Abstract
The first chapter gives a brief introduction of post translational modifications (PTMs) of proteins including histones and non-histones such as p53, together with modifications on DNA and cellular RNA, termed the core of “epigenetics”. Over 200 proteins PTMs have been identified until now. Among them, the modifications especially acylation on lysine (Kacyl) are important, involved in many cellular process and various diseases. Notably, their biological functions are regulated by ‘writers’ (adding specific modification), ‘erasers’ (removing PTM), and ‘readers’ that bind to the specific mark. More comprehensive understanding of the PTMs and their regulators would offer insights of the roles and help designing drugs. Thus, development of facial and efficient method for detection of deacylation activity (eraser) and identification of reader of Kacyl will be highly useful. A number of methods were used to prepare deacylation activity assay such as high-performance liquid chromatography (HPLC) and antibody. However, their widespread applications have been hindered by laborious preparation work and multistep protocols. One-step fluorescence-based method has the advantage of high sensitivity and facileness in a continuous manner. Affinity-based-probes (AfBPs) is a powerful tool to study transient protein-ligand interactions and identify the regulators, which utilizes photo-cross-linking approach to convert noncovalent protein-ligand interactions into irreversible chemical linkages.The second chapter introduces a model probe HRM for one-step fluorescence signal generation based on the intramolecular exchange reaction. This model strategy is used to develop sing-step fluorescent probe for histone deacetylases (HDACs) activity assay. The normally used fluorescent probes with the strategies of “masking group”, “fluorescence resonance energy transfer (FRET)” and “direct substitution” are not suitable to design probes for HDACs because the modification like Kac is installed on the side chain of lysine and the aliphatic amide structure in the substrate will not allow π-conjugation of the group to a fluorophore and modulate the electronic state directly. Here HRM contains three parts: 1. a -Boc protected lysine which serves as a model of Kacyl modification and a substrate of HDACs, 2. a hydrophilic and flexible aminoethoxyl linker which increases solubility of probe and offers flexibility for intramolecular reaction, 3. a NBD-O dye for fluorescence signal generation. The fluoresce turn-on can be triggered by spontaneous intramolecular exchange reaction of NBD-O with released free amine -NH2 which is removed under acid condition as a model of enzymatic reaction.
Chapter 3 moves on to design of single-step fluorescent probes for detection and proteomic profiling of HDACs. They are HT1, HT2 and HTP. Instead of HRM, HT1 and HT2 were featured with acetylation of lysine (Kac) directly. HT1 and HT2 contains different linker length between Kac group and the NBD-O moiety. Results indicated that there is no difference between HT1 and HT2. These two probes are capable of monitoring HDAC activity in a continuous manner. The fluorescence increment could be up to 50-fold, which was better than the existing one-step HDAC fluorescent probes. In addition, inhibition assay of HDACs can be conducted using HT2. More importantly, the probe HTP has multiple functions which can be used to 1) report the enzymatic activity of HDACs; 2) conduct proteomic profiling in complex cellular environment; 3) identify epigenetic readers and erasers and differentiate between them.
Chapter 4 continues with the detection of decrotonylaiton and delipoylaiton activity of HDACs using one-step fluorescent probes. In this chapter, we first designed probe KTcr-I and KTcr-II for decrotonylation activity detection. Peptide sequence-dependent effect was observed. KTcr-I can be recognized by Sirt2 more effectively, while KTcr-II was found to preferentially react with HDAC3. To study the delipoylation activity of HDACs, we next synthesized probe KTlip. It was suitable for profiling delipoylation activity of enzymes. Combined with results of a series of lipoylated peptides (KAlip-1 to KAlip-11) and affinity-based-probe KTP-lip and KPlip, we identified Sirt2 as a new robust delipoylase. The efficiency (Kcat × Km-1) of Sirt2 to remove lipoyl group of DLAT K259 of PDH complex was approximately 426-fold of that of Sirt4 in vitro.
Chapter 5 provides a summary of the above fundamental principles of design, application and key new features, and suggests directions for future works.
| Date of Award | 1 Jun 2018 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Hongyan SUN (Supervisor) |
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