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Double-sieving-defective aminoacyl-tRNA synthetase causes protein mistranslation and affects cellular physiology and development.

Lu J, Bergert M, Walther A, Suter B - Nat Commun (2014)

Bottom Line: At the cellular level, the mutations reduce cell proliferation and promote cell death.Our results also reveal the particular importance of the first amino-acid recognition sieve.Overall, these findings provide new mechanistic insights into how malfunctioning of aaRSs can cause diseases.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology, University of Bern, Baltzerstrasse 4, Bern 3012, Switzerland.

ABSTRACT
Aminoacyl-tRNA synthetases (aaRSs) constitute a family of ubiquitously expressed essential enzymes that ligate amino acids to their cognate tRNAs for protein synthesis. Recently, aaRS mutations have been linked to various human diseases; however, how these mutations lead to diseases has remained unclear. In order to address the importance of aminoacylation fidelity in multicellular organisms, we generated an amino-acid double-sieving model in Drosophila melanogaster using phenylalanyl-tRNA synthetase (PheRS). Double-sieving-defective mutations dramatically misacylate non-cognate Tyr, induce protein mistranslation and cause endoplasmic reticulum stress in flies. Mutant adults exhibit many defects, including loss of neuronal cells, impaired locomotive performance, shortened lifespan and smaller organ size. At the cellular level, the mutations reduce cell proliferation and promote cell death. Our results also reveal the particular importance of the first amino-acid recognition sieve. Overall, these findings provide new mechanistic insights into how malfunctioning of aaRSs can cause diseases.

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Related in: MedlinePlus

Sieving-defective mutations in Drosophila PheRS.(a,c) Structure-based alignment of PheRS sequences using the PROMALS3D method. The sequences of prokaryotes and archaea/eukaryotes are shown in grey and blue, respectively. The C-terminal region of α-PheRS is listed in a, and the red Ala is the amino-acid residue substituted by Gly. Domain B3-4 of β-PheRS is shown in c. Residues crucial for editing, the ones at the entrance of the editing pocket (red) and the ones at the activation centre (black), are indicated. DROME, Drosophila melanogaster; HUMAN, Homo sapiens; PYRHO, P. horikoshii; THET8, T. thermophilus; ECOLI, E. coli. (b,d) Structure of the Drosophila PheRS catalytic module (first sieve) and domain B3-4 (second sieve) modelled by UCSF Chimera. The residues highlighted in red in b,d correspond to the red Ala in a,c, respectively. The catalytic module (b; domain B6-7 in light blue and domain A1-2 in grey) was modelled on the human PheRS (PDB: 3L4G). Domain B3-4 was modelled on P. horikoshii PheRS (d; PDB: 2CX1). (e,f) The Phe and Tyr aminoacylation activities of PheRS variants were determined in vitro. (e) PheRS variants are still active in Phe aminoacylation. However, while the enzymatic activity of the βA158W mutant is normal, the activity of the αA456G mutant is somewhat reduced. (f) Tyr mis-aminoacylation activity of PheRS variants. Wild-type PheRS produced very low levels of TCA-insoluble Tyr and the single mutant αA456G and βA158W produced only slightly elevated levels. In contrast, the αA456G, βA158W ‘double-mutant’ protein dramatically misacylated Tyr. Values are the means of three independent experiments. Error bars show s.d.
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f2: Sieving-defective mutations in Drosophila PheRS.(a,c) Structure-based alignment of PheRS sequences using the PROMALS3D method. The sequences of prokaryotes and archaea/eukaryotes are shown in grey and blue, respectively. The C-terminal region of α-PheRS is listed in a, and the red Ala is the amino-acid residue substituted by Gly. Domain B3-4 of β-PheRS is shown in c. Residues crucial for editing, the ones at the entrance of the editing pocket (red) and the ones at the activation centre (black), are indicated. DROME, Drosophila melanogaster; HUMAN, Homo sapiens; PYRHO, P. horikoshii; THET8, T. thermophilus; ECOLI, E. coli. (b,d) Structure of the Drosophila PheRS catalytic module (first sieve) and domain B3-4 (second sieve) modelled by UCSF Chimera. The residues highlighted in red in b,d correspond to the red Ala in a,c, respectively. The catalytic module (b; domain B6-7 in light blue and domain A1-2 in grey) was modelled on the human PheRS (PDB: 3L4G). Domain B3-4 was modelled on P. horikoshii PheRS (d; PDB: 2CX1). (e,f) The Phe and Tyr aminoacylation activities of PheRS variants were determined in vitro. (e) PheRS variants are still active in Phe aminoacylation. However, while the enzymatic activity of the βA158W mutant is normal, the activity of the αA456G mutant is somewhat reduced. (f) Tyr mis-aminoacylation activity of PheRS variants. Wild-type PheRS produced very low levels of TCA-insoluble Tyr and the single mutant αA456G and βA158W produced only slightly elevated levels. In contrast, the αA456G, βA158W ‘double-mutant’ protein dramatically misacylated Tyr. Values are the means of three independent experiments. Error bars show s.d.

