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P. Bargagna-Mohan, A. Hamza, Y.-E. Kim, Y. K. Ho, J. Liu, R. M. Evans, C.-G. Zhan, K. B. Kim, R. Mohan; Withaferin A Is a Chemical Genetic Probe of the Intermediate Filament Protein Vimentin. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1742.
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© ARVO (1962-2015); The Authors (2016-present)
The prototypic withanolide, WFA, which is found abundantly in the Indian medicinal plant Withania somnifera, is a potent inhibitor of angiogenesis. To understand the mode of action of this natural product, we exploited the chemical genetic approach that affords the isolation of small molecule binding target(s). The purpose of our work was to identify and characterize the biological target of WFA.
We recently synthesized a biotinylated WFA analog and demonstrated that this affinity probe binds to a 56 kDa protein in human umbilical vein endothelial cells (Yokota et al., Bioorg. Med. Chem. Lett. 2006). Here we employed Neutravidin affinity chromatography to isolate this protein from bovine aortic endothelial cells and characterize it by liquid chromatography mass spectrometry. Additionally, we have employed an array of experimental methods, which include competitive ligand binding analysis, western blotting, transfection analysis, protein transduction, transmission electron microscopy, immunofluorescence, flow cytometry and molecular modeling using x-ray crystallography coordinates of vimentin and WFA to investigate the binding mode of WFA with tetrameric and filamentous vimentin.
We demonstrate that WFA binds to tetrameric vimentin by covalently modifying the cysteine residue in its conserved α-helical coiled coil 2B domain. WFA binding to vimentin tetramers induces filamentous aggregation in vitro and this drug-induced phenotype in cell cultures manifests as punctate cytoplasmic aggregates that co-stain strongly for vimentin and actin. WFA’s potent dominant-negative effect on F-actin is dependent on vimentin expression, a drug-induced activity that leads to cellular apoptosis.
Our findings identify WFA as a novel chemical genetic probe of vimentin functions, and illuminate a potential new molecular target for withanolide-based therapeutics for the treatment of angioproliferative and malignant diseases.
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