- +/+ -/- +/+ -/- +/+ -/- brain CI/CS CIV/CS CI/CS CIV/CS 200 Muscle Brain 150 100 muscle 50 ** ** 0 +/+ -/- +/+ -/- +/+ -/- +/+ -/- CI/CS CIV/CS CI/CS CIV/CS 200 Heart colon 150 100 ** 50 0 ** p<0.0004 +/+ -/- +/+ -/- jejunum CI/CS CIV/CS -/- +/+ Western-blot analysis of COX assembly in ETHE1 KO mice BRAIN LIVER MUSCLE +/+ -/- +/+ -/- +/+ -/- S1 COI I dimension Heme A COI Cox10p Cox11p Heme A3 Cox14p ? Cu B Sco1p Cox17p Cox15p Sco2p COIV Pet100p Pet117p ? S2 COII Cu A Sco1p Cox17p SDH Pet191p Sco2p Surf1 COIII Oxa1p ? COVa, Vb, VIc, VIIa, VIII COVIb Lon Afg3 ? COVIIc Rca1 BRAIN S3 COVIIb COVIa +/+ S4 II dimension on Brain -/- COI Bioinformatic search ETHE1-like proteins are present in operons containing Rhodanese-like proteins or as Chimeric proteins ETHE1/Rhodanese Fusion events Operon Polaromonas sp. 54032671 9 F Bradyrhizobium japonicum 27376118 9 soxX soxY soxZ soxA ?? soxB ?? Legionella pneumophila 54293763 9 Legionella pneumophila 52841017 9 Danio rerio 47086181 9 Caenorhabditis elegans 17538952 9 Apis mellifera 48140497 9 Drosophila melanogaster 24652740 9 Drosophila melanogaster 27819799 9 Zn-hydrolase domain Anopheles gambiae 31210533 9 Anopheles gambiae 55240363 9 Rhodanese-related sulfurtransferase domain ‏ Oryza sativa 56202167 9 ?? Domain of unknown function Oryza sativa 34910930 9 ‏ Arabidopsis thaliana 12324040 9 Predicted permease Arabidopsis thaliana 1644427 9 Gluthation S-transferase ‏ Arabidopsis thaliana 22655048 9 Sulfur oxidase gene cluster Xenopus laevis 27371293 9 Pan troglodytes 55649315 9 Homo sapiens ETHE1 Mus musculus 53236965 9 Rattus norvegicus 27676450 9 Burkholderia cepacia 46322841 9 F Burkholderia cepacia 46317848 9 F Ralstonia eutropha 45515657 9 Ralstonia metallidurans 48771425 9 Nostoc punctiforme 23128919 9 F ?? ?? Synechococcus elongatus 56750614 9 Nostoc sp. 17231396 9 Anabaena variabilis 45507470 9 Synechocystis sp. 16330445 9 ?? Thermosynechococcus elongatus 22299169 9 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 Thiosulphate in urines 20000 18000 16000 2500 14000 12000 2000 10000 1500 8000 6000 /mg creatinine 1000 /mg creatinine 4000 500 nmol nmol 2000 0 0 CTR EE-Patients CTR KO mice 200 and tissues 160 120 M µ 80 40 0 Szabo, Nat Rev Drug Discov 2007 +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- brain liver muscle kidney serum H2S concentration in tissues of ETHE1 knockout mice MUSCLE LIVER 2500,0 3500,0 3000,0 2000,0 p<0.0007 2500,0 p=0.00007 1500,0 2000,0 1500,0 1000,0 1000,0 500,0 500,0 0,0 0,0 CTR KO CTR KO BRAIN 90,0 80,0 70,0 ) p<0.0016 M 60,0 n ( 50,0 S 40,0 2 H 30,0 20,0 10,0 0,0 CTR KO H2S inhibits cytochrome c oxidase (COX) Szabo, Nat Rev Drug Discov 2007 H2S inhibits enzymatic activities 120 120 100 100 80 MUSCLE 80 COX/CS SCAD in liver BRAIN 60 60 LIVER 40 4residual activity 0 NaCN residual activity 20 20 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 0 200 400 600 800 1000 1200 NaSH (µM) NaSH (µM) 120 100 CI/CS 80 CS CI and CS in muscle 60 40 residual activity 