Aip1 destabilizes cofilin-saturated actin filaments by severing and accelerating monomer dissociation from ends.

BACKGROUND: Depolymerization of actin filaments is vital for the morphogenesis of dynamic cytoskeletal arrays and actin-dependent cell motility. Cofilin is necessary for actin disassembly in cells, and it severs filaments most efficiently at low cofilin to actin ratios, whereas higher concentrations of cofilin suppress severing. However, the cofilin concentration in thymocytes is too high to allow the severing of single-actin filaments. RESULTS: We observed that filaments sever efficiently in thymus cytosol. We identified Aip1 as a critical factor responsible for the severing and destabilization of actin filaments even in the presence of high amounts of cofilin. By fluorescence resonance energy transfer (FRET)-based spectroscopy and single-filament imaging of actin, we show that, besides driving the rapid severing of cofilin-actin filaments, Aip1 also augments the monomer dissociation rate at both the barbed and pointed ends of actin. Our results also demonstrate that Aip1 does not cap the barbed ends of actin filaments, as was previously thought. CONCLUSIONS: Our results indicate that Aip1 is a cofilin-dependent actin depolymerization factor and not a barbed-end-capping factor as was previously thought. Aip1 inverts the rules of cofilin-mediated actin disassembly such that increasing ratios of cofilin to actin now result in filament destabilization through faster severing and accelerated monomer loss from barbed and pointed ends. Aip1 therefore offers a potential control point for disassembly mechanisms in cells to switch from a regime of cofilin-saturation and stabilization to one that favors fast disassembly and destabilization.

Simultaneous inhibition of key growth pathways in melanoma cells and tumor regression by a designed bidentate constrained helical peptide.

Protein-protein interactions are part of a large number of signaling networks and potential targets for drug development. However, discovering molecules that can specifically inhibit such interactions is a major challenge. S100B, a calcium-regulated protein, plays a crucial role in the proliferation of melanoma cells through protein-protein interactions. In this article, we report the design and development of a bidentate conformationally constrained peptide against dimeric S100B based on a natural tight-binding peptide, TRTK-12. The helical conformation of the peptide was constrained by the substitution of α-amino isobutyric acid--an amino acid having high helical propensity--in positions which do not interact with S100B. A branched bidentate version of the peptide was bound to S100B tightly with a dissociation constant of 8 nM. When conjugated to a cell-penetrating peptide, it caused growth inhibition and rapid apoptosis in melanoma cells. The molecule exerts antiproliferative action through simultaneous inhibition of key growth pathways, including reactivation of wild-type p53 and inhibition of Akt and STAT3 phosphorylation. The apoptosis induced by the bidentate constrained helix is caused by direct migration of p53 to mitochondria. At moderate intravenous dose, the peptide completely inhibits melanoma growth in a mouse model without any significant observable toxicity. The specificity was shown by lack of ability of a double mutant peptide to cause tumor regression at the same dose level. The methodology described here for direct protein-protein interaction inhibition may be effective for rapid development of inhibitors against relatively weak protein-protein interactions for de novo drug development.