title: Proteomic and metabolomic analyses of mitochondrial complex I-deficient mouse model generated by spontaneous B2 short interspersed nuclear element (SINE) insertion into NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) gene.
abstract: Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4(fky), the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4(fky/fky) mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4(fky/fky) mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "N assembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/NAD(+) ratio that inhibits mitochondrial fatty acid β-oxidation.
authors.affiliation: Molecular Medicine Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.
authors.forename: Dillon W
authors.initials: DW
authors.lastname: Leong
authors.forename: Jasper C
authors.initials: JC
authors.lastname: Komen
authors.forename: Chelsee A
authors.initials: CA
authors.lastname: Hewitt
authors.forename: Estelle
authors.initials: E
authors.lastname: Arnaud
authors.forename: Matthew
authors.initials: M
authors.lastname: McKenzie
authors.forename: Belinda
authors.initials: B
authors.lastname: Phipson
authors.forename: Melanie
authors.initials: M
authors.lastname: Bahlo
authors.forename: Adrienne
authors.initials: A
authors.lastname: Laskowski
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Kinkel
authors.forename: Gayle M
authors.initials: GM
authors.lastname: Davey
authors.forename: William R
authors.initials: WR
authors.lastname: Heath
authors.forename: Anne K
authors.initials: AK
authors.lastname: Voss
authors.forename: René P
authors.initials: RP
authors.lastname: Zahedi
authors.forename: James J
authors.initials: JJ
authors.lastname: Pitt
authors.forename: Roman
authors.initials: R
authors.lastname: Chrast
authors.forename: Albert
authors.initials: A
authors.lastname: Sickmann
authors.forename: Michael T
authors.initials: MT
authors.lastname: Ryan
authors.forename: Gordon K
authors.initials: GK
authors.lastname: Smyth
authors.forename: David R
authors.initials: DR
authors.lastname: Thorburn
authors.forename: Hamish S
authors.initials: HS
authors.lastname: Scott
mesh_terms: D000818:Animals; D001665:Binding Sites; D004251:DNA Transposable Elements; D042967:Electron Transport Complex I / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007888:Leigh Disease / Q000201:enzymology* / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D055432:Metabolomics / Q000379:methods; D051379:Mice; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D009154:Mutation; D009243:NAD / Q000235:genetics / Q000378:metabolism*; D009245:NADH Dehydrogenase / Q000235:genetics / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D012326:RNA Splicing / Q000235:genetics
chemical_list: D004251:DNA Transposable Elements; C534449:Ndufs4 protein, mouse; D009243:NAD; D009245:NADH Dehydrogenase; D042967:Electron Transport Complex I; C427643:NDUFS4 protein, human