Recessive Mitochondrial Diseases

Transcription

1 Recessive Mitochondrial Diseases Normal at birth, lactic acidosis, brain, heart or liver disease, early death What to do?

2 Blue Native Gel Electrophoresis (Immunoblot Analysis)

3 BN-PAGE: A biochemical fingerprint Isolated complex IV (COX) and I account for about half of patients

4 Patient E 143B Rho o Is the defect in mtdna or ndna? N N + G418 resistance Fusion cell N N No mtdna Grown in medium + uridine Puromycin resistance COX Activity (Average % Control) Selection for fusion cell by Puromycin, G418, and absence of uridine 0 Patient E+ 143B rho0 Patient E Patient cells are defective in ndna because defect was rescued by wild type ndna

5 Functional complementation cloning Retroviral expression of candidate genes that we know about from yeast (SURF1, SCO1, SCO2, pet191, COX10, COX11, COX15, COX16, COX17, COX18, COX19, Oxa1) Candidate gene A Normal human chromosomes IRES GF P HyTK A a a Patient cell line

6 Functional complementation by microcell-mediated chromosome transfer Doherty et al.,mammalian genome,(2003)

7 Experimental procedure Candidate Chromosome Candidate Region Candidate Gene Chromosome Transfer Genotyping (Microsatellite) Search known MT targeting genes Selection (40 clones) Genotyping (SNP-Affymetrix) RT-PCR sequencing cdna COX Activity Found region of interest? No Yes Found mutation? No Yes None Rescued? Most Try other methods (irradiation, ) Overexpression

8 Genotyping of selected clones by affymetrix (SNP) Clone 10- Rescued Clone 16- Not Rescued Clone 23- Rescued Clone 43- Not Rescued 17q22 17q

9 COX Structure Consists of 13 subunits 3 mtdna-encoded (copper, heme moieties) 10 nuclear-encoded - function? IMS matrix COX I COX II

10 Cytochrome c oxidase reaction cytochrome c Cu A Cu A Subunit II heme a Cu B heme a Subunit I heme a3 -Cu B heme a 3 O 2

11 Leigh Syndrome Subacute neurodegenerative disease Bilaterally symmetric lesions in brainstem, basal ganglia, spinal cord Vascular proliferation, gliosis, neuronal loss, demyelination, cystic cavitation

12 Analysis of SURF1 and COX Leigh Syndrome patients have no immunodectable SURF1 protein Incomplete COX assembly results in proteolysis of free subunits

13 Assembly intermediate in all SURF1 cases

14 Genotype-phenotype relationships Assembly factors for COX SURF1- Leigh Syndrome, leukodystrophy, villous atrophy SCO2- hypertrophic cardiomyopathy, encephalopathy SCO1- hepatopathy, ketoacidotic coma COX10- leukodystrophy, tubulopathy, hypertrophic cardiomyopathy, Leigh Syndrome, anemia COX15 - hypertrophic cardiomyopathy, Leigh Syndrome LRPPRC- Leigh Syndrome (FC)

15 A big question in this field Why do mutations in ubiquitously expressed housekeeping genes cause tissue-specific clinical phenotypes?

16 SCO2 MLLLTRSPTAWHRLSQLKPPVLPGTLGGQALHLRSWLLSRQGPAETGGQGQPQGPGLRTRLLITGLF GAGLGGAWLALRAEKERLQQQKRTEALRQAAVGQGDFHLLDHRGRARCKADFRGQWVLMYFGFT HCPDICPDELEKLVQVVRQLEAEPGLPPVQPVFITVDPERDDVEAMARYVQDFHPRLLGLTGSTKQV AQASHSYRVYYNAGPKDEDQDYIVDHSIAIYLLNPDGLFTDYYGRSRSAEQISDSVRRHMAAFRSVL S R90X E140K L151P R171W hypertrophic cardiomyopathy SCO1 MAMLVLVPGRVMRPLGGQLWRFLPRGLEFWGPAEGTARVLLRQFCARQAEAWRASGRPGYCLGTR PLSTARPPPPWSQKGPGDSTRPSKPGPVSWKSLAITFAIGGALLAGMKHVKKEKAEKLEKERQRHIG KPLLGGPFSLTTHTGERKTDKDYLGQWLLIYFGFTHCPDVCPEELEKMIQVVDEIDSITTLPDLTPLFISI DPERDTKEAIANYVKEFSPKLVGLTGTREEVDQVARAYRVYYSPGPKDEDEDYIVDHTIIMYLIGPDGE FLDYFGQNKRKGEIAASIATHMRPYRKKS P174L hepatopathy Sco1 and Sco2 are paralogous copper metallochaperones

