Functional targeting of the TGF-ßR1 kinase domain and downstream signaling: A role for the galloyl moiety of green tea-derived catechins in ES-2 ovarian clear cell carcinoma.

The galloyl moiety is a specific structural feature which dictates, in part, the chemopreventive properties of diet-derived catechins. In ovarian cancer cells, galloylated catechins were recently demonstrated to target the transforming growth factor (TGF)-ß-mediated control of the epithelial-mesenchymal transition process. The specific impact of the galloyl moiety on such signaling, however, remains poorly understood. Here, we questioned whether the sole galloyl moiety interacted with TGF-ß-receptors to alter signal transduction and chemotactic migratory response in an ES-2 serous carcinoma-derived ovarian cancer cell model. In line with the LogP and LogS values of the tested molecules, we found that TGF-ß-induced Smad-3 phosphorylation and cell migration were optimally inhibited, provided that the lateral aliphatic chain of the galloyl moiety reached 8-10 carbons. Functional inhibition of the TGF-ß receptor (TGF-ßR1) kinase activity was supported by surface plasmon resonance assays showing direct physical interaction between TGF-ßR1 and the galloyl moiety. In silico molecular docking analysis predicted a model where galloylated catechins may bind TGF-ßR1 within its adenosine triphosphate binding cleft in a site analogous to that of Galunisertib, a selective adenosine triphosphate-mimetic competitive inhibitor of TGF-ßR1. In conclusion, our data suggest that the galloyl moiety of the diet-derived catechins provides specificity of action to galloylated catechins by positioning them within the kinase domain of the TGF-ßR1 in order to antagonize TGF-ß-mediated signaling that is required for ovarian cancer cell invasion and metastasis.

A family portrait: structural comparison of the Whirly proteins from Arabidopsis thaliana and Solanum tuberosum.

DNA double-strand breaks are highly detrimental genomic lesions that routinely arise in genomes. To protect the integrity of their genetic information, all organisms have evolved specialized DNA-repair mechanisms. Whirly proteins modulate DNA repair in plant chloroplasts and mitochondria by binding single-stranded DNA in a non-sequence-specific manner. Although most of the results showing the involvement of the Whirly proteins in DNA repair have been obtained in Arabidopsis thaliana, only the crystal structures of the potato Whirly proteins WHY1 and WHY2 have been reported to date. The present report of the crystal structures of the three Whirly proteins from A. thaliana (WHY1, WHY2 and WHY3) reveals that these structurally similar proteins assemble into tetramers. Furthermore, structural alignment with a potato WHY2-DNA complex reveals that the residues in these proteins are properly oriented to bind single-stranded DNA in a non-sequence-specific manner.

A conserved lysine residue of plant Whirly proteins is necessary for higher order protein assembly and protection against DNA damage.

All organisms have evolved specialized DNA repair mechanisms in order to protect their genome against detrimental lesions such as DNA double-strand breaks. In plant organelles, these damages are repaired either through recombination or through a microhomology-mediated break-induced replication pathway. Whirly proteins are modulators of this second pathway in both chloroplasts and mitochondria. In this precise pathway, tetrameric Whirly proteins are believed to bind single-stranded DNA and prevent spurious annealing of resected DNA molecules with other regions in the genome. In this study, we add a new layer of complexity to this model by showing through atomic force microscopy that tetramers of the potato Whirly protein WHY2 further assemble into hexamers of tetramers, or 24-mers, upon binding long DNA molecules. This process depends on tetramer-tetramer interactions mediated by K67, a highly conserved residue among plant Whirly proteins. Mutation of this residue abolishes the formation of 24-mers without affecting the protein structure or the binding to short DNA molecules. Importantly, we show that an Arabidopsis Whirly protein mutated for this lysine is unable to rescue the sensitivity of a Whirly-less mutant plant to a DNA double-strand break inducing agent.

Purification, crystallization and preliminary X-ray diffraction analysis of the Whirly domain of StWhy2 in complex with single-stranded DNA.

StWhy1 and StWhy2 are members of the Whirly family of single-stranded DNA (ssDNA) binding proteins. To understand the mode of binding of the Whirly proteins to single-stranded DNA, crystals of the Whirly domains of both StWhy1 and StWhy2 in complex with single-stranded DNA were obtained by the hanging-drop vapour-diffusion method. The diffraction patterns of the StWhy1-ssDNA complex crystals displayed severe anisotropy and were of low resolution, making them unsuitable for structure determination. In contrast, the crystals of the StWhy2-ssDNA complex diffracted isotropically to 2.20 A resolution. The crystallization and data collection to 2.20 A resolution of StWhy2 in the free form are also reported.