Limits...
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

Exposure to BN and BC194 results in vascular defects and mis-patterning.(a) Representative confocal images (20x magnification) of zebrafish ISVs at 24 and 48 hpe. Zebrafish embryos were incubated in egg water at 25 °C until 8–12 somites of age. The embryos were then manually dechorionated and incubated in egg water containing the indicated compounds at 28.5 °C until the images were taken. Calibration bar represents 100 μm. (b,c) Quantification of ectopic branching (b) and aberrant patterning (c) of ISVs. Embryos were incubated with the indicated compounds (5 μM) or DMSO until images were taken by fluorescent microscopy at 24 and 48 hpe. Aberrant structures were then counted within a region encompassing 5 ISVs anterior and posterior from the end of the yolk extension; mean ± SEM, n ≥ 8, #*p < 0.0001 relative to DMSO at 24 and 48 hpe respectively (one-way ANOVA, Tukey Test). ‡BN-treated fish rarely survived to 48 hpe. (d) RT-qPCR values for the expression amino acid starvation response genes asns, gpt2, and eif4ebp1. Dechorionated embryos (15 somites) were incubated in egg water containing the indicated compounds for 24 hours. Total RNA was extracted by trizol/chloroform and the expression levels relative to actb were determined using the ΔΔCT method; mean ± SEM, n = 3, ****p < 0.0001 relative to DMSO (two-way ANOVA, Tukey Test). See Supplementary Figure S7 for physiological and toxicity information.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4536658&req=5

f5: Exposure to BN and BC194 results in vascular defects and mis-patterning.(a) Representative confocal images (20x magnification) of zebrafish ISVs at 24 and 48 hpe. Zebrafish embryos were incubated in egg water at 25 °C until 8–12 somites of age. The embryos were then manually dechorionated and incubated in egg water containing the indicated compounds at 28.5 °C until the images were taken. Calibration bar represents 100 μm. (b,c) Quantification of ectopic branching (b) and aberrant patterning (c) of ISVs. Embryos were incubated with the indicated compounds (5 μM) or DMSO until images were taken by fluorescent microscopy at 24 and 48 hpe. Aberrant structures were then counted within a region encompassing 5 ISVs anterior and posterior from the end of the yolk extension; mean ± SEM, n ≥ 8, #*p < 0.0001 relative to DMSO at 24 and 48 hpe respectively (one-way ANOVA, Tukey Test). ‡BN-treated fish rarely survived to 48 hpe. (d) RT-qPCR values for the expression amino acid starvation response genes asns, gpt2, and eif4ebp1. Dechorionated embryos (15 somites) were incubated in egg water containing the indicated compounds for 24 hours. Total RNA was extracted by trizol/chloroform and the expression levels relative to actb were determined using the ΔΔCT method; mean ± SEM, n = 3, ****p < 0.0001 relative to DMSO (two-way ANOVA, Tukey Test). See Supplementary Figure S7 for physiological and toxicity information.

Mentions: The angiogenesis-related effects of these compounds were assessed by exposing the embryos to 5 μM concentrations of the various macrolides, followed by screening using fluorescence microscopy for development defects in trunk inter-segmental vessels (ISVs) (Fig. 5a). Significant patterning defects were observed in response to either BN or BC194 treatment. Ectopic branches readily formed in the ISVs of embryos exposed to 5 μM BC194 for 48 hpe, but not in those of BN-treated embryos (Fig. 5b). By contrast, the ISVs in BN-exposed fish were often truncated or incomplete at both 24 and 48 hpe (Fig. 5c). Significantly, these effects were not observed when embryos were treated with the control BC220 macrolide or the DMSO carrier (Fig. 5a–c). One explanation for the diminished toxicity of BC194 relative to BN is that, as seen with in animal cell culture, BC194 has a reduced ability to induce amino acid starvation in the context of a whole animal model. To test this hypothesis, quantitative real time PCR was performed on total RNA prepared from embryos treated with equivalent (5 μM) concentrations of BN, BC194, and BC220, and the DMSO vehicle. The ATF4 target genes asns, gpt2, and eif4ebp1 served as AAS markers43. Consistent with our model, BN caused a significant increase in the expression of the markers asns and gpt2, while BC194 did not (Fig. 5d). Thus, BC194’s induction of ectopic ISV formation in the zebrafish is not dependent on the induction of the amino acid starvation response.


