Untargeted proteomics greatly aides the functional diagnosis of mitochondrial aminoacyl-tRNA synthetases (ARS2s)
Biochemical/Metabolic and Therapeutics
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Introduction:
: MItochondria have their own protein translation machinery to synthesize the respiratory chain subunit proteins that are encoded on mitochondrial DNA. This includes proteins to charge the corresponding mitochondrial tRNAs with their cognate amino acid, which includes the mitochondrial aminoacyl-tRNA synthetases (ARS2 proteins), and the GatCAB system for tRNAGln, in total encompassing 22 nuclear encoded proteins. The disorders of each of these proteins present with a wide variety of clinical symptoms and represent a relatively frequent group of mitochondrial disorders. They are generally recognized on genomic sequencing, but unfortunately, variants of unknown significance are present in more than half of all cases. These cases require functional assays to verify variant pathogenicity. Unfortunately, these disorders are known to rarely provide abnormalities on traditional respiratory chain enzyme assays.
We aimed to identify an efficient diagnostic system, we aimed to evaluate utility of a series of functional assays for the identification of pertinent functional abnormalities in mitochondrial function in ARS2 defects.
Methods:
Method: We evaluated a series of 34 fibroblast cell lines with bi-allelic variants in an ARS2 gene by respiratory chain enzyme assay, blue native PAGE with in-gel enzyme activity staining, and complex I analysis, all existing clinical assays. In addition, we analyzed by specific western blot the amount of ARS2 protein. We performed label-free mass-spectrometry based whole cell proteomics and analyzed the relative abundance of the ARS2 protein, with 35% abundance of control as a cutoff for protein absence, and the relative abundance of each of the respiratory chain complexes derived by analysis of all its subunits in comparison to a set of normal controls. Proteomics was able to identify 21 of 22 ARS2 proteins.
Results:
Respiratory chain enzyme assays showed a deficiency of either complex I or complex IV in only 15% of cases, all borderline results. An abnormal pattern was shown on blue native PAGE with in-gel activity staining in 21% of cases and on complex I assembly assay in 24% of cases. Combined, an abnormality in any current clinical assays was present in only 45% of cases. A decrease of the ARS2 protein was found by western blot in 63% of cases. Proteomics showed decreased complex I abundance in 32% of cases and complex IV in 24% of cases, but also identified a decreased ARS2 protein abundance in 72% of cases. In total, proteomics showed a diagnostic abnormality in 82% of cases, and when combined with traditional assays, an abnormality was found in 94% of cases. Diagnostic challenges in proteomics were encountered with individual cell lines harboring defects in VARS2, DARS2, and GATCab complex proteins.
Conclusion:
Proteomics as a single assay outperformed all current clinical assays. Using a minimally invasive tissue of skin biopsy derived fibroblasts allowed diagnostic functional abnormalities in the vast majority of cases. The use of untargeted whole cell proteomics also lends itself to additional discovery studies in cases where a mitochondrial defect is excluded.
: MItochondria have their own protein translation machinery to synthesize the respiratory chain subunit proteins that are encoded on mitochondrial DNA. This includes proteins to charge the corresponding mitochondrial tRNAs with their cognate amino acid, which includes the mitochondrial aminoacyl-tRNA synthetases (ARS2 proteins), and the GatCAB system for tRNAGln, in total encompassing 22 nuclear encoded proteins. The disorders of each of these proteins present with a wide variety of clinical symptoms and represent a relatively frequent group of mitochondrial disorders. They are generally recognized on genomic sequencing, but unfortunately, variants of unknown significance are present in more than half of all cases. These cases require functional assays to verify variant pathogenicity. Unfortunately, these disorders are known to rarely provide abnormalities on traditional respiratory chain enzyme assays.
We aimed to identify an efficient diagnostic system, we aimed to evaluate utility of a series of functional assays for the identification of pertinent functional abnormalities in mitochondrial function in ARS2 defects.
Methods:
Method: We evaluated a series of 34 fibroblast cell lines with bi-allelic variants in an ARS2 gene by respiratory chain enzyme assay, blue native PAGE with in-gel enzyme activity staining, and complex I analysis, all existing clinical assays. In addition, we analyzed by specific western blot the amount of ARS2 protein. We performed label-free mass-spectrometry based whole cell proteomics and analyzed the relative abundance of the ARS2 protein, with 35% abundance of control as a cutoff for protein absence, and the relative abundance of each of the respiratory chain complexes derived by analysis of all its subunits in comparison to a set of normal controls. Proteomics was able to identify 21 of 22 ARS2 proteins.
Results:
Respiratory chain enzyme assays showed a deficiency of either complex I or complex IV in only 15% of cases, all borderline results. An abnormal pattern was shown on blue native PAGE with in-gel activity staining in 21% of cases and on complex I assembly assay in 24% of cases. Combined, an abnormality in any current clinical assays was present in only 45% of cases. A decrease of the ARS2 protein was found by western blot in 63% of cases. Proteomics showed decreased complex I abundance in 32% of cases and complex IV in 24% of cases, but also identified a decreased ARS2 protein abundance in 72% of cases. In total, proteomics showed a diagnostic abnormality in 82% of cases, and when combined with traditional assays, an abnormality was found in 94% of cases. Diagnostic challenges in proteomics were encountered with individual cell lines harboring defects in VARS2, DARS2, and GATCab complex proteins.
Conclusion:
Proteomics as a single assay outperformed all current clinical assays. Using a minimally invasive tissue of skin biopsy derived fibroblasts allowed diagnostic functional abnormalities in the vast majority of cases. The use of untargeted whole cell proteomics also lends itself to additional discovery studies in cases where a mitochondrial defect is excluded.