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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

Comparison of WT, Q184K and R263Q mt-AspRSs by SAXS.(A) Overlay of normalized SEC absorbance profiles and SAXS curves recorded for each absorbance peak. The WT protein (plots in red) forms essentially a dimer present in peak 3 (93% of the sample), while mutants Q184K (in orange) and R263Q (in violet) also form entities of higher molecular sizes that elute faster in peak 1 (11%, 2% of overall population, respectively) and peak 2 (23%, 9%, respectively). (B) SAXS profiles of AspRS populations in peaks 1, 2 and 3. For clarity of presentation, the curves are offset. (C) Modeling of mt-AspRS dimer in peak 3 by atomic models generated from the X-ray structure of mt-AspRS (PDBid: 4AH6) to which flexible C-terminal tails were added. In this example, data from Q184K mutant are represented as orange crosses and a typical profile calculated from one of conformers generated under SAXS data constrains by DADIMODO is shown in black. Eight conformations of C-terminal extensions with lowest Chi values (see also Supplementary Fig. 5) are depicted: yellow and cyan tails belong to orange and blue subunit, respectively. The gray region corresponds to the tag used for affinity purification.
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f5: Comparison of WT, Q184K and R263Q mt-AspRSs by SAXS.(A) Overlay of normalized SEC absorbance profiles and SAXS curves recorded for each absorbance peak. The WT protein (plots in red) forms essentially a dimer present in peak 3 (93% of the sample), while mutants Q184K (in orange) and R263Q (in violet) also form entities of higher molecular sizes that elute faster in peak 1 (11%, 2% of overall population, respectively) and peak 2 (23%, 9%, respectively). (B) SAXS profiles of AspRS populations in peaks 1, 2 and 3. For clarity of presentation, the curves are offset. (C) Modeling of mt-AspRS dimer in peak 3 by atomic models generated from the X-ray structure of mt-AspRS (PDBid: 4AH6) to which flexible C-terminal tails were added. In this example, data from Q184K mutant are represented as orange crosses and a typical profile calculated from one of conformers generated under SAXS data constrains by DADIMODO is shown in black. Eight conformations of C-terminal extensions with lowest Chi values (see also Supplementary Fig. 5) are depicted: yellow and cyan tails belong to orange and blue subunit, respectively. The gray region corresponds to the tag used for affinity purification.

Mentions: Mutants Q184K and R263Q, showing respectively the highest impact on protein solubility in cellular extracts (either whole cells or enriched mitochondria) and the highest impact on aminoacylation in vitro23, were compared to that of the WT enzyme by SAXS analyses. Each sample was subjected to size exclusion chromatography (HPLC-SEC) directly upstream the SAXS cell to separate aggregates of various sizes from oligomers, allowing individual characterization of each species. The WT enzyme eluted from the SEC column essentially as a single population of particles (UV-absorbance peak 3 in Fig. 5A) while both mutants also contained a significant amount of high molecular-weight particles eluting earlier as peaks 1 and 2 (Fig. 5A). The X-ray scattering signal recorded during the elution of these three protein samples revealed that each peak was composed of well-defined particles yielding similar SAXS curves (Fig. 5B).


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)

Comparison of WT, Q184K and R263Q mt-AspRSs by SAXS.(A) Overlay of normalized SEC absorbance profiles and SAXS curves recorded for each absorbance peak. The WT protein (plots in red) forms essentially a dimer present in peak 3 (93% of the sample), while mutants Q184K (in orange) and R263Q (in violet) also form entities of higher molecular sizes that elute faster in peak 1 (11%, 2% of overall population, respectively) and peak 2 (23%, 9%, respectively). (B) SAXS profiles of AspRS populations in peaks 1, 2 and 3. For clarity of presentation, the curves are offset. (C) Modeling of mt-AspRS dimer in peak 3 by atomic models generated from the X-ray structure of mt-AspRS (PDBid: 4AH6) to which flexible C-terminal tails were added. In this example, data from Q184K mutant are represented as orange crosses and a typical profile calculated from one of conformers generated under SAXS data constrains by DADIMODO is shown in black. Eight conformations of C-terminal extensions with lowest Chi values (see also Supplementary Fig. 5) are depicted: yellow and cyan tails belong to orange and blue subunit, respectively. The gray region corresponds to the tag used for affinity purification.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Comparison of WT, Q184K and R263Q mt-AspRSs by SAXS.(A) Overlay of normalized SEC absorbance profiles and SAXS curves recorded for each absorbance peak. The WT protein (plots in red) forms essentially a dimer present in peak 3 (93% of the sample), while mutants Q184K (in orange) and R263Q (in violet) also form entities of higher molecular sizes that elute faster in peak 1 (11%, 2% of overall population, respectively) and peak 2 (23%, 9%, respectively). (B) SAXS profiles of AspRS populations in peaks 1, 2 and 3. For clarity of presentation, the curves are offset. (C) Modeling of mt-AspRS dimer in peak 3 by atomic models generated from the X-ray structure of mt-AspRS (PDBid: 4AH6) to which flexible C-terminal tails were added. In this example, data from Q184K mutant are represented as orange crosses and a typical profile calculated from one of conformers generated under SAXS data constrains by DADIMODO is shown in black. Eight conformations of C-terminal extensions with lowest Chi values (see also Supplementary Fig. 5) are depicted: yellow and cyan tails belong to orange and blue subunit, respectively. The gray region corresponds to the tag used for affinity purification.
Mentions: Mutants Q184K and R263Q, showing respectively the highest impact on protein solubility in cellular extracts (either whole cells or enriched mitochondria) and the highest impact on aminoacylation in vitro23, were compared to that of the WT enzyme by SAXS analyses. Each sample was subjected to size exclusion chromatography (HPLC-SEC) directly upstream the SAXS cell to separate aggregates of various sizes from oligomers, allowing individual characterization of each species. The WT enzyme eluted from the SEC column essentially as a single population of particles (UV-absorbance peak 3 in Fig. 5A) while both mutants also contained a significant amount of high molecular-weight particles eluting earlier as peaks 1 and 2 (Fig. 5A). The X-ray scattering signal recorded during the elution of these three protein samples revealed that each peak was composed of well-defined particles yielding similar SAXS curves (Fig. 5B).

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