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2-(m-Azidobenzoyl)taxol binds differentially to distinct ?-tubulin isotypes.

ABSTRACT: There are seven ?-tubulin isotypes present in distinct quantities in mammalian cells of different origin. Altered expression of ?-tubulin isotypes has been reported in cancer cell lines resistant to microtubule stabilizing agents (MSAs) and in human tumors resistant to Taxol. To study the relative binding affinities of MSAs, tubulin from different sources, with distinct ?-tubulin isotype content, were specifically photolabeled with a tritium-labeled Taxol analog, 2-(m-azidobenzoyl)taxol, alone or in the presence of MSAs. The inhibitory effects elicited by these MSAs on photolabeling were distinct for ?-tubulin from different sources. To determine the exact amount of drug that binds to different ?-tubulin isotypes, bovine brain tubulin was photolabeled and the isotypes resolved by high-resolution isoelectrofocusing. All bands were analyzed by mass spectrometry following cyanogen bromide digestion, and the identity and relative quantity of each ?-tubulin isotype determined. It was found that compared with other ?-tubulin isotypes, ?III-tubulin bound the least amount of 2-(m-azidobenzoyl)taxol. Analysis of the sequences of ?-tubulin near the Taxol binding site indicated that, in addition to the M-loop that is known to be involved in drug binding, the leucine cluster region of ?III-tubulin contains a unique residue, alanine, at 218, compared with other isotypes that contain threonine. Molecular dynamic simulations indicated that the frequency of Taxol-accommodating conformations decreased dramatically in the T218A variant, compared with other ?-tubulins. Our results indicate that the difference in residue 218 in ?III-tubulin may be responsible for inhibition of drug binding to this isotype, which could influence downstream cellular events.

SUBMITTER: Yang CH

PROVIDER: S-EPMC5056068 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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2-(m-Azidobenzoyl)taxol binds differentially to distinct β-tubulin isotypes.

Yang Chia-Ping Huang CH Yap Eng-Hui EH Xiao Hui H Fiser Andras A Horwitz Susan Band SB

Proceedings of the National Academy of Sciences of the United States of America 20160920 40

There are seven β-tubulin isotypes present in distinct quantities in mammalian cells of different origin. Altered expression of β-tubulin isotypes has been reported in cancer cell lines resistant to microtubule stabilizing agents (MSAs) and in human tumors resistant to Taxol. To study the relative binding affinities of MSAs, tubulin from different sources, with distinct β-tubulin isotype content, were specifically photolabeled with a tritium-labeled Taxol analog, 2-(m-azidobenzoyl)taxol, alone o ...[more]

PMID: 27651486

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Project description:BackgroundThe multi-tubulin hypothesis proposes that each tubulin isotype performs a unique role, or subset of roles, in the universe of microtubule function(s). To test this hypothesis, we are investigating the functions of the recently discovered, noncanonical β-like tubulins (BLTs) of the ciliate, Tetrahymena thermophila. Tetrahymena forms 17 distinct microtubular structures whose assembly had been thought to be based on single α- and β-isotypes. However, completion of the macronuclear genome sequence of Tetrahymena demonstrated that this ciliate possessed a β-tubulin multigene family: two synonymous genes (BTU1 and BTU2) encode the canonical β-tubulin, BTU2, and six genes (BLT1-6) yield five divergent β-tubulin isotypes. In this report, we examine the structural features and functions of two of the BLTs (BLT1 and BLT4) and compare them to those of BTU2.Methodology/principal findingsWith respect to BTU2, BLT1 and BLT4 had multiple sequence substitutions in their GTP-binding sites, in their interaction surfaces, and in their microtubule-targeting motifs, which together suggest that they have specialized functions. To assess the roles of these tubulins in vivo, we transformed Tetrahymena with expression vectors that direct the synthesis of GFP-tagged versions of the isotypes. We show that GFP-BLT1 and GFP-BLT4 were not detectable in somatic cilia and basal bodies, whereas GFP-BTU2 strongly labeled these structures. During cell division, GFP-BLT1 and GFP-BLT4, but not GFP-BTU2, were incorporated into the microtubule arrays of the macronucleus and into the mitotic apparatus of the micronucleus. GFP-BLT1 also participated in formation of the microtubules of the meiotic apparatus of the micronucleus during conjugation. Partitioning of the isotypes between nuclear and ciliary microtubules was confirmed biochemically.Conclusion/significanceWe conclude that Tetrahymena uses a family of distinct β-tubulin isotypes to construct subsets of functionally different microtubules, a result that provides strong support for the multi-tubulin hypothesis.

Dataset Information

2-(m-Azidobenzoyl)taxol binds differentially to distinct ?-tubulin isotypes.

Publications

2-(m-Azidobenzoyl)taxol binds differentially to distinct β-tubulin isotypes.

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