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Tubulin from Hey cells by reverse-phase HPLC coupled to ESI ion-trap MS is analyzed to confirm that the sequence of human betaV-tubulin entered in the NCBI protein database is correct beyond the C- terminal sequence. The results demonstrate that the human betaV-tubulin sequence present in the NCBI protein database is effectively expressed at the protein level in epithelial cells.

The liquid chromotography/electrospray ionization mass spectrometry analysis of native tubulins in Taxol-stabilized microtubules from parental and Taxol/epothilone-resistant human cancer cell lines are described. This method allows the direct determination of tubulin isotype composition, including post-translational modifications and mutations occurring throughout the entire protein.

Research delineates expression of tubulin mutations at the protein level and their role in drug resistance. Results indicate that in the cell lines exhibiting resistance to microtubule-stabilizing drugs such as taxol, betaIII-tubulin expression is observed, which represents tubulin isotype-related resistance marker.

The antitumor drug Taxol stabilizes microtubules and reduces their dynamicity, promoting mitotic arrest and cell death. Upon assembly of the α/β-tubulin heterodimer, GTP bound to β-tubulin is hydrolyzed to GDP reaching a steady-state equilibrium between free tubulin dimers and microtubules. The binding of Taxol to β-tubulin in the polymer results in cold-stable microtubules at the expense of tubulin dimers, even in the absence of exogenous GTP. However, there is little biochemical insight into the mechanism(s) by which Taxol stabilizes microtubules. Here, we analyze the structural changes occurring in both β- and α-tubulin upon microtubule stabilization by Taxol. Hydrogen/deuterium exchange (HDX) coupled to liquid chromatography-electrospray ionization MS demonstrated a marked reduction in deuterium incorporation in both β-and α-tubulin when Taxol was present. Decreased local HDX in peptic peptides was mapped on the tubulin structure and revealed both expected and new dimer-dimer interactions. The increased rigidity in Taxol microtubules was distinct from and complementary to that due to GTP-induced polymerization. The Taxol-induced changes in tubulin conformation act against microtubule depolymerization in a precise directional way. These results demonstrate that HDX coupled to liquid chromatography-electrospray ionization MS can be effectively used to study conformational effects induced by small ligands on microtubules. The present study also opens avenues for locating drug and protein binding sites and for deciphering the mechanisms by which their interactions alter the conformation of microtubules and tubulin dimers. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is an apicomplexan of both medical and veterinary importance which is classified as an NIH Category B priority pathogen. It is best known for its ability to cause congenital infection in immune competent hosts and encephalitis in immune compromised hosts. The highly stable and specialized microtubule-based cytoskeleton participates in the invasion process. The genome encodes three isoforms of both alpha- and beta-tubulin and we show that the tubulin is extensively altered by specific post-translational modifications (PTMs) in this paper. T. gondii tubulin PTMs were analyzed by mass spectrometry and immunolabeling using specific antibodies. The PTMs identified on alpha-tubulin included acetylation of Lys40, removal of the last C-terminal amino acid residue Tyr453 (detyrosinated tubulin) and truncation of the last five amino acid residues. Polyglutamylation was detected on both alpha- and beta-tubulins. An antibody directed against mammalian alpha-tubulin lacking the last two C-terminal residues (Delta2-tubulin) labeled the apical region of this parasite. Detyrosinated tubulin was diffusely present in subpellicular microtubules and displayed an apparent accumulation at the basal end. Methylation, a PTM not previously described on tubulin, was also detected. Methylated tubulins were not detected in the host cells, human foreskin fibroblasts, suggesting that this may be a modification specific to the Apicomplexa.