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)

Exposure to BN and BC194 results in vascular defects and mis-patterning.(a) Representative confocal images (20x magnification) of zebrafish ISVs at 24 and 48 hpe. Zebrafish embryos were incubated in egg water at 25 °C until 8–12 somites of age. The embryos were then manually dechorionated and incubated in egg water containing the indicated compounds at 28.5 °C until the images were taken. Calibration bar represents 100 μm. (b,c) Quantification of ectopic branching (b) and aberrant patterning (c) of ISVs. Embryos were incubated with the indicated compounds (5 μM) or DMSO until images were taken by fluorescent microscopy at 24 and 48 hpe. Aberrant structures were then counted within a region encompassing 5 ISVs anterior and posterior from the end of the yolk extension; mean ± SEM, n ≥ 8, #*p < 0.0001 relative to DMSO at 24 and 48 hpe respectively (one-way ANOVA, Tukey Test). ‡BN-treated fish rarely survived to 48 hpe. (d) RT-qPCR values for the expression amino acid starvation response genes asns, gpt2, and eif4ebp1. Dechorionated embryos (15 somites) were incubated in egg water containing the indicated compounds for 24 hours. Total RNA was extracted by trizol/chloroform and the expression levels relative to actb were determined using the ΔΔCT method; mean ± SEM, n = 3, ****p < 0.0001 relative to DMSO (two-way ANOVA, Tukey Test). See Supplementary Figure S7 for physiological and toxicity information.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Exposure to BN and BC194 results in vascular defects and mis-patterning.(a) Representative confocal images (20x magnification) of zebrafish ISVs at 24 and 48 hpe. Zebrafish embryos were incubated in egg water at 25 °C until 8–12 somites of age. The embryos were then manually dechorionated and incubated in egg water containing the indicated compounds at 28.5 °C until the images were taken. Calibration bar represents 100 μm. (b,c) Quantification of ectopic branching (b) and aberrant patterning (c) of ISVs. Embryos were incubated with the indicated compounds (5 μM) or DMSO until images were taken by fluorescent microscopy at 24 and 48 hpe. Aberrant structures were then counted within a region encompassing 5 ISVs anterior and posterior from the end of the yolk extension; mean ± SEM, n ≥ 8, #*p < 0.0001 relative to DMSO at 24 and 48 hpe respectively (one-way ANOVA, Tukey Test). ‡BN-treated fish rarely survived to 48 hpe. (d) RT-qPCR values for the expression amino acid starvation response genes asns, gpt2, and eif4ebp1. Dechorionated embryos (15 somites) were incubated in egg water containing the indicated compounds for 24 hours. Total RNA was extracted by trizol/chloroform and the expression levels relative to actb were determined using the ΔΔCT method; mean ± SEM, n = 3, ****p < 0.0001 relative to DMSO (two-way ANOVA, Tukey Test). See Supplementary Figure S7 for physiological and toxicity information.
Mentions: The angiogenesis-related effects of these compounds were assessed by exposing the embryos to 5 μM concentrations of the various macrolides, followed by screening using fluorescence microscopy for development defects in trunk inter-segmental vessels (ISVs) (Fig. 5a). Significant patterning defects were observed in response to either BN or BC194 treatment. Ectopic branches readily formed in the ISVs of embryos exposed to 5 μM BC194 for 48 hpe, but not in those of BN-treated embryos (Fig. 5b). By contrast, the ISVs in BN-exposed fish were often truncated or incomplete at both 24 and 48 hpe (Fig. 5c). Significantly, these effects were not observed when embryos were treated with the control BC220 macrolide or the DMSO carrier (Fig. 5a–c). One explanation for the diminished toxicity of BC194 relative to BN is that, as seen with in animal cell culture, BC194 has a reduced ability to induce amino acid starvation in the context of a whole animal model. To test this hypothesis, quantitative real time PCR was performed on total RNA prepared from embryos treated with equivalent (5 μM) concentrations of BN, BC194, and BC220, and the DMSO vehicle. The ATF4 target genes asns, gpt2, and eif4ebp1 served as AAS markers43. Consistent with our model, BN caused a significant increase in the expression of the markers asns and gpt2, while BC194 did not (Fig. 5d). Thus, BC194’s induction of ectopic ISV formation in the zebrafish is not dependent on the induction of the amino acid starvation response.

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