CONICET | Buscador de Institutos y Recursos Humanos

Post-translational modifications (PTMs) of proteins affect almost all cellular processes. Among them, acetylation of lysines (K) has emerged recently as a reversible and dynamic PTM with important biological functions. The 14-3-3 proteins regulate the function and subcellular localization of thousands of proteins, through interaction with specific phospho-serine and threonine residues. The acetyla- tion of a specific K (K49) which is part of the 14-3-3 binding pocket and is essential for its function, results in an inactive 14-3-3. To study its regulation and the crosstalk mechanism between acetylation-phosphorylation, we produced an antibody to recognize the above-mentioned AcK49 in 14-3-3. A chemically synthetized 15 amino acid peptide, including the acetylated K49, and conserved in all 14-3-3 paralogs, was coupled to Bovine Serum Albumin (BSA) as carrier. This antigen was used to immunize rabbits in a pathogen-free animal facility (ISAL, UNL-CONICET). After 4 immunizations, blood was drawn from the animals, and the serum obtained by centrifugation. The later was tested by Dot- and Western blot against different 14-3-3 constructions. It recognized all the acetylated peptide or complete 14-3-3 forms (the antigen, an unconjugated synthetic peptide and a recombinant peptide coupled to Histone H3, all corresponding to the same sequence in 14-3-3, and in vitro acetylated 14-3-3), but not the non-acetylated recombinant 14-3-3. The serum did not recognize the recombinant peptide-H3 when blocked with the synthetic peptide. As expected, the preimmune serum did not recognize the antigen. This was the first step in a project to study 14-3-3 regulation by acetylation, which means regulation of regulators. We produced and characterized a polyclonal antibodythat recognizes site-specifically the acetylation of 14-3-3 on its K49, which at the best of our knowledge is not commercially available.

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