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

赖氨酸或组蛋白脱乙酰酶(HDAC)是转录的中心调节者，它催化组蛋白赖氨酸残基上的乙酰基的去除，从而导致染色质区域的失活。HDAC因其在各种癌症中普遍的去调节作用而被认为是药物靶标，但其生物化学研究因多蛋白复合体的形成而受阻，该复合体影响酶的活性和特异性。在前一个项目中，我们已经在已知乙酰化位点的多肽文库中探索了HDAC捕获氨基酸AsuHd。我们发现了包括转录因子NF-kB在内的HDAC6新的潜在结合蛋白，并研究了这些蛋白之间的功能串扰。然而，多肽只反映了一小部分乙酰化的蛋白质，如果诉诸于多肽探针，HDAC与其蛋白质靶标之间的许多重要相互作用可能会丢失。在这个后续的提案中，我们计划将我们的方法从多肽扩展到捕捉HDAC的蛋白质。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复合体的位置特异性招募和相邻修饰的影响。利用基因编码的AsuHd，我们计划探索潜在的含AsuHd的重组蛋白，并与多肽探针进行比较。此外，我们计划合成和基因编码带有光不稳定保护基团的AsuHd衍生物，允许用光激活HDAC捕获蛋白。利用基因编码的AsuHd和光笼中的衍生物，我们进一步计划建立一个系统，用于监测活细胞中HDAC抑制剂的活性，并通过Förster共振荧光寿命成像能量转移(Flim-FRET)进行实时监测。

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).