Annalia Focà

The three members (GADD45α, GADD45β, and GADD45γ) of the growth arrest and DNA damage-inducible 45 (GADD45) protein family are involved in a myriad of diversified cellular functions. With the aim of unravelling analogies and differences, we performed comparative biochemical and biophysical analyses on the three proteins. The characterization and quantification of their binding to the MKK7 kinase, a validated functional partner of GADD45β, indicate that GADD45α and GADD45γ are strong interactors of the kinase. Despite their remarkable sequence similarity, the three proteins present rather distinct biophysical properties. Indeed, while GADD45β and GADD45γ are marginally stable at physiological temperatures, GADD45α presents the Tm value expected for a protein isolated from a mesophilic organism. Surprisingly, GADD45α and GADD45β, when heated, form high-molecular weight species that exhibit features (ThT binding and intrinsic label-free UV/visible fluorescence) proper of amyloid-like aggregates. Cell viability studies demonstrate that they are endowed with a remarkable toxicity against SHSY-5Y and HepG2 cells. The very uncommon property of GADD45β to form cytotoxic species in near-physiological conditions represents a puzzling finding with potential functional implications. Finally, the low stability and/or the propensity to form toxic species of GADD45 proteins constitute important features that should be considered in interpreting their many functions.

Nodal is a potent embryonic morphogen belonging to the TGF-β superfamily. Typically, it also binds to the ALK4/ActRIIB receptor complex in the presence of the co-receptor Cripto-1. Nodal expression is physiologically restricted to embryonic tissues and human embryonic stem cells, is absent in normal cells but re-emerges in several human cancers, including melanoma, breast, and colon cancer. Our aim was to obtain mAbs able to recognize Nodal on a major CBR (Cripto-Binding-Region) site and to block the Cripto-1-mediated signalling. To achieve this, antibodies were raised against hNodal(44–67) and mAbs generated by the hybridoma technology. We have selected one mAb, named 3D1, which strongly associates with full-length rhNodal (K_D 1.4 nM) and recognizes the endogenous protein in a panel of human melanoma cell lines by western blot and FACS analyses. 3D1 inhibits the Nodal-Cripto-1 binding and blocks Smad2/3 phosphorylation. Data suggest that inhibition of the Nodal-Cripto-1 axis is a valid therapeutic approach against melanoma and 3D1 is a promising and interesting agent for blocking Nodal-Cripto mediated tumor development. These findings increase the interest for Nodal as both a diagnostic and prognostic marker and as a potential new target for therapeutic intervention.