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2C

22. Carlos Moreno Yruela

University of Copenhagen
Interrogating substrate specificity of human histone deacetylases using peptide microarrays
Copenhagen, Denmark
Ever since SPOT synthesis was developed in 1990, for the synthesis of peptide arrays on a cellulose membrane, it has served as a critical efficient tool for the identification of enzyme substrates and ligands for disruption of protein–protein interactions. Here, we wish to investigate the human histone deacetylase (HDAC) enzymes by taking advantage of the high-throughput and minute material consumption of SPOT synthesis and CelluSPOT arrays, in order to define substrate recognition for each of the HDAC isoforms. Different zinc-binding groups have been successfully incorporated into the peptides in the form of unnatural amino acids, thus, increasing affinity for the active site of HDACs. Preliminary data shows selective binding of isolated HDACs to the modified peptides, with differences in affinity depending on the zinc-binding group and the substrate sequence. This methodology will be employed for substrate screening, also including neighboring post-translational modifications, and for pull-down of HDAC-containing complexes from cell lysates.