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.

Organic acidurias: Major gaps, new challenges, and a yet unfulfilled promise

“…Evidence for sustained mitochondrial pathology was found in Gcdh −/− mice, showing enlarged mitochondria with reduced electron density during induced metabolic crises, 59 and in HeLa cells transfected with mutant GCDH, revealing elongation of mitochondria and disturbed inner mitochondrial membrane organization. 60 Progression of mitochondrial dysmorphology and dysfunction was also demonstrated in liver, kidney, skeletal and heart muscle of MMA and PA patients, showing multiple deficiencies of respiratory chain complexes and thus partially resembling mitochondrial DNA depletion syndromes. In analogy, reduced cytochrome c oxidase was shown in Mut −/− mice, being accompanied by progressive organ dysfunction and the formation of megamitochondria.…”

Naturally-Occurring Rare Mutations Cause Mild to Catastrophic Effects in the Multifunctional and Cancer-Associated NQO1 Protein

“…The molecular mechanisms by which missense mutations cause loss-of-function are many-fold [24] and include accelerated protein degradation [5,9,11,25,26], enhanced protein aggregation [14,15,21], catalytic and regulatory alterations [21,[27][28][29] and altered biomacromolecular interactions [10,30]. Importantly, the common molecular origin for all these coexisting mechanisms appears to be the structural and energetic perturbation caused by missense mutations [5,8,11,19,24,26].…”

An RNAi Screen for Genes Required for Growth ofDrosophilaWing Tissue

“…This enzyme is a homotetramer that localizes to the mitochondrial matrix and is involved in lysine and tryptophan metabolic processes (Lenich and Goodman 1986; Goodman et al 1995; Schmiesing et al 2014). In a number of different human populations, alleles of GCDH cause the metabolic disorder glutaric acidemia type I, an early-onset neurodegenerative disorder (Table 3) (Goodman et al 1995; Hedlund et al 2006; Schmiesing et al 2017; Schmiesing et al 2018). In Drosophila , expression of CG9547 is upregulated in response to starvation and oxidative stress, and its knockdown altered eye growth (Fujikawa et al 2009; Gruenewald et al 2009; Pletcher et al 2019).…”