Cucurbitacin I elicits the formation of actin/phospho-myosin II co-aggregates by stimulation of the RhoA/ROCK pathway and inhibition of LIM-kinase.

Cucurbitacins are cytotoxic triterpenoid sterols isolated from plants. One of their earliest cellular effect is the aggregation of actin associated with blockage of cell migration and division that eventually lead to apoptosis. We unravel here that cucurbitacin I actually induces the co-aggregation of actin with phospho-myosin II. This co-aggregation most probably results from the stimulation of the Rho/ROCK pathway and the direct inhibition of the LIMKinase. We further provide data that suggest that the formation of these co-aggregates is independent of a putative pro-oxidant status of cucurbitacin I. The results help to understand the impact of cucurbitacins on signal transduction and actin dynamics and open novel perspectives to use it as drug candidates for cancer research.

Probing interactions of tubulin with small molecules, peptides, and protein fragments by solution nuclear magnetic resonance.

The description of the molecular mechanisms of interaction between tubulin or microtubules and partners at atomic scale is expected to have critical impacts on the understanding of basic physiological processes. This information will also help the design of future drug candidates that may be used to fight various pathologies such as cancer or neurological diseases. For these reasons, this aspect of tubulin research has been tackled since the seventies using many different methods and at different scales. NMR appears as a unique approach to provide, with atomic resolution, the solution structure and dynamical properties of tubulin/microtubule partners in free and bound states. Though tubulin is not directly amenable to solution NMR, the NMR ligand-based experiments allow one to obtain valuable data on the molecular mechanisms that sustain structure-function relationship, in particular atomic details on the partner binding site. We will first describe herein some basic principles of solution NMR spectroscopy that should not be missed for a comprehensive reading of NMR reports. A series of results will then be presented to illustrate the wealth and variety of NMR experiments and how this approach enlightens tubulin/microtubules interaction with partners.

The stathmin-derived I19L peptide interacts with FtsZ and alters its bundling.

FtsZ is a prokaryotic tubulin-like protein. Despite a low degree of sequence identity with tubulin, it presents the same folding pattern and some similar functions, notably in cell division. Indeed, FtsZ and tubulin polymerize to form bundles and microtubules, respectively, which are essential for cell cytokinesis. We previously demonstrated that peptides derived from the N-terminal stathmin domain interact with tubulin and impede microtubule formation. We demonstrated here that I19L, the most efficient of these peptides, also alters FtsZ bundling assembly in vitro. STD-NMR and TRNOESY experiments revealed that I19L interacts with FtsZ and folds upon its binding but in a way different from what we observed with tubulin. These NMR data were used in molecular modeling calculations to propose models of the I19L-FtsZ complex. Interestingly, two models, consistent with NMR data, show an interaction of I19L near the T7 loop or near the GTP binding site of FtsZ, explaining the modifications of the bundling assembly observed with this peptide. The fine analysis of the structural differences of the complexes of I19L with FtsZ and tubulin should help for the rational development of new specific antibiotic agents.

Benomyl and colchicine synergistically inhibit cell proliferation and mitosis: evidence of distinct binding sites for these agents in tubulin.

Benomyl, a tubulin-targeted antimitotic antifungal agent, belongs to the benzimidazole group of compounds, which are known to inhibit the binding of colchicine to tubulin. Therefore, benomyl was thought to bind at or near the colchicine-binding site on tubulin. However, recent mutational studies in yeast and fluorescence studies involving competitive binding of benomyl and colchicine on goat brain tubulin suggested that benomyl may bind to tubulin at a site distinct from the colchicine-binding site. We set out to examine whether colchicine and benomyl bind to tubulin at distinct sites using a human cervical cancer (HeLa) cell line with the thinking that these agents should exert either additive or synergistic activity on cell proliferation if their binding sites on tubulin are different. We found that benomyl and colchicine synergistically inhibited the proliferation of HeLa cells and blocked their cell cycle progression at mitosis. The synergistic activity of benomyl and colchicine was also apparent from their strong depolymerizing effects on both the spindle and interphase microtubules when used in combinations, providing further evidence that these agents bind to tubulin at different sites. Using NMR spectroscopy, we finally demonstrated that benomyl and colchicine bind to tubulin at different sites and that the binding of colchicine seems to positively influence the binding of benomyl to tubulin and vice versa. Further, an analysis of the saturation transfer difference NMR data yielded an interesting insight into the colchicine-tubulin interaction. The data presented in this study provided a mechanistic understanding of the synergistic effects of benomyl and colchicine on HeLa cell proliferation.

N-terminal stathmin-like peptides bind tubulin and impede microtubule assembly.

Microtubules are major cytoskeletal components involved in numerous cellular functions such as mitosis, cell motility, or intracellular traffic. These cylindrical polymers of alphabeta-tubulin assemble in a closely regulated dynamic manner. We have shown that the stathmin family proteins sequester tubulin in a nonpolymerizable ternary complex, through their stathmin-like domains (SLD) and thus contribute to the regulation of microtubule dynamics. We demonstrate here that short peptides derived from the N-terminal part of SLDs impede tubulin polymerization with various efficiencies and that phosphorylation of the most potent of these peptides reduces its efficiency as in full-length stathmin. To understand the mechanism of action of these peptides, we undertook a NMR-based structural analysis of the peptide-tubulin interaction with the most efficient peptide (I19L). Our results show that, while disordered when free in solution, I19L folds into a beta-hairpin upon binding to tubulin. We further identified, by means of saturation transfer difference NMR, hydrophobic residues located on the beta2-strand of I19L that are involved in its tubulin binding. These structural data were used together with tubulin atomic coordinates from the tubulin/RB3-SLD crystal structure to model the I19L/tubulin interaction. The model agrees with I19L acting through an autonomous tubulin capping capability to impede tubulin polymerization and provides information to help understand the variation of efficiency against tubulin polymerization among the peptides tested. Altogether these results enlighten the mechanism of tubulin sequestration by SLDs, while they pave the way for the development of protein-based compounds aimed at interfering with tubulin polymerization.