Structural and Biochemical Studies of Histone Acetyltransferases and Sirtuin Enzymes

Acetyltransferases;Sirtuins;X-ray Crystallography;Biophysics

Moreno, Alison EWolberger, Cynthia ;

Johns Hopkins University

关键词: Acetyltransferases;    Sirtuins;    X-ray Crystallography;    Biophysics;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/39377/MORENO-DISSERTATION-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Lysine acetylation is an abundant and reversible post-translational modification that regulates a diverse range of biological processes. Dedicated enzyme families exist that catalyze and remove acetyl modifications from lysine, and dysregulation of so-called HATs (Histone AcetylTransferases) and HDACs (Histone DeACetylases) has been implicated in cancer and other human diseases. This dissertation focuses on structural and biochemical characterization of two classes of enzymes that regulate lysine acetylation. First, Gcn5 is a highly conserved acetyltransferase associated with the SAGA transcriptional co-activator. While Gcn5 is catalytically active on its own, its specificity and overall activity changes when it associates with three additional SAGA subunits, Ada2, Ada3, and Sgf29, which are collectively referred to as the Histone AcetylTransferase (HAT) module. Sgf29 contains a tandem Tudor domain that specifically recognizes H3K4 trimethylation (H3K4me3), but how this interaction modulates the acetyltransferase activity of Gcn5 is still unknown. Here, I have studied the interplay between H3K4 trimethyl recognition and acetylation by Gcn5, and have developed a new technique for monitoring crosstalk between lysine acetylation and other histone post-translational modifications. Whereas Gcn5 is only slightly faster on H3K4me3 compared to unmodified nucleosomes, H3K4 trimethyl recognition by Sgf29 promotes processive acetylation by the HAT module, explaining how co-localization between hyperacetylation and H3K4 trimethylation is established at gene promoters on a mechanistic level. Sirtuins, which catalyze NAD+-dependent lysine deacylation, represent the second class of enzymes studied in this dissertation. In light of recent studies demonstrating that many sirtuins remove other acyl modifications than acetylation, we revisited the lysine specificity of four bacterial and archaeal enzymes originally identified as deacetylases using biochemistry and x-ray crystallography. Out of the four sirtuins tested, three preferentially removed acyl modifications other than acetylation from lysine. Crystal structures of Sir2Af1 bound to a succinylated peptide and Sir2Af2 bound to a myristoylated peptide revealed structural determinants of acyl chain specificity, including an unexpected conformational change allowing Sir2Af2 to accommodate a myristoyl chain in its active site. These results identify desuccinylation and demyristoylation as activities catalyzed by archaeal sirtuins, and emphasize the importance of structural and biochemical studies to our understanding of sirtuin specificity.