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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

BN and BC194 exhibit comparable inhibition of angiogenesis at sub-toxic levels.(a) Quantification of HUVEC branching at varying concentrations of BN, BC194, and BC220. HUVECs were plated on Matrigel in full EGM-2 (2% FBS) media and exposed to the indicated concentrations compounds. Cells were fixed after 4–8 h and stained with Oregon Green 488 Phalloidin. Numbers represent percentage of control (0 nM) samples, mean ± SEM, n = 3 *p < 0.05 relative to 0 nM (one-way ANOVA, Tukey Test for each compounds). No significant differences were observed between 0.1, 1, and 10 nM samples within the same treatment or between BN and BC194-treated samples at the same concentrations (t-test for each treatment pair, not indicated in figure). (b) The effects of BN and BC194 on in vivo angiogenesis in a CAM assay. Fertilized chicken embryos were cultured ex-ova for 10 days after which compounds at the indicated concentrations were applied to gelform sponges on the CAM. Images were taken daily over 72 h and quantified as the change in vascularity score over this entire period; mean ± SEM, n ≥ 12, *p < 0.05 relative to PBS (one-way ANOVA, Tukey Test). (c) Aminoacylation activity data for R442A TARS. Numbers represent the formation of Thr-tRNAThr per active site (pmol/pmol) over time; mean ± SEM, n = 3. (d,e) Quantification of HUVEC branching (d) change in CAM vascularity (e) in response to exogenous wildtype TARS, the catalytically R442A TARS, and LARS; mean ± SEM, n = 4, *p < 0.05 (Tube Formation), n ≥ 12, *p < 0.01 (CAM). (f) Quantification of migrated cells exposed to 5 or 25 nM BC194 after 5 and 24 h in the donut assay; mean ± SEM, n ≥ 6, *p < 0.01 relative to DMSO of same time point (one-way ANOVA, Tukey Test). (g) Image of cells at the leading edge of the migratory boundary after 24 h exposure to DMSO (left) or 25 nM BC194 (right). See also Supplementary Figure S4, S5, and S6 for representative images.
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f4: BN and BC194 exhibit comparable inhibition of angiogenesis at sub-toxic levels.(a) Quantification of HUVEC branching at varying concentrations of BN, BC194, and BC220. HUVECs were plated on Matrigel in full EGM-2 (2% FBS) media and exposed to the indicated concentrations compounds. Cells were fixed after 4–8 h and stained with Oregon Green 488 Phalloidin. Numbers represent percentage of control (0 nM) samples, mean ± SEM, n = 3 *p < 0.05 relative to 0 nM (one-way ANOVA, Tukey Test for each compounds). No significant differences were observed between 0.1, 1, and 10 nM samples within the same treatment or between BN and BC194-treated samples at the same concentrations (t-test for each treatment pair, not indicated in figure). (b) The effects of BN and BC194 on in vivo angiogenesis in a CAM assay. Fertilized chicken embryos were cultured ex-ova for 10 days after which compounds at the indicated concentrations were applied to gelform sponges on the CAM. Images were taken daily over 72 h and quantified as the change in vascularity score over this entire period; mean ± SEM, n ≥ 12, *p < 0.05 relative to PBS (one-way ANOVA, Tukey Test). (c) Aminoacylation activity data for R442A TARS. Numbers represent the formation of Thr-tRNAThr per active site (pmol/pmol) over time; mean ± SEM, n = 3. (d,e) Quantification of HUVEC branching (d) change in CAM vascularity (e) in response to exogenous wildtype TARS, the catalytically R442A TARS, and LARS; mean ± SEM, n = 4, *p < 0.05 (Tube Formation), n ≥ 12, *p < 0.01 (CAM). (f) Quantification of migrated cells exposed to 5 or 25 nM BC194 after 5 and 24 h in the donut assay; mean ± SEM, n ≥ 6, *p < 0.01 relative to DMSO of same time point (one-way ANOVA, Tukey Test). (g) Image of cells at the leading edge of the migratory boundary after 24 h exposure to DMSO (left) or 25 nM BC194 (right). See also Supplementary Figure S4, S5, and S6 for representative images.

Mentions: Previous studies indicate that BN and BC194 are both potent inhibitors of angiogenesis253446. We wondered whether the anti-angiogenic effect is a direct consequence of the induction of the amino acid starvation response in endothelial cells, leading to their self-destruction through apoptosis, or is instead a reflection of another physiological effect. The ability of all three macrolides to inhibit HUVEC branching at sub-toxic concentrations was therefore tested in the endothelial tube formation assay, a cellular model of angiogenesis (Fig. 4a, S4a,c). At concentrations as low as 1 nM, significant decreases in branch formation were observed for both BN (1) and BC194 (2) relative to uninhibited conditions. Notably, these concentrations are 10- and 100- fold below the lowest BN and BC194 concentrations required to induce eIF2α phosphorylation (Fig. 3). Exposure to BC220 (3) at these same concentrations had no effect on tube formation (Fig. 4a). BN and BC194 thus exhibit comparable potencies in inhibiting angiogenesis, despite the differences between their interactions with the TARS active site.