17 Mitochondrial Copper Delivery (in yeast)

18 A few observations. yeast only need one SCO protein both human SCOs are copper binding proteins, Cu(I) and Cu(II) binding necessary for function SCO1 metallation is COX17 dependent, P174L mutant defective in COX17 transfer, but not copper binding copper rescue of COX (high [ ], extended culture) in SCO1 and SCO2 patients, not in yeast so things are organized differently in mammals

19 Dominant negative effects of SCO1 and SCO2: nonoverlapping cooperative functions Sco2 Sco1

20 SCO2-independent assembly of COX in liver, Mutant protein barely detectable

21 Very low COX in SCO2 heart, SCO1 liver heart liver

22 Copper deficiency in SCO patient tissues copper content in neonatal liver of SCO2 patients enough to rescue, not in muscle SCO1 patient severely copper deficient, exacerbate COX defect

23 Cellular Copper Metabolism Intracellular copper metabolism

24 Copper deficiency is proportional to efflux uptake efflux

25 Conclusions SCOs are bifunctional molecules with roles in mitochondrial copper delivery and cellular copper homeostasis Hypothesis: thiol redox status of SCO1, modulated by SCO2, acts as a signal to communicate cellular copper status Disease implications: SCO2-independent COX assembly in liver due to relatively high copper content in neonates, copper deficiency exacerbates COX deficiency in SCO1, should mimic nutritional copper deficiency, cause of anemia in COX10 patients?

26 48 (28) Mitochondrial Translation 29 (14)

27 Mitochondrial Translation Screen

28 Patient Information Pedigree 1 Intra-uterine growth retardation, microcephaly, severe lactic acidosis, liver failure Muscle 600g supernatant showed decreased COX activity, Co I was normal, Co II and Co III were increased Suspected mtdna depletion Pedigree 2 Intra-uterine growth retardation, microcephaly, liver failure Normal mtdna levels and no rearrangements Low COX I and COX II, slightly reduced COX IV Low levels of Core 2 Possible nuclear origin

29 EFG1 patients N174S S321P STOP

30 Over-expression of EFG1 rescues translation defect and COX activity Global translation defect, not uniform Complexes not equally affected COX activity increased to ~77% of control In-vitro translation rate increased 2-fold

31 Translation elongation factors in control tissues 1:1 1:7 1:1 1:1 EFTu : EFTs mito muscle, liver, fibroblasts 1:1 mito heart 1:7 prokaryotes 8:1 yeast no EFTs

32 Levels of OXPHOS Complexes in control tissues ralative ratios Complex I Complex II Complex III Complex IV Complex V Muscle Heart Liver Fibroblasts Steady-state level of EFG1 inversely correlated with levels of the OPHOS complexes

33 Tissue Specificity: heart is spared, liver and fibroblasts severely deficient in Complexes I, IV

34 EFG1 is stabilized in heart, undetectable in liver EFTu 4X increased in heart

35 Genotype-phenotype relationships Mitochondrial translation factors EFG1- hepatopathy, Leigh Syndrome EFTs- encephalopathy, hypertrophic cardiomyopathy EFTu- encephalopathy MRPS16- dysgenesis corpus callosum, dysmorphy paraplegin- hereditary spastic paraplegia

36 Evolution of Complex I: Mergers and acquisitions

37 Genetic complementation of CI patients with assembly defects

38 Many Genetic Complementation groups, no common cause of enzyme deficiency (Six different complementation groups in patients from 6 pedigrees) A Be Br Bo F L M A Be Br ND ND ND + Bo F + + L + M

39 Génolevures:Genomic Exploration of the Hemiascomycete Yeasts

40 Genome subtraction Genolevures: complete genomic sequence of four different yeast species. -S. cerevisiae -Candida glabrata -Kluyveromyces lactis? Site compares each one to the others, and to Saccharomyces cerevisiae, using BLAST the ORF s are grouped into families of related proteins. Two of the yeasts have Complex I - Yarrowia lipolytica and Debaryomyces hansenii. The others do not.? -Yarrowia lipolytica -Debaryomyces hansenii

