Disease-causing mutations affecting surface residues of mitochondrial glutaryl-CoA dehydrogenase impair stability, heteromeric complex formation, and mitochondria architecture

TY - JOUR

T1 - Disease-causing mutations affecting surface residues of mitochondrial glutaryl-CoA dehydrogenase impair stability, heteromeric complex formation, and mitochondria architecture

AU - Schmiesing, Jessica

AU - Lohmöller, Benjamin

AU - Schweizer, Michaela

AU - Tidow, Henning

AU - Gersting, Søren W

AU - Muntau, Ania C

AU - Braulke, Thomas

AU - Mühlhausen, Chris

N1 - © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

PY - 2017/2/1

Y1 - 2017/2/1

N2 - 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.

AB - 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.

U2 - 10.1093/hmg/ddw411

DO - 10.1093/hmg/ddw411

M3 - SCORING: Journal article

C2 - 28062662

VL - 26

SP - 538

EP - 551

JO - HUM MOL GENET

JF - HUM MOL GENET

SN - 0964-6906

IS - 3

ER -