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

赖氨酸或组蛋白去乙酰化酶（hdac）是转录的中心调节剂，通过催化从组蛋白赖氨酸残基上去除乙酰基团，从而促进染色质区域的失活。由于hdac在各种类型的癌症中普遍失调，因此被认为是药物靶点，但其生化研究受到多蛋白复合物的形成的阻碍，这些复合物影响了酶的活性和特异性。在之前的项目中，我们在已知乙酰化位点衍生的肽库中探索了捕获hdac的氨基酸AsuHd。我们发现了新的潜在的HDAC6结合蛋白，包括转录因子NF-kB，并研究了这些蛋白之间的功能串扰。然而，肽只反映了乙酰化蛋白的一小部分，如果采用肽探针，hdac和它们的蛋白靶标之间的许多重要相互作用可能会丢失。在这个后续提案中，我们计划将我们的方法从肽扩展到hdac捕获蛋白。在四个子项目中，我们计划使用蛋白质半合成来生成含有asuhd的核小体，并探索HDAC复合物的位点特异性募集和相邻修饰的影响。与肽探针相比，我们计划利用基因编码的AsuHd来探索含有AsuHd的潜在重组蛋白。此外，我们计划合成并遗传编码具有光不稳定保护基团的AsuHd衍生物，允许光激活hdac捕获蛋白。利用遗传编码的AsuHd和光笼衍生物，我们进一步计划建立一个系统，通过荧光寿命成像（FLIM-FRET） Förster共振能量转移实时监测活细胞中HDAC抑制剂的活性。

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).