Mentions: On the basis of previous results from Escherichia coli, Thermus thermophilus and Pyrococcus horikoshii242526, we first predicted potential crucial amino-acid residues for aminoacylation fidelity in Drosophila PheRS. We used the knowledge about the secondary structure to align the sequences for both subunits of the selected prokaryotic and archaeal/eukaryal PheRS28. The sequences are highly conserved within the archaea/eukaryotes and more diverse between the bacterial and archaeal/eukaryal species (Fig. 2a–d). Proper Phe specificity for the activation step is controlled by the amino-acid recognition sieve. A294 in E. coli and A463 in P. horikoshii are the key residues in α-PheRS to ensure substrate specificity because they restrict the space of the pocket to the size of Phe12. Replacing this Ala by the smaller Gly enlarges the Phe-binding pocket, making it more permissive to receive the non-cognate Tyr, thereby enhancing Tyr misactivation. Despite the high sequence divergence between the bacterial and the archaeal/eukaryal α-subunits, we identified the highly conserved A456 in Drosophila α-PheRS as the homologous position (Fig. 2a,b). To produce a PheRS enzyme with an increased activation error rate, we therefore introduced the A456G mutation into the Drosophila α-PheRS gene.


Double-sieving-defective aminoacyl-tRNA synthetase causes protein mistranslation and affects cellular physiology and development.

Lu J, Bergert M, Walther A, Suter B - Nat Commun (2014)

Sieving-defective mutations in Drosophila PheRS.(a,c) Structure-based alignment of PheRS sequences using the PROMALS3D method. The sequences of prokaryotes and archaea/eukaryotes are shown in grey and blue, respectively. The C-terminal region of α-PheRS is listed in a, and the red Ala is the amino-acid residue substituted by Gly. Domain B3-4 of β-PheRS is shown in c. Residues crucial for editing, the ones at the entrance of the editing pocket (red) and the ones at the activation centre (black), are indicated. DROME, Drosophila melanogaster; HUMAN, Homo sapiens; PYRHO, P. horikoshii; THET8, T. thermophilus; ECOLI, E. coli. (b,d) Structure of the Drosophila PheRS catalytic module (first sieve) and domain B3-4 (second sieve) modelled by UCSF Chimera. The residues highlighted in red in b,d correspond to the red Ala in a,c, respectively. The catalytic module (b; domain B6-7 in light blue and domain A1-2 in grey) was modelled on the human PheRS (PDB: 3L4G). Domain B3-4 was modelled on P. horikoshii PheRS (d; PDB: 2CX1). (e,f) The Phe and Tyr aminoacylation activities of PheRS variants were determined in vitro. (e) PheRS variants are still active in Phe aminoacylation. However, while the enzymatic activity of the βA158W mutant is normal, the activity of the αA456G mutant is somewhat reduced. (f) Tyr mis-aminoacylation activity of PheRS variants. Wild-type PheRS produced very low levels of TCA-insoluble Tyr and the single mutant αA456G and βA158W produced only slightly elevated levels. In contrast, the αA456G, βA158W ‘double-mutant’ protein dramatically misacylated Tyr. Values are the means of three independent experiments. Error bars show s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263187&req=5

