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Neurodegenerative disease-associated mutants of a human mitochondrial aminoacyl-tRNA synthetase present individual molecular signatures.

Sauter C, Lorber B, Gaudry A, Karim L, Schwenzer H, Wien F, Roblin P, Florentz C, Sissler M - Sci Rep (2015)

Bottom Line: The effects of these mutations on the structure and function of the enzymes remain to be established.Mutations with mild effects on solubility occur in patients as allelic combinations whereas those with strong effects on solubility or on aminoacylation are necessarily associated with a partially functional allele.The fact that all mutations show individual molecular and cellular signatures and affect amino acids only conserved in mammals, points towards an alternative function besides aminoacylation.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité de l'ARN, CNRS, Université de Strasbourg, IBMC, 15 rue René Descartes, 67084 STRASBOURG Cedex, France.

ABSTRACT
Mutations in human mitochondrial aminoacyl-tRNA synthetases are associated with a variety of neurodegenerative disorders. The effects of these mutations on the structure and function of the enzymes remain to be established. Here, we investigate six mutants of the aspartyl-tRNA synthetase correlated with leukoencephalopathies. Our integrated strategy, combining an ensemble of biochemical and biophysical approaches, reveals that mutants are diversely affected with respect to their solubility in cellular extracts and stability in solution, but not in architecture. Mutations with mild effects on solubility occur in patients as allelic combinations whereas those with strong effects on solubility or on aminoacylation are necessarily associated with a partially functional allele. The fact that all mutations show individual molecular and cellular signatures and affect amino acids only conserved in mammals, points towards an alternative function besides aminoacylation.

No MeSH data available.


Related in: MedlinePlus

Solubility of WT versus mutant mt-AspRSs in cellulo.(A) Flowchart of experimental procedure. (B) Representative western blots of WT and mutant mt-AspRS (detected with anti-Flag antibodies) in soluble and insoluble fractions, within whole cells or enriched mitochondria. Detections of SOD2 (a mitochondrial matrix protein) and prohibitin (a mitochondrial membrane protein) were performed as loading controls for the soluble and the insoluble fractions, respectively. Three sets of independent experiments for whole cells and three sets of independent experiments for enriched mitochondria were analyzed. (C) Relative abundance of mutant proteins as compared to WT mt-AspRS in soluble (top) and insoluble (bottom) fractions. Relative amounts of polypeptides were estimated as described in the methods section and normalized according to the WT mt-AspRS, arbitrarily set to a value of 1 in both soluble and insoluble fractions. Errors bars illustrate the standard deviations calculated from the three sets of independent experiments. *p < 0.05 and **p ≤ 0.01 based on Student’s t-test.
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f4: Solubility of WT versus mutant mt-AspRSs in cellulo.(A) Flowchart of experimental procedure. (B) Representative western blots of WT and mutant mt-AspRS (detected with anti-Flag antibodies) in soluble and insoluble fractions, within whole cells or enriched mitochondria. Detections of SOD2 (a mitochondrial matrix protein) and prohibitin (a mitochondrial membrane protein) were performed as loading controls for the soluble and the insoluble fractions, respectively. Three sets of independent experiments for whole cells and three sets of independent experiments for enriched mitochondria were analyzed. (C) Relative abundance of mutant proteins as compared to WT mt-AspRS in soluble (top) and insoluble (bottom) fractions. Relative amounts of polypeptides were estimated as described in the methods section and normalized according to the WT mt-AspRS, arbitrarily set to a value of 1 in both soluble and insoluble fractions. Errors bars illustrate the standard deviations calculated from the three sets of independent experiments. *p < 0.05 and **p ≤ 0.01 based on Student’s t-test.

Mentions: WT and mutants of mt-AspRS were expressed in BHK21 cells and their abundance in soluble and insoluble fraction in either whole cell extracts or enriched mitochondria was determined by western blot (Fig. 4B). Histograms displaying relative amounts of mt-AspRS in the various fractions are shown in Fig. 4C. Similar trends were found in whole cell extracts and enriched mitochondria: mutant L613F is slightly more soluble than the WT mt-AspRS, L626Q is not significantly impacted, and R58G, T136S and R263Q are slightly less soluble. The most statistically significant decrease in solubility (with p-values ≤ 0.01) was only observed for mutant Q184K.


