The Natural Product Cucurbitacin E Inhibits Depolymerization of Actin Filaments

Abstract

Although small molecule actin modulators have been widely used as research tools, only one cell permeable small molecule inhibitor of actin depolymerization (jasplakinolide) is commercially available. We report that the natural product cucurbitacin E inhibits actin depolymerization and show that its mechanism of action is different from jasplakinolide. In assays using pure fluorescently labeled actin, cucurbitacin E specifically affected depolymerization without affecting polymerization. It inhibited actin depolymerization at sub-stoichiometric concentrations up to 1:6 cucurbitacin:actin E. Cucurbitacin E specifically binds to filamentous actin (F-actin) forming a covalent bond at residue Cys257, but not to monomeric actin (G-actin). Based on its compatibility with phalloidin staining, we show that cucurbitacin E occupies a different binding site on actin filaments. Using loss of fluorescence after localized photoactivation, we found that cucurbitacin E inhibited actin depolymerization in live cells. Cucurbitacin E is a widely available plant-derived natural product, making it a useful tool to study actin dynamics in cells and actin-based processes such as cytokinesis.

Investigating the Role of F-Actin in Human Immunodeficiency Virus Assembly by Live-Cell Microscopy

“…We conclude that HIV-1 assembly in jasplakinolide-treated cells proceeded with normal rates during the phase of F-actin filament thickening and then ceased completely upon actin network collapse. This was supported by analyses of cells treated with cucurbitacin E, which has been reported to inhibit F-actin depolymerization by a different mechanism of action (38). Again, Gag assembly in the early phase of cucurbitacin E treatment proceeded with a similar rate (k ϭ 0.0054 Ϯ 0.0028 s Ϫ1 ) as that in control cells ( Fig.…”

Cucurbitacin covalent bonding to cysteine thiols: the filamentous-actin severing protein Cofilin1 as an exemplary target

“…The effect of cucurbitacin E on increasing cellular F-actin has recently been attributed to its direct conjugation with Cys257 on polymerized actin, but not monomeric globular actin (G-actin) [16]. An additional actin regulator identified as a potential target is the F-actin severing protein Cofilin1, which was isolated as a biotin-linked cucurbitacin E interacting protein [12].…”

“…Increasing Cofilin1 concentrations revealed an efficient effect of shifting actin from P to S fractions at 5 μM. To test the possibility that cucurbitacin E binds covalently to Cofilin1 as it does for actin [16], a range of cucurbitacin E (CuE) concentrations at molar ratios up to 1:100 (relative to constant 5 μM Cofilin1) were incubated with purified Cofilin1 protein for 16 h and examined for their ability to induce a Cofilin1 mobility shift on 12% Bis-Tris polyacrylamide gels. Although low cucurbitacin E concentrations had no obvious effect, reduced Cofilin1 mobility was clearly evident at 1:50 and 1:100 molar ratios (Figure 3B).…”

“…Cucurbitacin analogues also have marked effects on the actin cytoskeleton, which in turn affects processes such as cell motility, tumour cell invasion and metastasis [12-15]. In fact, the ability of cucurbitacin E to inhibit filamentous actin (F-actin) depolymerisation led to the suggestion that it would be useful as a tool to study actin dynamics and actin-based processes in live cells [16]. …”

Cucurbitacin E suppresses cytokine expression in human Jurkat T cells through down-regulating the NF-κB signaling

“…The anticancer activity of CucE has been proposed to be mediated by inhibiting signal transducer and activator of transcription 3 phosphorylation in cancer cells [5]. Recent studies showed that CucE, like other cucurbitacins [6,7], disrupts the actin cytoskeleton in cells leading to cell cycle arrest at G 2 /M phases [3,8,9], suggesting that actin is one of the potential targets for these compounds. It has been found that activation of cofilin by cucurbitacins may be an important event in damaging the actin cytoskeleton [10][11][12][13].…”