title: Disease-causing mutations affecting surface residues of mitochondrial glutaryl-CoA dehydrogenase impair stability, heteromeric complex formation and mitochondria architecture.
abstract: The neurometabolic disorder glutaric aciduria type 1 (GA1) is caused by mutations in the GCDH gene encoding the mitochondrial matrix protein glutaryl-CoA dehydrogenase (GCDH), which forms homo- and heteromeric complexes. Twenty percent of all pathogenic mutations affect single amino acid residues on the surface of GCDH resulting in a severe clinical phenotype. We report here on heterologous expression studies of 18 missense mutations identified in GA1 patients affecting surface amino acids. Western blot and pulse chase experiments revealed that the stability of half of the GCDH mutants was significantly reduced. In silico analyses showed that none of the mutations impaired the 3D structure of GCDH. Immunofluorescence co-localisation studies in HeLa cells demonstrated that all GCDH mutants were correctly translocated into mitochondria. Surprisingly, the expression of p.Arg88Cys GCDH as well as further substitutions by alanine, lysine, or methionine but not histidine or leucine resulted in the disruption of mitochondrial architecture forming longitudinal structures composed of stacks of cristae and partial loss of the outer mitochondrial membrane. The expression of mitochondrial fusion or fission proteins was not affected in these cells. Bioluminescence resonance energy transfer analyses revealed that all GCDH mutants exhibit an increased binding affinity to electron transfer flavoprotein beta, whereas only p.Tyr155His GCDH showed a reduced interaction with dihydrolipoamide succinyl transferase. Our data underscore the impact of GCDH protein interactions mediated by amino acid residues on the surface of GCDH required for proper enzymatic activity.
authors.affiliation: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Schmiesing
authors.affiliation: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Lohmöller
authors.affiliation: Center of Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Michaela
authors.initials: M
authors.lastname: Schweizer
authors.affiliation: The Hamburg Centre for Ultrafast Imaging & Department of Chemistry, University of Hamburg, Hamburg, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Tidow
authors.affiliation: Department of Molecular Pediatrics, Dr. von Hauner Childrens Hospital, Ludwig-Maximilians-University, Munich, Germany and.
authors.forename: Søren W
authors.initials: SW
authors.lastname: Gersting
authors.affiliation: University Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Ania C
authors.initials: AC
authors.lastname: Muntau
authors.affiliation: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Braulke
authors.affiliation: Department of Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.|University Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Chris
authors.initials: C
authors.lastname: Mühlhausen
mesh_terms: D000592:Amino Acid Metabolism, Inborn Errors / Q000235:genetics* / Q000473:pathology; D019943:Amino Acid Substitution / Q000235:genetics; D001928:Brain Diseases, Metabolic / Q000235:genetics* / Q000473:pathology; D004795:Enzyme Stability / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D050770:Glutaryl-CoA Dehydrogenase / Q000737:chemistry / Q000172:deficiency* / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000235:genetics* / Q000473:pathology; D063154:Mitochondrial Dynamics / Q000235:genetics; D020125:Mutation, Missense / Q000235:genetics; D011487:Protein Conformation; D055503:Protein Multimerization / Q000235:genetics
chemical_list: D050770:Glutaryl-CoA Dehydrogenase