20 0 0 2 4 6 8 10 12 NaSH (µM) Conclusions 1 •The pathogenetic mechanism of Ethylmalonic Encephalopathy (EE) relies on high levels of H2S, that are toxic for both COX and SCAD •H2S can act as a vasodilator thus explaining the acrocyanosis and can be toxic for the microvessels thus explaining the petechiae Which is the role of ETHE1???? H2S (sulfide) is the first inorganic substrate for mitochondrial respiratory chain in mammals SQR SDO H2S RSSH+H2O+O2 H2SO3 sulfide quinone sulfur dioxygenase oxidoreductase TST sulfur tranferase H2S2O3 H2S2O3 (thiosulfate) H2O H2SO3 (sulfite) O2 SDO TST/Rhodanese (SDO: Sulfur Dioxygenase) (Thiosulfate Sulfur Transferase) 2H2S R -SSH R -SSH (hydrogen sulfide) (persulfide) (persulfide) e- S S O2 2H2O R (SQR: Sulfide quinone matrix Oxidoreductase) SQR UQ inner mitochondrial - - membrane e e UQH2 Cyt c intermembrane space CIII e- CIV SDO activity in liver of ETHE1 -/- homogenates 10 Sulfide ) 9 8 7 mol/mg/min 6 n 5 4 Liver 3 -/- +/+ ETHE1 SDO activity ( 2 1 SDH 0 +/+ -/- liver homogenate H2S2O3 H2SO3 (sulfite) ETHE1 Rhodanese (SDO: Sulfur Dioxygenase) (Thiosulfate Sulfur Transferase) R -SSH R -SSH H2S (persulfide) (persulfide) e- S S O2 2H2O R (SQR: Sulfide quinone matrix Oxidoreductase) SQR UQ inner mitochondrial - - membrane e e UQH2 Cyt c intermembrane space CIII e- CIV Conclusions 2 .ETHE1, encodes a mitochondrial sulfur dioxygenase that takes part in aerobic energetic exploitation of, and detoxification from, H2S .EE is the first example of a mitochondrial disorder caused by genetically determined poisoning of the respiratory chain .The creation of organ-specific conditional KO animals, can be useful to identify the main source of H2S generation in EE, e.g. exogenous absorption from the gut flora vs. endogenous production H2S-producing gut flora and mucosal COX 101-103 cfu/ml 1011-1012 cfu/ml 104-107 cfu/ml H&E COX WT KO KO WT .The toxic mechanism underpinning Ethylmalonic Encephalopathy makes effective therapy a realistic goal: for instance antibiotic control of H2S-producing bacterial flora vs. H2S neutralization using GSH as a sulfide acceptor Final considerations and future perspectives A specific treatment against the proliferation of H2S-producing anaerobic bacterial flora in the large intestine leads to substantial improvement of the clinical conditions in experimental murine EE (1) the diffusion of H2S through the colonic mucosa could be a major, but not the only cause of disease in Ethe1-/- mice and patients (2) the restriction of bacterial production of H2S and its neutralization by NAC, may be therapeutically beneficial in the mouse, and possibly human, disease condition. THANK YOU!! Unit of Molecular Neurogenetics Massimo Zeviani Barbara Garavaglia Eleonora Lamantea Carlo Viscomi Ivano DiMeo Rossana Mineri Egill Briem Collaborations Tatjana Hildebrandt Mike Levitt Marco Rimoldi Alessandro Prelle Gigliola Fagiolari Cecilia Tiveron All the clinicians world-wide who referred the patients to our Centre.