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Aminoacyl-tRNA synthetase dependent angiogenesis revealed by a bioengineered macrolide inhibitor.

Mirando AC, Fang P, Williams TF, Baldor LC, Howe AK, Ebert AM, Wilkinson B, Lounsbury KM, Guo M, Francklyn CS - Sci Rep (2015)

Bottom Line: These include angiogenesis, and human threonyl-tRNA synthetase (TARS) represents a potent pro-angiogenic AARS.Recently, a less toxic variant (BC194) was identified that potently inhibits angiogenesis.Bioengineered natural products are thus useful tools in unmasking the cryptic functions of conventional enzymes in the regulation of complex processes in higher metazoans.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Vermont.

ABSTRACT
Aminoacyl-tRNA synthetases (AARSs) catalyze an early step in protein synthesis, but also regulate diverse physiological processes in animal cells. These include angiogenesis, and human threonyl-tRNA synthetase (TARS) represents a potent pro-angiogenic AARS. Angiogenesis stimulation can be blocked by the macrolide antibiotic borrelidin (BN), which exhibits a broad spectrum toxicity that has discouraged deeper investigation. Recently, a less toxic variant (BC194) was identified that potently inhibits angiogenesis. Employing biochemical, cell biological, and biophysical approaches, we demonstrate that the toxicity of BN and its derivatives is linked to its competition with the threonine substrate at the molecular level, which stimulates amino acid starvation and apoptosis. By separating toxicity from the inhibition of angiogenesis, a direct role for TARS in vascular development in the zebrafish could be demonstrated. Bioengineered natural products are thus useful tools in unmasking the cryptic functions of conventional enzymes in the regulation of complex processes in higher metazoans.

No MeSH data available.


Related in: MedlinePlus

Structure of TARS-BC194 complex.(a) Two-dimensional scheme of TARS-BC194 interactions. H-bonding residues are shown as sticks. Hydrophobic interacting residues are shown in grey. (b) Structure superimposition of TARS-BC194 (green) and TARS-BN (grey) complexes. The protein is shown in ribbon cartoon representation, and the bound BC194 and BN are shown as pink and blue sticks, respectively. (c) Close up view of BC194 (green) and BN (grey) binding site residues. The five shared H-bonds are shown as black dash lines. The 2 BN-specific interactions are shown as blue dash lines, while the corresponding distances in BC194 structure are indicated in pink. (d) Close up view of threonine binding interactions. Interactions are shown as dashed lines. (e,f) The effects of BC194 (e) and BN (f) treatment on a cell-free translation system. Rabbit reticulocyte lysate (RRL) was incubated with 0.02 mg/ml luciferase mRNA and translation of luciferase enzyme was quantified in a luminescence assay. Serial diluted BC194 and borrelidin (2.5 nM - 25 μM) was added to inhibit the translation of luciferase mRNA; mean ± SEM, n = 3.
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f2: Structure of TARS-BC194 complex.(a) Two-dimensional scheme of TARS-BC194 interactions. H-bonding residues are shown as sticks. Hydrophobic interacting residues are shown in grey. (b) Structure superimposition of TARS-BC194 (green) and TARS-BN (grey) complexes. The protein is shown in ribbon cartoon representation, and the bound BC194 and BN are shown as pink and blue sticks, respectively. (c) Close up view of BC194 (green) and BN (grey) binding site residues. The five shared H-bonds are shown as black dash lines. The 2 BN-specific interactions are shown as blue dash lines, while the corresponding distances in BC194 structure are indicated in pink. (d) Close up view of threonine binding interactions. Interactions are shown as dashed lines. (e,f) The effects of BC194 (e) and BN (f) treatment on a cell-free translation system. Rabbit reticulocyte lysate (RRL) was incubated with 0.02 mg/ml luciferase mRNA and translation of luciferase enzyme was quantified in a luminescence assay. Serial diluted BC194 and borrelidin (2.5 nM - 25 μM) was added to inhibit the translation of luciferase mRNA; mean ± SEM, n = 3.

Mentions: The molecular cloning of the BN biosynthetic operon from Streptomyces parvulus Tu405536 permitted novel variants of BN to be produced through biosynthetic engineering3334. In BC194, a cyclobutane ring replaces the pendant C17 cyclopentane ring (2, Fig. 1). Relative to other less effective variants, BC194 retained potent inhibition of angiogenesis while possessing substantially reduced toxicity towards endothelial cells34. As a first step towards understanding the molecular basis of these effects, we co-crystallized BC194 with a fragment of human TARS comprising the catalytic and anticodon binding domains, and solved the structure to a resolution of 2.8 Å (Table S1). The structures of BN and BC194 differ at position C17, with BN containing a pendant cyclopentanecarboxylic acid ring, and BC194 a cyclobutanecarboxylic acid ring (Fig. 1). BC194 binding to the TARS active site is stabilized by numerous Van der Waals interactions and five distinct enzyme-compound hydrogen bonds (Fig. 2a). In addition, BC194 induces a conformation of TARS close to that of BN – TARS complex, with an r.m.s.d. of 0.62 Å between superimposed BC194 and BN – TARS complex structures (for all 402 Ca’s in TARS) (Fig. 2b)37. In a global structural sense, BN and BC194 act to stabilize the same conformational state for TARS, with potential consequences for secondary functions (vide infra).


