The important anticancer drug Taxol (paclitaxel) binds to tubulin in a stoichiometric ratio and promotes its assembly into microtubules. The conformation of microtubule-bound drug has been the subject of intense study, and various suggestions have been made. In this work we present experimental and theoretical evidence that Taxol adopts a T-shaped conformation when it is bound to tubulin.
Metal response element-binding transcription factor-1 (MTF-1) is a six-zinc finger protein that plays an essential role in activating metallothionein expression in response to the heavy metals zinc and cadmium. Low affinity interactions between zinc and specific zinc fingers in MTF-1 reversibly regulate its binding to the metal response elements in the mouse metallothionein-I promoter. This study examined the subcellular distribution and DNA binding activity of MTF-1 in cells treated with zinc or cadmium. Immunoblot analysis of cytosolic and nuclear extracts demonstrated that in untreated cells, about 83% of MTF-1 is found in the cytosolic extracts and is not activated to bind to DNA. In sharp contrast, within 30 min of zinc treatment (100 microM), MTF-1 is detected only in nuclear extracts and is activated to bind to DNA. The activation to bind to DNA and nuclear translocation of MTF-1 occurs in the absence of increased MTF-1 content in the cell. Furthermore, immunocytochemical localization and immunoblotting assays demonstrated that zinc induces the nuclear translocation of MTF-1-FLAG, expressed from the cytomegalovirus promoter in transiently transfected dko7 (MTF-1 double knockout) cells. Immunoblot analysis of cytosolic and nuclear extracts from cadmium-treated cells demonstrated that concentrations of cadmium (10 microM) that actively induce metallothionein gene expression cause only a small increase in the amount of nuclear MTF-1. In contrast, an overtly toxic concentration of cadmium (50 microM) rapidly induced the complete nuclear translocation and activation of DNA binding activity of MTF-1. These studies are consistent with the hypothesis that MTF-1 serves as a zinc sensor that responds to changes in cytosolic free zinc concentrations. In addition, these data suggest that cadmium activation of metallothionein gene expression may be accompanied by only small changes in nuclear MTF-1.
The important anticancer drug Taxol ® (paclitaxel, PTX) owes its unique activity to its ability to bind to tubulin in a stoichiometric ratio and promote its assembly into microtubules. The conformation of the microtubule-bound drug has been the focus of numerous research efforts, since the inability of polymerized tubulin to form crystals precludes structure proof by X-ray crystallography. Likewise, although the αβ-tubulin dimer structure has been solved by electron crystallography, the 3.7 Å resolution is too low to permit direct determination of either ligand conformation or binding pose. In this study we present experimental results from 2 H{ 19 F} REDOR NMR that provide direct confirmation that paclitaxel adopts a T-shaped conformation when it is bound to tubulin.
The important anticancer drug paclitaxel binds to the -subunit of the R -tubulin dimer in the microtubule in a stoichiometric ratio, promoting microtubule polymerization and stability. The conformation of microtubule-bound drug has been the subject of intense study, and various suggestions have been proposed. In previous work we presented experimental and theoretical evidence that paclitaxel adopts a T-shaped conformation when it is bound to tubulin. In this study we report additional experimental data and calculations that delineate the allowable parameters for effective paclitaxel-tubulin interactions.
A fluorescent probe has been attached to the carboxy terminus of the α-subunit of α,β-tubulin by an enzymatic reaction followed by a chemical reaction. The unnatural amino acid 3-formyltyrosine is attached to the carboxy terminus of α-tubulin through the use of the enzyme tubulin tyrosine ligase. The aromatic aldehyde of the unnatural amino acid serves as an orthogonal electrophile that specifically reacts with a fluorophore containing an aromatic hydrazine functional group, which in this case is 7-hydrazino-4-methyl coumarin. Conditions for covalent bond formation between the unnatural amino acid and the fluorophore are mild, allowing fluorescently labeled tubulin to retain its ability to assemble into microtubules. A key feature of the labeling reaction is that it produces a red shift in the fluorophore's absorption and emission maxima, accompanied by an increase in its quantum yield; thus, fluorescently labeled protein can be observed in the presence of unreacted fluorophore. Both the enzymatic and coupling reaction can occur in living cells. The approach presented here should be applicable to a wide variety of in vitro systems.
Tubulin, the basic component of microtubules, is present in most eukaryotic cells as multiple gene products, called isotypes. The major tubulin isotypes are highly conserved in terms of structure and drug binding capabilities. The tubulin isotype βVI, however, is significantly divergent from the other isotypes in sequence, assembly properties and function. It is the major β-tubulin isotype of hematopoietic tissue and forms the microtubules of platelet marginal bands. The interaction of the major tubulin isotypes βI, βII, βIII and βIV with antimicrotubule drugs has been widely studied, but little is known about the drug binding properties of tubulin isotype βVI. In this investigation, we characterized the activity of various colchicine-site ligands with tubulin isolated from Gallus gallus erythrocytes (CeTb), which is ~95% βVI. Colchicine binding is thought to be a universal property of higher eukaryotic tubulin; however, we were unable to detect colchicine binding to CeTb under any experimental conditions. Podophyllotoxin and nocodazole, other colchicine-site ligands with divergent structures, were able to inhibit paclitaxel-induced CeTb assembly. Surprisingly, the colchicine isomer allocolchicine also inhibited CeTb assembly and displayed measurable, moderate affinity for CeTb (Ka = 0.18 × 10 5 M −1 vs. 5.0 × 10 5 M −1 for bovine brain tubulin). Since allocolchicine and colchicine differ in their C ring structures, the two C-ring colchicine analogues were also tested for CeTb binding. Kinetic experiments indicate that thiocolchicine and chlorocolchicine bind to CeTb, but very slowly and with low affinity. Molecular modeling of CeTb identified five divergent amino acid residues within 6 Å of the colchicine binding site compared to βI, βII, and βIV; three of these amino acids are also altered in βIII-tubulin. Interestingly, the altered amino acids are in the vicinity of the A ring region of the colchicine binding site rather than the C ring region. We propose that the amino acid differences in the binding site constrict the A ring binding domain in CeTb, which interferes with the positioning of the trimethoxyphenyl A ring and prevents C ring binding site interactions from efficiently occurring. Allocolchicine is able to accommodate the altered binding mode because of its smaller ring size and more flexible C ring substituents. The sequence of the colchicine binding domain of CeTb βVI-isotype is almost identical to that of it human hematopoietic counterpart. Thus, through analysis of the interactions of ligands with CeTb, it may be possible to discover colchicine site ligands that specifically target tubulin in human hematopoietic cells. † Financial support was provided by the National Institutes Health (Grant No. CA-69571).*Corresponding Author: Phone: 607-777-2927. Fax: 607-777-4478., sbane@binghamton.edu. SUPPORTING INFORMATION AVAILABLE The inhibition of paclitaxel induced CeTb assembly by allocolchicine, an illustration of three dimensional alignment of tubulin-bound colchicine and podophyllotoxin, an...
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