41 Candidates Family Accession Number and Info Mitopred Percentage Comments GLC 1123.txt >gi gb AAH ZZZ3 protein [Homo sapiens] Non-Mitochondrial -- Unknown function GLC 1233.txt GLC 1790.txt GLC 1807.txt GLC 1819 GLC 1838.txt GLC 1858.txt GLC 2835.txt GLC 295.txt GLC 3105 GLC 3118 >gi ref NP_ putative nucleic acid binding protein RY-1 [Homo sapiens] >gi ref NP_ hypothetical protein LOC91942 [Homo sapiens] >gi ref XP_ hypothetical protein XP_ [Homo sapiens] >gi ref NP_ PRO1853 protein isoform 1 [Homo sapiens] >gi emb CAG OXCT 3-oxoacid CoA transferase [Homo sapiens] >gi gb AAH OXCT2 3-oxoacid CoA transferase [Homo sapiens] >gi gb AAH Hypothetical protein MGC40214 [Homo sapiens] >gi dbj BAB unnamed protein product [Homo sapiens] >gi gb AAH Stomatin (EPB72)-like 2 [Homo sapiens] >gi ref NP_ chromosome 10 open reading frame 65 [Homo sapiens] >gi gb AAH Leucine zipper-ef-hand containing transmembrane protein 1 [Homo sapiens] >gi gb AAH hydroxyisobutyrate dehydrogenase [Homo sapiens] Mitochondrial 84.60% Unknown function Mitochondrial % B17.2 -like protein Mitochondrial 84.60% B17.2 like protein Mitochondrial 92.30% Unknown function - two transcripts known to exist - highly evolutionarily conserved Mitochondrial 92.30% 3-oxoacid CoA transferase Mitochondrial 84.60% 3-oxoacid CoA transferase Non-Mitochondrial -- Unknown function Non-Mitochondrial -- Unknown function Mitochondrial 92.30% Prohibitin domain, stomatin-like 2, associated with lipid rafts, Ubiquitously expressed Mitochondrial 92.30% Weakly similar to dihydrolipicolinate synthase - enzyme for lysine synthesis in micro-organisms and plants Mitochondrial 84.60% LetmI- Wolf -hirschorn syndrome microdeletion - severe growth and mental retardation, hypotonia, seizures and facial dysmorphy. S.cerevisiae homologue YOL0127 involved in mitochondrial K+ homostasis Mitochondrial % 3-hydroxyisobutyrate dehydrogenase GLC 488.txt >gi gb AAF AF201943_1 HAH-P [Homo sapiens] Mitochondrial % amino apidate - semialdehyde dehydrogenase - phosphopantetheinyl transferase. Mainly cytosolic but puts 4'phosphopantetheine moiety on ACP of mitochondria GLC 555.txt >gi dbj BAA unnamed protein product [Homo sapiens] Mitochondrial 84.60% Unknown function GLC 577.txt >gi ref NP_ death-associated protein 3; mitochondrial 28S ribosomal protein S29 [Homo sapiens] Mitochondrial 99.00% Overexpression changes mito- structure. Increase in fragmentation, positive mediator of cell death

42 B17.2L: A structural gene paralogue

43 B17.2L is ubiquitously expressed, but tissuespecific differences

44 B17.2L mutation Homozygous C182T transition (exon 2) of the mrna from patient A. Predicts R45X in the protein (169 aa, 19.8kDa) C P1 Predicted Protein MGWSQDLFRALWRSLSREVKEHVGTDQFGNKYYYIPQYKNWRGQTI*EKRIVEAANKKEV DYEAGDIPTEWEAWIRRTRKTPPTMEEILKNEKHREEIKIKSQDFYEKEKLLSKETSEEL LPPPVQTQIKGHASAPYFGKEEPSVAPSSTGKTFQPGSWMPRDGKSHNQ (Turned out to be hemizygous)

45 Complex I assembly and activity are rescued by retroviral expression of the cdna Associated with reappearance of the protein at near wild-type levels

46 B17.2L associates with a subassembly of Complex I in skeletal muscle in patients with assembly defects anti-nd1 anti-b17.2l

47 The subassembly is a common intermediate in several patients Patient A does not accumulate this subcomplex anti-nd1 anti-b17.2l

48 Phenotype similar to Vanishing White Matter Disease

49 Take home messages We can reject the hypothesis that tissue-specificity is a simple function of high energy demands Tissue-specific organization and regulation of mitochondrial translation factors, different response to mutations, bifunctional proteins Very likely modifier genes