Aminoacyl-tRNA synthetase dependent angiogenesis revealed by a bioengineered macrolide inhibitor.

Mirando AC, Fang P, Williams TF, Baldor LC, Howe AK, Ebert AM, Wilkinson B, Lounsbury KM, Guo M, Francklyn CS - Sci Rep (2015)

Structure of TARS-BC194 complex.(a) Two-dimensional scheme of TARS-BC194 interactions. H-bonding residues are shown as sticks. Hydrophobic interacting residues are shown in grey. (b) Structure superimposition of TARS-BC194 (green) and TARS-BN (grey) complexes. The protein is shown in ribbon cartoon representation, and the bound BC194 and BN are shown as pink and blue sticks, respectively. (c) Close up view of BC194 (green) and BN (grey) binding site residues. The five shared H-bonds are shown as black dash lines. The 2 BN-specific interactions are shown as blue dash lines, while the corresponding distances in BC194 structure are indicated in pink. (d) Close up view of threonine binding interactions. Interactions are shown as dashed lines. (e,f) The effects of BC194 (e) and BN (f) treatment on a cell-free translation system. Rabbit reticulocyte lysate (RRL) was incubated with 0.02 mg/ml luciferase mRNA and translation of luciferase enzyme was quantified in a luminescence assay. Serial diluted BC194 and borrelidin (2.5 nM - 25 μM) was added to inhibit the translation of luciferase mRNA; mean ± SEM, n = 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4536658&req=5

f2: Structure of TARS-BC194 complex.(a) Two-dimensional scheme of TARS-BC194 interactions. H-bonding residues are shown as sticks. Hydrophobic interacting residues are shown in grey. (b) Structure superimposition of TARS-BC194 (green) and TARS-BN (grey) complexes. The protein is shown in ribbon cartoon representation, and the bound BC194 and BN are shown as pink and blue sticks, respectively. (c) Close up view of BC194 (green) and BN (grey) binding site residues. The five shared H-bonds are shown as black dash lines. The 2 BN-specific interactions are shown as blue dash lines, while the corresponding distances in BC194 structure are indicated in pink. (d) Close up view of threonine binding interactions. Interactions are shown as dashed lines. (e,f) The effects of BC194 (e) and BN (f) treatment on a cell-free translation system. Rabbit reticulocyte lysate (RRL) was incubated with 0.02 mg/ml luciferase mRNA and translation of luciferase enzyme was quantified in a luminescence assay. Serial diluted BC194 and borrelidin (2.5 nM - 25 μM) was added to inhibit the translation of luciferase mRNA; mean ± SEM, n = 3.
Mentions: The molecular cloning of the BN biosynthetic operon from Streptomyces parvulus Tu405536 permitted novel variants of BN to be produced through biosynthetic engineering3334. In BC194, a cyclobutane ring replaces the pendant C17 cyclopentane ring (2, Fig. 1). Relative to other less effective variants, BC194 retained potent inhibition of angiogenesis while possessing substantially reduced toxicity towards endothelial cells34. As a first step towards understanding the molecular basis of these effects, we co-crystallized BC194 with a fragment of human TARS comprising the catalytic and anticodon binding domains, and solved the structure to a resolution of 2.8 Å (Table S1). The structures of BN and BC194 differ at position C17, with BN containing a pendant cyclopentanecarboxylic acid ring, and BC194 a cyclobutanecarboxylic acid ring (Fig. 1). BC194 binding to the TARS active site is stabilized by numerous Van der Waals interactions and five distinct enzyme-compound hydrogen bonds (Fig. 2a). In addition, BC194 induces a conformation of TARS close to that of BN – TARS complex, with an r.m.s.d. of 0.62 Å between superimposed BC194 and BN – TARS complex structures (for all 402 Ca’s in TARS) (Fig. 2b)37. In a global structural sense, BN and BC194 act to stabilize the same conformational state for TARS, with potential consequences for secondary functions (vide infra).

Bottom Line: These include angiogenesis, and human threonyl-tRNA synthetase (TARS) represents a potent pro-angiogenic AARS.Recently, a less toxic variant (BC194) was identified that potently inhibits angiogenesis.Bioengineered natural products are thus useful tools in unmasking the cryptic functions of conventional enzymes in the regulation of complex processes in higher metazoans.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Vermont.

ABSTRACT
Aminoacyl-tRNA synthetases (AARSs) catalyze an early step in protein synthesis, but also regulate diverse physiological processes in animal cells. These include angiogenesis, and human threonyl-tRNA synthetase (TARS) represents a potent pro-angiogenic AARS. Angiogenesis stimulation can be blocked by the macrolide antibiotic borrelidin (BN), which exhibits a broad spectrum toxicity that has discouraged deeper investigation. Recently, a less toxic variant (BC194) was identified that potently inhibits angiogenesis. Employing biochemical, cell biological, and biophysical approaches, we demonstrate that the toxicity of BN and its derivatives is linked to its competition with the threonine substrate at the molecular level, which stimulates amino acid starvation and apoptosis. By separating toxicity from the inhibition of angiogenesis, a direct role for TARS in vascular development in the zebrafish could be demonstrated. Bioengineered natural products are thus useful tools in unmasking the cryptic functions of conventional enzymes in the regulation of complex processes in higher metazoans.

No MeSH data available.


Related in: MedlinePlus