Glutaryl-CoA Dehydrogenase Misfolding in Glutaric Acidemia Type 1

Abstract

Glutaric acidemia type 1 (GA1) is a neurotoxic metabolic disorder due to glutaryl-CoA dehydrogenase (GCDH) deficiency. The high number of missense variants associated with the disease and their impact on GCDH activity suggest that disturbed protein conformation can affect the biochemical phenotype. We aimed to elucidate the molecular basis of protein loss of function in GA1 by performing a parallel analysis in a large panel of GCDH missense variants using different biochemical and biophysical methodologies. Thirteen GCDH variants were investigated in regard to protein stability, hydrophobicity, oligomerization, aggregation, and activity. An altered oligomerization, loss of protein stability and solubility, as well as an augmented susceptibility to aggregation were observed. GA1 variants led to a loss of enzymatic activity, particularly when present at the N-terminal domain. The reduced cellular activity was associated with loss of tetramerization. Our results also suggest a correlation between variant sequence location and cellular protein stability (p < 0.05), with a more pronounced loss of protein observed with variant proximity to the N-terminus. The broad panel of variant-mediated conformational changes of the GCDH protein supports the classification of GA1 as a protein-misfolding disorder. This work supports research toward new therapeutic strategies that target this molecular disease phenotype.

Compilation of Genotype and Phenotype Data in GCDH-LOVD for Variant Classification and Further Application

“…The crystal structure of human GCDH complexed with alternate substrate 4-nitrobutyryl-CoA has been determined and is available in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (PDB) (PDB code: 1SIR) [32]. Several studies used this model to investigate potential effects of GCDH missense variants on protein folding and stability, encompassing novel variants identified in a patient as well as large sets of already known variants and genotypes [12,28,29,44,45]. A consistent assessment of these results is challenging because different approaches were used.…”

“…Multiple investigators performed in vitro GCDH enzyme activity analysis in heterologous cells (E. coli or COS-7) [9,11,27]. Furthermore, a considerable number of studies addressed the additional potential impact of missense variants on GCDH function [28][29][30][31][32]. Thus, PS3 was used as supporting evidence from functional studies (PS3_Supporting) if only one study showed a residual mutant enzyme activity of 0-50% measured in vitro in comparison to the wild-type enzyme activity.…”