Neurodegenerative disease-associated mutants of a human mitochondrial aminoacyl-tRNA synthetase present individual molecular signatures.

Sauter C, Lorber B, Gaudry A, Karim L, Schwenzer H, Wien F, Roblin P, Florentz C, Sissler M - Sci Rep (2015)

Solubility of WT versus mutant mt-AspRSs in cellulo.(A) Flowchart of experimental procedure. (B) Representative western blots of WT and mutant mt-AspRS (detected with anti-Flag antibodies) in soluble and insoluble fractions, within whole cells or enriched mitochondria. Detections of SOD2 (a mitochondrial matrix protein) and prohibitin (a mitochondrial membrane protein) were performed as loading controls for the soluble and the insoluble fractions, respectively. Three sets of independent experiments for whole cells and three sets of independent experiments for enriched mitochondria were analyzed. (C) Relative abundance of mutant proteins as compared to WT mt-AspRS in soluble (top) and insoluble (bottom) fractions. Relative amounts of polypeptides were estimated as described in the methods section and normalized according to the WT mt-AspRS, arbitrarily set to a value of 1 in both soluble and insoluble fractions. Errors bars illustrate the standard deviations calculated from the three sets of independent experiments. *p < 0.05 and **p ≤ 0.01 based on Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664897&req=5

f4: Solubility of WT versus mutant mt-AspRSs in cellulo.(A) Flowchart of experimental procedure. (B) Representative western blots of WT and mutant mt-AspRS (detected with anti-Flag antibodies) in soluble and insoluble fractions, within whole cells or enriched mitochondria. Detections of SOD2 (a mitochondrial matrix protein) and prohibitin (a mitochondrial membrane protein) were performed as loading controls for the soluble and the insoluble fractions, respectively. Three sets of independent experiments for whole cells and three sets of independent experiments for enriched mitochondria were analyzed. (C) Relative abundance of mutant proteins as compared to WT mt-AspRS in soluble (top) and insoluble (bottom) fractions. Relative amounts of polypeptides were estimated as described in the methods section and normalized according to the WT mt-AspRS, arbitrarily set to a value of 1 in both soluble and insoluble fractions. Errors bars illustrate the standard deviations calculated from the three sets of independent experiments. *p < 0.05 and **p ≤ 0.01 based on Student’s t-test.
Mentions: WT and mutants of mt-AspRS were expressed in BHK21 cells and their abundance in soluble and insoluble fraction in either whole cell extracts or enriched mitochondria was determined by western blot (Fig. 4B). Histograms displaying relative amounts of mt-AspRS in the various fractions are shown in Fig. 4C. Similar trends were found in whole cell extracts and enriched mitochondria: mutant L613F is slightly more soluble than the WT mt-AspRS, L626Q is not significantly impacted, and R58G, T136S and R263Q are slightly less soluble. The most statistically significant decrease in solubility (with p-values ≤ 0.01) was only observed for mutant Q184K.

Bottom Line: The effects of these mutations on the structure and function of the enzymes remain to be established.Mutations with mild effects on solubility occur in patients as allelic combinations whereas those with strong effects on solubility or on aminoacylation are necessarily associated with a partially functional allele.The fact that all mutations show individual molecular and cellular signatures and affect amino acids only conserved in mammals, points towards an alternative function besides aminoacylation.

View Article: PubMed Central - PubMed

Affiliation: Architecture et Réactivité de l'ARN, CNRS, Université de Strasbourg, IBMC, 15 rue René Descartes, 67084 STRASBOURG Cedex, France.

ABSTRACT
Mutations in human mitochondrial aminoacyl-tRNA synthetases are associated with a variety of neurodegenerative disorders. The effects of these mutations on the structure and function of the enzymes remain to be established. Here, we investigate six mutants of the aspartyl-tRNA synthetase correlated with leukoencephalopathies. Our integrated strategy, combining an ensemble of biochemical and biophysical approaches, reveals that mutants are diversely affected with respect to their solubility in cellular extracts and stability in solution, but not in architecture. Mutations with mild effects on solubility occur in patients as allelic combinations whereas those with strong effects on solubility or on aminoacylation are necessarily associated with a partially functional allele. The fact that all mutations show individual molecular and cellular signatures and affect amino acids only conserved in mammals, points towards an alternative function besides aminoacylation.

No MeSH data available.


Related in: MedlinePlus