Examining the protective roles of sirtuin 2 against oxidative stress-induced histone damage and construction of degenerate peptide libraries for high-throughput screening of inhibitors of histone methylation "readers"

Abstract

Histone posttranslational modifications (PTMs) are covalent chemical modifications occurring on histones after their biosynthesis, and are responsible for regulating fundamental cellular events, such as gene transcription and chromatin remodeling. To date, more than 20 different PTMs have been identified to covalently modify histones, especially on their unstructured N-terminal tails. Except for the several well-characterized modifications such as acetylation and methylation, most of the PTMs are still poorly understood. Normally, histone PTMs are added and removed by their “writers” and “erasers”, respectively, and serve as docking platforms to recruit their effector proteins (“readers”) to alter chromatin architecture and conduct the chromatin-templated processes. Given the significant roles of histone PTMs in many essential cellular processes, dysregulating or misreading of histone PTMs has been implicated in the development of severe human diseases such as Alzheimer’s disease and cancer. Therefore, histone-modifying enzymes as well as their “readers” have become potential drug targets. A ROS-induced histone lysine gamma-oxonoanoylation (Kgon) has been recently discovered as a novel histone PTM. Under oxidative stress, one lipid-derived electrophile (LDE), 4-oxo-2-nonenal (4-ONE), could target histone lysine residues in an enzyme-independent manner to form Kgon and interfere with histone assembly into nucleosomes. However, whether Kgon causes irreversible damage or is regulated by enzymes “erasing” this modification remained unclear. In this thesis, I demonstrate that human Sirt2 is responsible for catalyzing the removal of histone Kgon. Among the tested human sirtuins, I observed robust deacylase activity of Sirt2 toward Kgon-modified histone peptides in vitro. By employing alkynyl-4-ONE as a chemical reporter for Kgon, I demonstrate that Sirt2 is responsible for the removal of histone Kgon in cells. Moreover, by developing a ketone-reactive chemical probe which could detect Kgon, I discovered that histone Kgon generated from endogenous 4-ONE in stimulated macrophages could be regulated by perturbing the activity of Sirt2. In this study, I describe a new regulation mechanism of LDE-derived histone PTMs and the protective roles of Sirt2 against oxidative stress-induced histone damage as a histone Kgon “eraser”. In the aim to develop specific inhibitors targeting histone-modifying enzymes and “readers”, considerable progress has been made toward inhibiting epigenetic targets such as methyltransferases, acetyltransferases, and deacetylases. However, due to the low in vitro affinity of methylation “readers” for their native substrates, it was inherently difficult to generate high-affinity chemical ligands. Moreover, with the high structural similarity of methylation “readers” within certain families, achieving selectivity is also a significant challenge for developing inhibitors. In this thesis, I describe an innovative strategy to develop D-peptide inhibitors against histone methylation “readers”. My approach includes “on-bead-one-sequence” library construction, high-throughput on-bead screening, and partial Edman degradation/mass spectrometry validations. Compared with small molecule inhibitors, peptide-based antagonists have advantages for targeting a relatively large interaction surface; in addition, by using D-amino acids as building blocks, the peptide inhibitors possess high stability to overcome protease-induced digestion in cells. With this high-throughput screening approach, two histone methylation “readers”, SPIN1 and HP1, have been targeted for inhibitor screening. Upon the screening against HP1, a consistent four- D-amino acid pattern inhibitor has been discovered.