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Proc Natl Acad Sci U S A. 2016 Oct 4;113(40):11294-11299. Epub 2016 Sep 20.

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

Author information

1
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461; Department of Obstetrics and Gynecology and Women's Health, Division of Gynecologic Oncology, Albert Einstein College of Medicine, Bronx, NY 10461; chia-ping.h.yang@einstein.yu.edu susan.horwitz@einstein.yu.edu.
2
Department of System and Computational Biology, Albert Einstein College of Medicine, Bronx, NY 10461.
3
Laboratory of Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine, Bronx, NY 10461.
4
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461; chia-ping.h.yang@einstein.yu.edu susan.horwitz@einstein.yu.edu.

Abstract

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 or in the presence of MSAs. The inhibitory effects elicited by these MSAs on photolabeling were distinct for β-tubulin from different sources. To determine the exact amount of drug that binds to different β-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 β-tubulin isotype determined. It was found that compared with other β-tubulin isotypes, βIII-tubulin bound the least amount of 2-(m-azidobenzoyl)taxol. Analysis of the sequences of β-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 βIII-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 β-tubulins. Our results indicate that the difference in residue 218 in βIII-tubulin may be responsible for inhibition of drug binding to this isotype, which could influence downstream cellular events.

KEYWORDS:

Taxol; drug binding; microtubule stabilizing agents; photolabeling; tubulin isotypes

PMID:
27651486
PMCID:
PMC5056068
DOI:
10.1073/pnas.1613286113
[Indexed for MEDLINE]
Free PMC Article

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