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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

The cytotoxicity of borrelidin is linked to the induction of the amino acid starvation response.(a–f) HUVEC cells grown in full serum media were exposed to the indicated concentrations of BN (a–c) or BC194 (d–f) and standardized to 0.05% DMSO. Cropped images from western blots of cell extracts were analyzed using antibodies recognizing phospho-eIF2α and cleaved-caspase 3 with β-tubulin as a loading control. Full images of blots can be found in Supplementary Figures S2a and S2b. Quantification of phospho-eIF2α and cleaved-caspase 3 for BN (b,c) and BC194 (e,f) relative to β-tubulin; mean ± SEM, n ≥ 3, *p < 0.05 relative to 0 nM (one-way ANOVA, Tukey Test). The apparent drop in the levels of phospho-eif2α as BC194 increased to 1000 nM and the exclusion of 1000 nM BN-treated data (designated by #) are due to the fact that endothelial cells exposed to high macrolide concentrations were visually apoptotic, making estimations of total protein loaded difficult. As such, we believe that the variability observed in these data at higher concentrations was more related to the severe status of the cells rather than a change in the amino acid starvation response. (g,h) Western blot (g) quantification of eif2α amounts relative to β-tubulin for both BN- and BC194-treated cells (100 nM) in the presence of various threonine concentrations (0–10 mM); mean ± SEM, n ≥ 3, *p < 0.05 relative to 0 mM threonine (one-way ANOVA, Tukey Test), #p < 0.01 between compounds at same threonine concentration (two-way ANOVA, Sidak Test). Full images of blots can be found in Supplementary Figure S2c. See also Supplementary Figure S1 for cell cycle, proliferation effects, and Supplementary Figure S3 for BC220 data.
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f3: The cytotoxicity of borrelidin is linked to the induction of the amino acid starvation response.(a–f) HUVEC cells grown in full serum media were exposed to the indicated concentrations of BN (a–c) or BC194 (d–f) and standardized to 0.05% DMSO. Cropped images from western blots of cell extracts were analyzed using antibodies recognizing phospho-eIF2α and cleaved-caspase 3 with β-tubulin as a loading control. Full images of blots can be found in Supplementary Figures S2a and S2b. Quantification of phospho-eIF2α and cleaved-caspase 3 for BN (b,c) and BC194 (e,f) relative to β-tubulin; mean ± SEM, n ≥ 3, *p < 0.05 relative to 0 nM (one-way ANOVA, Tukey Test). The apparent drop in the levels of phospho-eif2α as BC194 increased to 1000 nM and the exclusion of 1000 nM BN-treated data (designated by #) are due to the fact that endothelial cells exposed to high macrolide concentrations were visually apoptotic, making estimations of total protein loaded difficult. As such, we believe that the variability observed in these data at higher concentrations was more related to the severe status of the cells rather than a change in the amino acid starvation response. (g,h) Western blot (g) quantification of eif2α amounts relative to β-tubulin for both BN- and BC194-treated cells (100 nM) in the presence of various threonine concentrations (0–10 mM); mean ± SEM, n ≥ 3, *p < 0.05 relative to 0 mM threonine (one-way ANOVA, Tukey Test), #p < 0.01 between compounds at same threonine concentration (two-way ANOVA, Sidak Test). Full images of blots can be found in Supplementary Figure S2c. See also Supplementary Figure S1 for cell cycle, proliferation effects, and Supplementary Figure S3 for BC220 data.

Mentions: We hypothesized that the ability of the two compounds to induce cell cycle arrest is linked to how effectively each macrolide induces amino acid starvation. Accumulation of uncharged-tRNA within the cell arising from the inhibition of aminoacylation increases uncharged tRNA levels, which activates the EIFAK4 (GCN2) translational control kinase3940. Activation of EIFAK4 increases the phosphorylation of the downstream translational initiation factor eIF2α on Ser51, triggering additional ER stress and unfolded protein response pathways39. BN is known to de-repress expression of amino acid biosynthetic genes in yeast, a signature of GCN2 activity41. Particularly when coupled to the unfolded protein response, amino acid starvation leads to cell cycle arrest and, in severe cases, the initiation of apoptosis424344. To confirm that the anti-proliferative effects of BN are linked to AAS pathways in animal cells, cultured HUVECs were exposed to increasing concentrations of BN or BC194, and then lysates from these cultures were probed for the starvation and apoptosis markers phospo-eIF2α and cleaved-caspase 3, respectively (Fig. 3a,d, S2a,b). The phosphorylation of eIF2α was induced at concentrations as low as 10 nM BN (Fig. 3b) but at least 10-fold higher concentrations of BC194 were required to generate an equivalent response (Fig. 3e). Likewise, the appearance of cleaved-caspase 3 at 100 nM BN (Fig. 3c) compared to 1000 nM for BC194 (Fig. 3f) demonstrates the greater potency of BN relative to BC194 in inducing apoptosis. BN therefore blocks progression through the cell cycle and induces amino acid starvation and apoptosis at 10-fold lower concentrations than were required for BC194.


