In Vitro Effect of Cryptophycin 52 on Microtubule Assembly and Tubulin: Molecular Modeling of the Mechanism of Action of a New Antimitotic Drug

Barbier, Pascale; Grégoire, Catherine; Devred, François; Sarrazin, M.; Peyrot, Vincent

doi:10.1021/bi010926z

Cited by 48 publications

(52 citation statements)

References 33 publications

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“…Ligands of the vinblastine domain bind as wedges at the interdimer tubulin interface in T 2 RB3 (65) and induce self-association of tubulin dimers leading to spirals (66) or single rings (67,68). Their curvature radii are compatible with the formation of tubulin complexes with the long C-terminal ␣-helix of SLDs (15,65).…”

Section: Discussionmentioning

confidence: 99%

Stathmin and Interfacial Microtubule Inhibitors Recognize a Naturally Curved Conformation of Tubulin Dimers

Barbier

Dorléans

Devred

et al. 2010

Journal of Biological Chemistry

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Tubulin is able to switch between a straight microtubule-like structure and a curved structure in complex with the stathminlike domain of the RB3 protein (T 2 RB3). GTP hydrolysis following microtubule assembly induces protofilament curvature and disassembly. The conformation of the labile tubulin heterodimers is unknown. One important question is whether free GDP-tubulin dimers are straightened by GTP binding or if GTP-tubulin is also curved and switches into a straight conformation upon assembly. We have obtained insight into the bending flexibility of tubulin by analyzing the interplay of tubulinstathmin association with the binding of several small molecule inhibitors to the colchicine domain at the tubulin intradimer interface, combining structural and biochemical approaches. The crystal structures of T 2 RB3 complexes with the chiral R and S isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl-carbamate, show that their binding site overlaps with colchicine ring A and that both complexes have the same curvature as unliganded T 2 RB3. The binding of these ligands is incompatible with a straight tubulin structure in microtubules. Analytical ultracentrifugation and binding measurements show that tubulin-stathmin associations (T 2 RB3, T 2 Stath) and binding of ligands (R, S, TN-16, or the colchicine analogue MTC) are thermodynamically independent from one another, irrespective of tubulin being bound to GTP or GDP. The fact that the interfacial ligands bind equally well to tubulin dimers or stathmin complexes supports a bent conformation of the free tubulin dimers. It is tempting to speculate that stathmin evolved to recognize curved structures in unassembled and disassembling tubulin, thus regulating microtubule assembly.Microtubules are essential for eukaryotic chromosome segregation, cellular architecture and intracellular trafficking, among other processes. Understanding microtubule dynamics, regulation, and organization requires knowledge of the nucleotide-regulated assembly switch of tubulin. Microtubules are hollow cylinders made of protofilaments of ␣␤-tubulin dimers in head to tail association, forming a pseudohelical lattice (1). The functional assembly-disassembly cycle of a ␣␤-tubulin molecule includes activation by GTP binding at the ␤-subunit, polymerization into microtubules, GTP hydrolysis at the ␤-␣ interdimer interface and depolymerization of GDP-tubulin, followed by replacement by GTP. Vectorial polymerization and GTP hydrolysis combine with tubulin structural plasticity in microtubule dynamics (2). Depolymerizing microtubule ends show characteristic curled protofilaments, whereas relatively straight sheets form at growing ends (3, 4). GDP-tubulin does not assemble into microtubules, but forms double rings (5), which also form upon microtubule depolymerization (6) and correspond to curved microtubule protofilaments (7,8). The tendency of GDP-tubulin to curve is thought to strain the microtubule lattice, causing disassembly when the terminal cap of GTP-bound tubulin...

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Section: Discussionmentioning

confidence: 99%

Stathmin and Interfacial Microtubule Inhibitors Recognize a Naturally Curved Conformation of Tubulin Dimers

Barbier

Dorléans

Devred

et al. 2010

Journal of Biological Chemistry

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“…We should also note the recent modeling study of Barbier et al (54), in which cryptophycin 52 was modeled into the ␣␤-heterodimer and into the ring oligomers that the drugtubulin complex forms. Although the model proposed by these workers placed cryptophycin 52 in the ϩ end binding domain, the depsipeptide was not as close to the exchangeable site as the model shown in Fig.…”

