Chemical biology study on the post-translational modifications of high mobility group (HMG) family proteins via protein chemical synthesis approach

Abstract

Most eukaryotic proteins undergo additional reversible or irreversible modification processes (known as the post-translational modifications, PTMs) after translation, by which the chemical repertoire constructed by the natural amino acids can be significantly extended. As one of the most important signal regulatory network, systematical analysis of the PTMs could provide deep insight into the protein functions, which is particularly important to comprehend the distinct mechanism between normal and abnormal cell activities. Although generating the protein with site-specific PTMs of interest is critical, it is still challenging for researchers to study the PTMs. Protein chemical synthesis assisted by chemoselective peptide ligation has emerged as an effective way to site-selectively and homogeneously introduce PTMs into proteins.

High-Mobility Group (HMG) protein family is a group of non-histone chromosomal proteins that are involved in the regulation of DNA-dependent processes such as transcription, replication, recombination, and repair. Here, we have successfully produced two members of HMG family with site-specific PTMs via Ser/Thr ligation and used them to study their function. This thesis is therefore divided into the following two parts.

In chapter 2, homogenous HMGA1a with site-specific PTMs was generated by the Ser/Thr ligation assisted protein total synthesis. We demonstrated that the triple phosphorylatated acidic tail interacts with the Lysine-Arginine (KR) clusters via electrostatic interaction, thereby inducing a conformational change and regulating the protein-protein interaction. Finally, a synthetic protein-affinity purification mass spectrometry (SP-AP-MS) methodology was introduced to profile the phosphorylation-regulated binders of HMGA1a, which supported that the HMGA1a phosphorylation may serve as an widespread regulatory mode for protein-protein interactions.

In chapter 3, we explored the potential application of Ser/Thr ligation and Cys/Pen ligation in protein semi-synthesis. To maximize the efficiency of chemical synthesis of proteins bearing PTMs, the semi-synthetic approach facilitated by chemical ligation offers an expedient solution. The success of stitching different peptide salicylaldehyde esters with the N-terminus of MBP, a 40 kDa-protein, exemplified the operability of Ser/Thr ligation and Cys/Pen ligation on large protein chemical synthesis. Furthermore, we introduced two purification strategies for separating the ligated product from the unconsumed starting materials without using HPLC purification. Finally, this methodology has enabled us to generate acetylated HMGB1, a 25 kDa proinflammatory protein. Further studies revealed that the acetylation may represent a regulatory switch to control the HMGB1’s signalling pathway by accelerating its degradation, consequently preventing cells from pyroptosis and lethality upon infectious injury.