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)

The cytotoxicity of borrelidin is linked to the induction of the amino acid starvation response.(a–f) HUVEC cells grown in full serum media were exposed to the indicated concentrations of BN (a–c) or BC194 (d–f) and standardized to 0.05% DMSO. Cropped images from western blots of cell extracts were analyzed using antibodies recognizing phospho-eIF2α and cleaved-caspase 3 with β-tubulin as a loading control. Full images of blots can be found in Supplementary Figures S2a and S2b. Quantification of phospho-eIF2α and cleaved-caspase 3 for BN (b,c) and BC194 (e,f) relative to β-tubulin; mean ± SEM, n ≥ 3, *p < 0.05 relative to 0 nM (one-way ANOVA, Tukey Test). The apparent drop in the levels of phospho-eif2α as BC194 increased to 1000 nM and the exclusion of 1000 nM BN-treated data (designated by #) are due to the fact that endothelial cells exposed to high macrolide concentrations were visually apoptotic, making estimations of total protein loaded difficult. As such, we believe that the variability observed in these data at higher concentrations was more related to the severe status of the cells rather than a change in the amino acid starvation response. (g,h) Western blot (g) quantification of eif2α amounts relative to β-tubulin for both BN- and BC194-treated cells (100 nM) in the presence of various threonine concentrations (0–10 mM); mean ± SEM, n ≥ 3, *p < 0.05 relative to 0 mM threonine (one-way ANOVA, Tukey Test), #p < 0.01 between compounds at same threonine concentration (two-way ANOVA, Sidak Test). Full images of blots can be found in Supplementary Figure S2c. See also Supplementary Figure S1 for cell cycle, proliferation effects, and Supplementary Figure S3 for BC220 data.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f3: The cytotoxicity of borrelidin is linked to the induction of the amino acid starvation response.(a–f) HUVEC cells grown in full serum media were exposed to the indicated concentrations of BN (a–c) or BC194 (d–f) and standardized to 0.05% DMSO. Cropped images from western blots of cell extracts were analyzed using antibodies recognizing phospho-eIF2α and cleaved-caspase 3 with β-tubulin as a loading control. Full images of blots can be found in Supplementary Figures S2a and S2b. Quantification of phospho-eIF2α and cleaved-caspase 3 for BN (b,c) and BC194 (e,f) relative to β-tubulin; mean ± SEM, n ≥ 3, *p < 0.05 relative to 0 nM (one-way ANOVA, Tukey Test). The apparent drop in the levels of phospho-eif2α as BC194 increased to 1000 nM and the exclusion of 1000 nM BN-treated data (designated by #) are due to the fact that endothelial cells exposed to high macrolide concentrations were visually apoptotic, making estimations of total protein loaded difficult. As such, we believe that the variability observed in these data at higher concentrations was more related to the severe status of the cells rather than a change in the amino acid starvation response. (g,h) Western blot (g) quantification of eif2α amounts relative to β-tubulin for both BN- and BC194-treated cells (100 nM) in the presence of various threonine concentrations (0–10 mM); mean ± SEM, n ≥ 3, *p < 0.05 relative to 0 mM threonine (one-way ANOVA, Tukey Test), #p < 0.01 between compounds at same threonine concentration (two-way ANOVA, Sidak Test). Full images of blots can be found in Supplementary Figure S2c. See also Supplementary Figure S1 for cell cycle, proliferation effects, and Supplementary Figure S3 for BC220 data.
Mentions: We hypothesized that the ability of the two compounds to induce cell cycle arrest is linked to how effectively each macrolide induces amino acid starvation. Accumulation of uncharged-tRNA within the cell arising from the inhibition of aminoacylation increases uncharged tRNA levels, which activates the EIFAK4 (GCN2) translational control kinase3940. Activation of EIFAK4 increases the phosphorylation of the downstream translational initiation factor eIF2α on Ser51, triggering additional ER stress and unfolded protein response pathways39. BN is known to de-repress expression of amino acid biosynthetic genes in yeast, a signature of GCN2 activity41. Particularly when coupled to the unfolded protein response, amino acid starvation leads to cell cycle arrest and, in severe cases, the initiation of apoptosis424344. To confirm that the anti-proliferative effects of BN are linked to AAS pathways in animal cells, cultured HUVECs were exposed to increasing concentrations of BN or BC194, and then lysates from these cultures were probed for the starvation and apoptosis markers phospo-eIF2α and cleaved-caspase 3, respectively (Fig. 3a,d, S2a,b). The phosphorylation of eIF2α was induced at concentrations as low as 10 nM BN (Fig. 3b) but at least 10-fold higher concentrations of BC194 were required to generate an equivalent response (Fig. 3e). Likewise, the appearance of cleaved-caspase 3 at 100 nM BN (Fig. 3c) compared to 1000 nM for BC194 (Fig. 3f) demonstrates the greater potency of BN relative to BC194 in inducing apoptosis. BN therefore blocks progression through the cell cycle and induces amino acid starvation and apoptosis at 10-fold lower concentrations than were required for BC194.

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