Section: Treatment 2)mentioning

confidence: 94%

“…The only interaction the two models seem to have in common is of a phenyl ring of the drug with Tyr-222, but even this involves different phenyl rings of the cryptophycins. Although the modeling methods used by Barbier et al (54) do not differ greatly from those used here, nuances in methodology clearly affect the precise orientation selected by the computational methods used to obtain docking results.…”

Section: Treatment 2)mentioning

confidence: 96%

Direct Photoaffinity Labeling by Dolastatin 10 of the Amino-terminal Peptide of β-Tubulin Containing Cysteine 12

Bai

Covell

Taylor

et al. 2004

Journal of Biological Chemistry

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The subunit protein of microtubules, the ␣␤-tubulin heterodimer, has a number of ligand-binding sites. These include the exchangeable and nonexchangeable GTP-binding sites and at least three reasonably well characterized sites that bind antimitotic drugs. The electron crystallographic model of the tubulin sheet polymer formed in the presence of zinc and paclitaxel and composed of antiparallel protofilaments has provided relatively detailed information about the locations of the nucleotide-binding sites and the paclitaxel site on the ␣␤-dimer (1).

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“…Tubulin was purified from lamb brain by sulfate fractionation and ion exchange chromatography. The protein was stored in liquid nitrogen and prepared as previously described (16). The labeling by tetraethyl rhodamine succinimidyl ester (Molecular Probes, Leiden, the Netherlands) of tubulin and microinjection into cells were done as previously described (8).…”

Section: Methodsmentioning

confidence: 99%

Antiangiogenic Concentrations of Paclitaxel Induce an Increase in Microtubule Dynamics in Endothelial Cells but Not in Cancer Cells

et al. 2005

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Microtubule-targeted drugs such as paclitaxel exhibit potent antiangiogenic activity at very low concentrations, but the mechanism underlying such an effect remains unknown. To understand the involvement of microtubules in angiogenesis, we analyzed the dynamic instability behavior of microtubules in living endothelial cells [human microvascular endothelial cells (HMEC-1) and human umbilical vascular endothelial cells (HUVEC)] following 4 hours of paclitaxel treatment. Unexpectedly, antiangiogenic concentrations of paclitaxel (0.1-5 nmol/L) strongly increased microtubule overall dynamicity in both HMEC-1 (86-193%) and HUVEC (54-83%). This increase was associated with increased microtubule growth and shortening rates and extents and decreased mean duration of pauses. The enhancement of microtubule dynamics by paclitaxel seemed to be specific to antiangiogenic concentrations and to endothelial cells. Indeed, cytotoxic concentration (100 nmol/L) of paclitaxel suppressed microtubule dynamics by 40% and 54% in HMEC-1 and HUVECs, respectively, as observed for all tested concentrations in A549 tumor cells. After 4 hours of drug incubation, antiangiogenic concentrations of paclitaxel that inhibited endothelial cell proliferation without apoptosis (1-5 nmol/L) induced a slight decrease in anaphase/metaphase ratio, which was more pronounced and associated with increased mitotic index after 24 hours of incubation. Interestingly, the in vitro antiangiogenic effect also occurred at 0.1 nmol/L paclitaxel, a concentration that did not alter mitotic progression and endothelial cell proliferation but was sufficient to increase interphase microtubule dynamics. Altogether, our results show that paclitaxel mediates antiangiogenesis by an increase in microtubule dynamics in living endothelial cells and suggest that the impairment of interphase microtubule functions is responsible for the inhibition of angiogenesis. (Cancer Res 2005; 65(6): 2433-40)

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In Vitro Effect of Cryptophycin 52 on Microtubule Assembly and Tubulin: Molecular Modeling of the Mechanism of Action of a New Antimitotic Drug

Cited by 48 publications

References 33 publications

Stathmin and Interfacial Microtubule Inhibitors Recognize a Naturally Curved Conformation of Tubulin Dimers

Stathmin and Interfacial Microtubule Inhibitors Recognize a Naturally Curved Conformation of Tubulin Dimers

Direct Photoaffinity Labeling by Dolastatin 10 of the Amino-terminal Peptide of β-Tubulin Containing Cysteine 12

Antiangiogenic Concentrations of Paclitaxel Induce an Increase in Microtubule Dynamics in Endothelial Cells but Not in Cancer Cells

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