We discovered that GAPDH binds, in vitro, with high affinity to the core ARE from tumor necrosis factor-α (TNF-α) mRNA via a two-step binding mechanism. We investigated the effect of a dimer-interface mutation on GAPDH oligomerization and RNA binding by fluorescence anisotropy, crystallography, small-angle x-ray scattering, nano-electrospray ionization native mass spectrometry, and hydrogendeuterium exchange mass spectrometry. We showed that the mutation promotes short-range and longrange dynamic changes in regions located at the dimer and tetramer interface and in the NAD+ binding site, suggesting that these regions are crucial for RNA binding. We have recently obtained hydrogendeuterium exchange mass spectrometry data on the GAPDH-RNA complex, providing the first “image” of the AU-rich TNF-α RNA binding site in GAPDH. We are currently investigating the effects of posttranslational modifications on GAPDH binding to RNA.

TNF-α is a cytokine regulating inflammation and immune system development. Defects in TNF-α production and signaling underlie a number of autoimmune associated diseases. Results from these studies will be crucial for the design of small molecules that can modulate the GAPDH-RNA interaction to target TNF-α-associated inflammatory diseases.

Contact : ibs.seminaires@ibs.fr