Probing lysine deacetylases with trapping proteins generated by protein semisynthesis and genetic code expansion

• Applicant: Professor Dr. Dirk Schwarzer
• Subject Area: Biochemistry ; Biological and Biomimetic Chemistry
• Term: since 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 394748945

Lysine or histone deacetylases (HDACs) are central regulators of transcription by catalyzing the removal of acetyl groups from lysine residues of histones, thereby contributing to the inactivation of chromatin regions. HDACs are considered as drug targets due to their common deregulation in various types of cancer, but their biochemical investigation is hampered by the formation of multi protein complexes which impact the activity and specificity of the enzymes. In the previous project we have explored the HDAC-trapping amino acid AsuHd in the context peptide libraries derived from known acetylation sites. We uncovered new potential binding proteins of HDAC6 including transcription factor NF-kB and investigated the functional cross talk between these proteins. However, peptides reflect only a minor fraction of an acetylated proteins and many vital interactions between HDACs and their protein targets might be lost if resorting to peptide probes. In this follow-up proposal we plan to extend our approach from peptides to HDAC-trapping proteins. In four sub-projects we plan to use protein semisynthesis for generating AsuHd-containing nucleosomes and explore the site-specific recruitment of HDAC complexes and the impact of adjacent modifications. With genetically encoded AsuHd we plan to explore the potential recombinant AsuHd-containing proteins in comparison to peptide probes. Furthermore, we plan to synthesize and genetically encode AsuHd derivatives with photo-labile protection groups allowing to activate HDAC-trapping proteins by light. With genetic encoded AsuHd and photo-caged derivatives we further plan to establish a system for monitoring the activity of HDAC inhibitors in live cells and real time with Förster resonance energy transfer by fluorescence lifetime imaging (FLIM-FRET).

• DFG Programme: Research Grants