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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

TARS is involved in vascular development in the zebrafish.(a) Representative confocal images (20× magnification) of control and TARS morphant zebrafish ISVs at 24 and 48 hpe. Embryos were injected with a TARS morpholino (1.5 μM) at the one to four cell stage. Fish were manually dechorionated at 24 h after fertilization and imaged at 24 and 48 hpe. Arrows and asterisks denote the location of ectopic branches and missing/incomplete vessels respectively. (b,c) Quantification of ectopic branching (b) and missing/incomplete ISVs (c) from control and morphant zebrafish within a region of encompassing five ISVs anterior and posterior to the yolk extension; mean ± SEM, n ≥ 29, #*p < 0.0001 relative to uninjected controls at 24 and 48 hpe respectively (one-way ANOVA, Tukey Test). See Supplementary Figure S10 for morpholino validation and morphological effects.
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f6: TARS is involved in vascular development in the zebrafish.(a) Representative confocal images (20× magnification) of control and TARS morphant zebrafish ISVs at 24 and 48 hpe. Embryos were injected with a TARS morpholino (1.5 μM) at the one to four cell stage. Fish were manually dechorionated at 24 h after fertilization and imaged at 24 and 48 hpe. Arrows and asterisks denote the location of ectopic branches and missing/incomplete vessels respectively. (b,c) Quantification of ectopic branching (b) and missing/incomplete ISVs (c) from control and morphant zebrafish within a region of encompassing five ISVs anterior and posterior to the yolk extension; mean ± SEM, n ≥ 29, #*p < 0.0001 relative to uninjected controls at 24 and 48 hpe respectively (one-way ANOVA, Tukey Test). See Supplementary Figure S10 for morpholino validation and morphological effects.

Mentions: Previous work demonstrated that point mutations in TARS can block the anti-angiogenic effects of BC194, thereby indicating that TARS is the specific target of BC19419. To confirm that the vessel patterning effects of BN and BC194 in zebrafish are a direct consequence of action on TARS, we reduced the levels of functional TARS by some 62% using a splice altering antisense morpholino oligonucleotide (MO) (Fig. S10a–c). Consistent with our hypothesis, morphant fish developed with significantly more ectopic branches and incomplete vessels than uninjected controls (Fig. 6a–c). Phenotypically, these effects on the vasculature were intermediate with respect to the previously shown (Fig. 5a) effects of BN and BC194. Notably, the TARS morphant fish did not exhibit the severe body morphology associated with BN toxicity (Fig. S10d). In summary, these results demonstrate that vascular development in the zebrafish is highly sensitive to alterations in the activity of TARS, induced either as a result of chemical inhibition or altered gene expression. We conclude that TARS has a direct role in vertebrate vascular development.


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)

TARS is involved in vascular development in the zebrafish.(a) Representative confocal images (20× magnification) of control and TARS morphant zebrafish ISVs at 24 and 48 hpe. Embryos were injected with a TARS morpholino (1.5 μM) at the one to four cell stage. Fish were manually dechorionated at 24 h after fertilization and imaged at 24 and 48 hpe. Arrows and asterisks denote the location of ectopic branches and missing/incomplete vessels respectively. (b,c) Quantification of ectopic branching (b) and missing/incomplete ISVs (c) from control and morphant zebrafish within a region of encompassing five ISVs anterior and posterior to the yolk extension; mean ± SEM, n ≥ 29, #*p < 0.0001 relative to uninjected controls at 24 and 48 hpe respectively (one-way ANOVA, Tukey Test). See Supplementary Figure S10 for morpholino validation and morphological effects.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: TARS is involved in vascular development in the zebrafish.(a) Representative confocal images (20× magnification) of control and TARS morphant zebrafish ISVs at 24 and 48 hpe. Embryos were injected with a TARS morpholino (1.5 μM) at the one to four cell stage. Fish were manually dechorionated at 24 h after fertilization and imaged at 24 and 48 hpe. Arrows and asterisks denote the location of ectopic branches and missing/incomplete vessels respectively. (b,c) Quantification of ectopic branching (b) and missing/incomplete ISVs (c) from control and morphant zebrafish within a region of encompassing five ISVs anterior and posterior to the yolk extension; mean ± SEM, n ≥ 29, #*p < 0.0001 relative to uninjected controls at 24 and 48 hpe respectively (one-way ANOVA, Tukey Test). See Supplementary Figure S10 for morpholino validation and morphological effects.
Mentions: Previous work demonstrated that point mutations in TARS can block the anti-angiogenic effects of BC194, thereby indicating that TARS is the specific target of BC19419. To confirm that the vessel patterning effects of BN and BC194 in zebrafish are a direct consequence of action on TARS, we reduced the levels of functional TARS by some 62% using a splice altering antisense morpholino oligonucleotide (MO) (Fig. S10a–c). Consistent with our hypothesis, morphant fish developed with significantly more ectopic branches and incomplete vessels than uninjected controls (Fig. 6a–c). Phenotypically, these effects on the vasculature were intermediate with respect to the previously shown (Fig. 5a) effects of BN and BC194. Notably, the TARS morphant fish did not exhibit the severe body morphology associated with BN toxicity (Fig. S10d). In summary, these results demonstrate that vascular development in the zebrafish is highly sensitive to alterations in the activity of TARS, induced either as a result of chemical inhibition or altered gene expression. We conclude that TARS has a direct role in vertebrate vascular development.

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