Identification of sirtuin 2 as a lysine de-fatty-acylase and in vitro study of impact of histone glutarylation on nucleosomes stability

Abstract

Posttranslational modifications (PTMs) of proteins occur on different amino acid residues after proteins biosynthesis in both prokaryotic and eukaryotic cells. Such modifications dramatically increase the functional diversity of the proteomes and involve in the regulation of almost all aspects of cell biology as well as pathogenesis. To date, hundreds of protein PTMs have been identified, however, only a small set of them have been extensively studied. My research during the past four years mainly focuses on: 1) the identification and characterization of Sirt2 as an enzyme that catalyzes the removal of lysine fatty-acylation; and 2) the investigation of the impacts of histone H4 Lys91 glutarylation (H4K91glu) on nucleosome stability.

In chapter 2, by employing integrative chemical biology approaches, I demonstrate that human Sirt2 catalyzes the hydrolysis of lysine fatty-acyl modifications in vitro and in cells. Using a photo-cross-linking based approach, I showe that Sirt2 poses a strongest affinity toward a histone H3 peptide carrying a myristoylation mark at Lys9 (H3K9myr) among all the sirtuins tested. In addition, this enzyme catalyzes the removal of myristoyl group from the H3K9myr peptide. I also use alk-14, a biorthogonal chemical reporter developed for the detection of protein fattyacylation, to monitor the level change of endogenous lysine fatty-acylation upon Sirt2 inhibition and knockdown. After metabolic labeling, the alk-14-labeled proteins are conjugated to a azide-rhodamine dye via Cu(I)-assisted azide-alkyne cycloaddition (CuAAC). The subsequent in-gel fluorescence scanning analysis shows significantly increased signals for both cytosolic proteins and core histones labeled by alk-14 in cells treated with Sirt2 inhibitor and siRNA, suggesting that Sirt2 is an endogenous lysine defatty-acylase in living cells.

Nucleosomes are the basic repeating units of eukaryotic chromatin. A nucleosome consists of two copies each of four core histones: H2A, H2B, H3 and H4. The nucleosome dynamics alters the DNA accessibility and thus involves in regulation of DNA-associated processes. Histone PTMs is one of the mechanisms that modulates the nucleosome dynamics. By directly changing the physicochemical properties around the modification sites, histone PTMs may affect the histone-histone and histone-DNA interactions involving the modified residues. To this date, various histone modifications have been identified to influence the nucleosome dynamics, including lysine/arginine methylation, lysine acetylation. As a new type of lysine PTM, lysine glutarylation (Kglu) was recently discovered on histones. But its biological significance as a histone PTM remains unknown. To study the impacts of histone glutarylation on nucleosome dynamics, the preparation of homogenous histone proteins with site-specific modification installed at stoichiometric level is required. In Chapter 3, I utilize the expressed protein ligation (EPL) to synthesize various histone proteins carrying lysine acetylation (Kac) or glutarylation (Kglu), including H2BK120glu, H3K122glu, H4K91glu and H4K91ac. The characterizations of these proteins are also described.

With a terminal carboxylate, histone Kglu alters the positive charge of lysine side chain into negative charge, and is therefore likely to cause significant structural changes to nucleosomes. In Chapter 4, I use the synthetic H4K91glu protein to study the impacts of this modification on nucleosome dynamics. Different biochemical and biophysical methods are applied and demonstrate that H4K91glu indeed influences the nucleosome stability by eliminating the interactions between H2A-H2B dimer and (H3-H4)2 tetramer.