The competition between the microtubule-stabilizing drug paclitaxel and tubulin acetyltransferases

Abstract

Microtubules are polymers of heterodimers (α-tubulin and β-tubulin) that form hollow tubes and act as major components of the eukaryotic cytoskeleton. Microtubules undergo post-translational modifications that may impact the structure and properties, such as stability and rigidity. Catalyzed by tubulin acetyltransferases, α-tubulin acetylation occurs in the hollow lumen of the microtubule and acts as a marker of stable cellular microtubules. To stabilize microtubules for in vitro studies, the chemotherapeutic drug paclitaxel is used as it binds to β-tubulin and prevents the depolymerization of microtubules. The sensitivity of cancer cells to paclitaxel was observed to be correlated to α-tubulin acetylation levels, yet the drug’s effect on acetyltransferase activity is uncertain. Through the preliminary structural analysis of the tubulin acetyltransferases’ binding pattern on microtubules by Dr. Ti’s lab, the enzymes were seen to share the same binding site as paclitaxel on β-tubulin. This project aims to illustrate the direct competition between paclitaxel and tubulin acetyltransferases. In this study, the presence of paclitaxel is hypothesized to reduce the frequency of tubulin acetyltransferases from binding to microtubules, thereby reducing the overall acetylation level. Recombinant tubulin and tubulin acetyltransferases were generated for in vitro reconstitution assays. TIRF microscopy was employed to visualize the change in acetyltransferase binding affinity in the presence of paclitaxel. Paclitaxel was observed to have increased the dissociation rate of tubulin acetyltransferases from microtubules. Furthermore, cancer cell-based assays were performed to assess endogenous acetyltransferase expression and acetylation levels. While the presence of paclitaxel was initially observed to have increased the cellular α-tubulin acetylation, the overall level eventually decreased at increased concentrations of paclitaxel. The expression levels of tubulin acetyltransferase were assessed, and no correlation was observed between the enzyme expression levels and changes in α-tubulin acetylation. Taken together, this study illustrated the impact of paclitaxel on tubulin acetyltransferase activity. The presence of paclitaxel had resulted in an increased dissociation rate of tubulin acetyltransferases and by extension, decreased the α-tubulin acetylation level. These results may provide the molecular basis for future dissection of the biological functions of tubulin acetyltransferases, as well as further understanding of the effects of paclitaxel.