Date of Completion

2017

Thesis Type

College of Arts and Science Honors

Department

Biochemistry

First Advisor

Karen Lounsbury

Second Advisor

Christopher Francklyn

Keywords

Threonyl-tRNA Synthetase, Biochemistry, Pharmacology, Borrelidin, endothelial cell migration, ovarian cancer

Abstract

Aminoacyl-tRNA synthetases (aaRs) have been known for their importance in protein synthesis. However, the Francklyn/Lounsbury lab discovered that some aaRs, like tyrosyl-tRNA synthetase and tryptophanyl-tRNA synthetase are linked to angiogenesis. Recently, the lab discovered that threonyl-tRNA synthetase (TARS) was an angiogenic factor that is linked to ovarian cancer. Studies have shown that a derivative of macrolide inhibitor Borrelidin, known as BC194, inhibits TARS angiogenic and functional activity and altering TARS conformation. Sites of BC19 were found and used to generate mutated version of human TARS. Q566W and H388A TARS mutations were investigated to see whether induced conformational change or loss of aminoacyl-transferase activity is the key to inhibit angiogenic activity respectively. Q566W and H388A plasmids were generated using site-directed mutagenesis. The plasmids were expressed in Escherichia coli cells and purified using sonication, protamine sulfate salting, His6tag affinity chromatography and Hydroxyapatite chromatography. Purified mutants were found to have a molecular weight of 83kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Following purification, TARS enzymes were applied to the in vitro tube assay using human umbilical vein endothelial cells to test angiogenic activity. Q566W promoted a similar number of pieces and branches as wild type TARS, while H388A had the greatest amount tube pieces, branches, and branching length. The study didn’t find a difference in angiogenic activity between the wild type and mutant TARS. However, the study shows that TARS has dose-dependent angiogenic activity and may link to α5β3 intergin migration signaling. More studies on angiogenic activity using integrin inhibitor dose response in vitro tube assay under TARS are needed to verify this mechanism.

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