Binding of Pro-Apoptotic Protein Bax to Cytoprotective UDCA and TUDCA

Abstract

Hydrophobic bile acids, such as deoxycholic acid (DCA), strongly induce apoptosis in both hepatic and non-hepatic cells while hydrophilic bile acids, like ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA), are cytoprotective and inhibit cell death. The mechanisms associated with these distinct effects are not entirely clear. However, the effect of hydrophilic bile acids seems to be related with the blockage of a series of processes that converge on mitochondrial damage. Bax is a pro-apoptotic protein that belongs to the superfamily of the Bcl-2 proteins and is involved in mitochondrial pore formation. Submicellar concentrations of cytoprotective bile acids have been shown to modulate Bax concentration in mitochondria, suggesting that these molecules may interact directly with the protein. In this study, our objective was to evaluate the affinity of bile acids to recombinant Bax protein, making use of fluorescence spectroscopy (FRET and fluorescence anisotropy), as well as Fluorescence Correlation Spectroscopy (FCS). Our results show that the cytoprotective bile acids UDCA and TUDCA associate with recombinant Bax protein with high affinity, while the cytotoxic bile acid DCA only seems to be able to adsorb to the protein with much lower affinity. Notably, the binding site for UDCA seems to be located in a hydrophobic pocket of the protein. This interaction could be responsible for the disruption of Bax translocation to the mitochondrial outer membrane in the presence of UDCA and/or TUDCA.Supported from FCT/Portugal (Projects PTDC/QUI-BIQ/119494/2010 and RECI/CTM-POL/0342/2012).T.S. and F.F. acknowledges FCT grants SFRH/BD/92398/2013 and SFRH/BPD/64320/2009