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)

BN and BC194 exhibit comparable inhibition of angiogenesis at sub-toxic levels.(a) Quantification of HUVEC branching at varying concentrations of BN, BC194, and BC220. HUVECs were plated on Matrigel in full EGM-2 (2% FBS) media and exposed to the indicated concentrations compounds. Cells were fixed after 4–8 h and stained with Oregon Green 488 Phalloidin. Numbers represent percentage of control (0 nM) samples, mean ± SEM, n = 3 *p < 0.05 relative to 0 nM (one-way ANOVA, Tukey Test for each compounds). No significant differences were observed between 0.1, 1, and 10 nM samples within the same treatment or between BN and BC194-treated samples at the same concentrations (t-test for each treatment pair, not indicated in figure). (b) The effects of BN and BC194 on in vivo angiogenesis in a CAM assay. Fertilized chicken embryos were cultured ex-ova for 10 days after which compounds at the indicated concentrations were applied to gelform sponges on the CAM. Images were taken daily over 72 h and quantified as the change in vascularity score over this entire period; mean ± SEM, n ≥ 12, *p < 0.05 relative to PBS (one-way ANOVA, Tukey Test). (c) Aminoacylation activity data for R442A TARS. Numbers represent the formation of Thr-tRNAThr per active site (pmol/pmol) over time; mean ± SEM, n = 3. (d,e) Quantification of HUVEC branching (d) change in CAM vascularity (e) in response to exogenous wildtype TARS, the catalytically R442A TARS, and LARS; mean ± SEM, n = 4, *p < 0.05 (Tube Formation), n ≥ 12, *p < 0.01 (CAM). (f) Quantification of migrated cells exposed to 5 or 25 nM BC194 after 5 and 24 h in the donut assay; mean ± SEM, n ≥ 6, *p < 0.01 relative to DMSO of same time point (one-way ANOVA, Tukey Test). (g) Image of cells at the leading edge of the migratory boundary after 24 h exposure to DMSO (left) or 25 nM BC194 (right). See also Supplementary Figure S4, S5, and S6 for representative images.
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Related In: Results  -  Collection

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f4: BN and BC194 exhibit comparable inhibition of angiogenesis at sub-toxic levels.(a) Quantification of HUVEC branching at varying concentrations of BN, BC194, and BC220. HUVECs were plated on Matrigel in full EGM-2 (2% FBS) media and exposed to the indicated concentrations compounds. Cells were fixed after 4–8 h and stained with Oregon Green 488 Phalloidin. Numbers represent percentage of control (0 nM) samples, mean ± SEM, n = 3 *p < 0.05 relative to 0 nM (one-way ANOVA, Tukey Test for each compounds). No significant differences were observed between 0.1, 1, and 10 nM samples within the same treatment or between BN and BC194-treated samples at the same concentrations (t-test for each treatment pair, not indicated in figure). (b) The effects of BN and BC194 on in vivo angiogenesis in a CAM assay. Fertilized chicken embryos were cultured ex-ova for 10 days after which compounds at the indicated concentrations were applied to gelform sponges on the CAM. Images were taken daily over 72 h and quantified as the change in vascularity score over this entire period; mean ± SEM, n ≥ 12, *p < 0.05 relative to PBS (one-way ANOVA, Tukey Test). (c) Aminoacylation activity data for R442A TARS. Numbers represent the formation of Thr-tRNAThr per active site (pmol/pmol) over time; mean ± SEM, n = 3. (d,e) Quantification of HUVEC branching (d) change in CAM vascularity (e) in response to exogenous wildtype TARS, the catalytically R442A TARS, and LARS; mean ± SEM, n = 4, *p < 0.05 (Tube Formation), n ≥ 12, *p < 0.01 (CAM). (f) Quantification of migrated cells exposed to 5 or 25 nM BC194 after 5 and 24 h in the donut assay; mean ± SEM, n ≥ 6, *p < 0.01 relative to DMSO of same time point (one-way ANOVA, Tukey Test). (g) Image of cells at the leading edge of the migratory boundary after 24 h exposure to DMSO (left) or 25 nM BC194 (right). See also Supplementary Figure S4, S5, and S6 for representative images.
Mentions: Previous studies indicate that BN and BC194 are both potent inhibitors of angiogenesis253446. We wondered whether the anti-angiogenic effect is a direct consequence of the induction of the amino acid starvation response in endothelial cells, leading to their self-destruction through apoptosis, or is instead a reflection of another physiological effect. The ability of all three macrolides to inhibit HUVEC branching at sub-toxic concentrations was therefore tested in the endothelial tube formation assay, a cellular model of angiogenesis (Fig. 4a, S4a,c). At concentrations as low as 1 nM, significant decreases in branch formation were observed for both BN (1) and BC194 (2) relative to uninhibited conditions. Notably, these concentrations are 10- and 100- fold below the lowest BN and BC194 concentrations required to induce eIF2α phosphorylation (Fig. 3). Exposure to BC220 (3) at these same concentrations had no effect on tube formation (Fig. 4a). BN and BC194 thus exhibit comparable potencies in inhibiting angiogenesis, despite the differences between their interactions with the TARS active site.

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