f2: Sieving-defective mutations in Drosophila PheRS.(a,c) Structure-based alignment of PheRS sequences using the PROMALS3D method. The sequences of prokaryotes and archaea/eukaryotes are shown in grey and blue, respectively. The C-terminal region of α-PheRS is listed in a, and the red Ala is the amino-acid residue substituted by Gly. Domain B3-4 of β-PheRS is shown in c. Residues crucial for editing, the ones at the entrance of the editing pocket (red) and the ones at the activation centre (black), are indicated. DROME, Drosophila melanogaster; HUMAN, Homo sapiens; PYRHO, P. horikoshii; THET8, T. thermophilus; ECOLI, E. coli. (b,d) Structure of the Drosophila PheRS catalytic module (first sieve) and domain B3-4 (second sieve) modelled by UCSF Chimera. The residues highlighted in red in b,d correspond to the red Ala in a,c, respectively. The catalytic module (b; domain B6-7 in light blue and domain A1-2 in grey) was modelled on the human PheRS (PDB: 3L4G). Domain B3-4 was modelled on P. horikoshii PheRS (d; PDB: 2CX1). (e,f) The Phe and Tyr aminoacylation activities of PheRS variants were determined in vitro. (e) PheRS variants are still active in Phe aminoacylation. However, while the enzymatic activity of the βA158W mutant is normal, the activity of the αA456G mutant is somewhat reduced. (f) Tyr mis-aminoacylation activity of PheRS variants. Wild-type PheRS produced very low levels of TCA-insoluble Tyr and the single mutant αA456G and βA158W produced only slightly elevated levels. In contrast, the αA456G, βA158W ‘double-mutant’ protein dramatically misacylated Tyr. Values are the means of three independent experiments. Error bars show s.d.
Mentions: On the basis of previous results from Escherichia coli, Thermus thermophilus and Pyrococcus horikoshii242526, we first predicted potential crucial amino-acid residues for aminoacylation fidelity in Drosophila PheRS. We used the knowledge about the secondary structure to align the sequences for both subunits of the selected prokaryotic and archaeal/eukaryal PheRS28. The sequences are highly conserved within the archaea/eukaryotes and more diverse between the bacterial and archaeal/eukaryal species (Fig. 2a–d). Proper Phe specificity for the activation step is controlled by the amino-acid recognition sieve. A294 in E. coli and A463 in P. horikoshii are the key residues in α-PheRS to ensure substrate specificity because they restrict the space of the pocket to the size of Phe12. Replacing this Ala by the smaller Gly enlarges the Phe-binding pocket, making it more permissive to receive the non-cognate Tyr, thereby enhancing Tyr misactivation. Despite the high sequence divergence between the bacterial and the archaeal/eukaryal α-subunits, we identified the highly conserved A456 in Drosophila α-PheRS as the homologous position (Fig. 2a,b). To produce a PheRS enzyme with an increased activation error rate, we therefore introduced the A456G mutation into the Drosophila α-PheRS gene.

Bottom Line: At the cellular level, the mutations reduce cell proliferation and promote cell death.Our results also reveal the particular importance of the first amino-acid recognition sieve.Overall, these findings provide new mechanistic insights into how malfunctioning of aaRSs can cause diseases.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology, University of Bern, Baltzerstrasse 4, Bern 3012, Switzerland.

ABSTRACT
Aminoacyl-tRNA synthetases (aaRSs) constitute a family of ubiquitously expressed essential enzymes that ligate amino acids to their cognate tRNAs for protein synthesis. Recently, aaRS mutations have been linked to various human diseases; however, how these mutations lead to diseases has remained unclear. In order to address the importance of aminoacylation fidelity in multicellular organisms, we generated an amino-acid double-sieving model in Drosophila melanogaster using phenylalanyl-tRNA synthetase (PheRS). Double-sieving-defective mutations dramatically misacylate non-cognate Tyr, induce protein mistranslation and cause endoplasmic reticulum stress in flies. Mutant adults exhibit many defects, including loss of neuronal cells, impaired locomotive performance, shortened lifespan and smaller organ size. At the cellular level, the mutations reduce cell proliferation and promote cell death. Our results also reveal the particular importance of the first amino-acid recognition sieve. Overall, these findings provide new mechanistic insights into how malfunctioning of aaRSs can cause diseases.

Show MeSH
Related in: MedlinePlus