Quantitative Acetylome Analysis Reveals the Roles of SIRT1 in Regulating Diverse Substrates and Cellular Pathways

Abstract

Despite of the progress in identifying many Lysine (Lys)1 acetylation is a dynamic, reversible, and evolutionarily conserved protein post-translational modification (PTM). After the discovery of Lys acetylation in histones more than forty years ago (1), early studies mainly focused on histones and transcription factors, establishing the modification's fundamental role in DNA-templated biological processes (2, 3). The discovery of Lys acetylation in tubulin and the presence of sirtuins in mitochondria argued that Lys acetylation may not be restricted to nuclei (4 -6). The complexity of Lys acetylomes outside the nuclei and the high abundance of the PTM in mitochondria revealed by proteomics studies suggested that the regulatory functions of this PTM may mirror those of protein phosphorylation (7-9).Lys acetylation is regulated by two groups of enzymes with opposing activities, lysine acetyltransferases and deacetylases (10). Sirtuins are a family of highly conserved NAD ϩ -dependent deacetylases (11,12). Numerous studies, mainly focusing on the family's founding member, SIRT1 in mammals or Sir2, the enzyme's homolog in yeast, show that sirtuins regulate diverse cellular functions and appear to affect a variety of aging-related diseases, such as cancer, metabolic diseases, and inflammation and are involved in pathways such as metabolisms, oxidative stress, DNA damage, cell cycle, and signaling (12-15). A number of protein substrates for SIRT1 have been identified including p53, DNA methyltransferase 1 (DNMT1), NF-B, forkhead transcription factors, PGC-1␣, and histones (16 -22). Despite significant progress in the past decade, the molecular mechanisms by which SIRT1 regulates cellular physiology are not well understood. A major hurdle in our understanding of SIRT1 biology is our incomplete knowledge of Lys acetylation proteins that mediate SIRT1 functions. Because SIRT1 is a lysine deacetylase, a major mechanism for SIRT1 to exert its functions should be its deacetylation activity. However, SIRT1-induced lysine acetylation proteins are far from complete. This knowledge limitation also exists with other deacetylases as well as with lysine acetyltransferases, representing a major challenge in Lys acetylation biology and in evaluating clinical compounds that target dysregulated Lysacetylation regulatory enzymes.Here we report the first proteomics quantification of Lys acetylation in response to a regulatory enzyme. Our study identified 4623 Lys acetylation sites from SIRT1 ϩ/ϩ and SIRT1 Ϫ/Ϫ MEF cells, among which 4130 Lys acetylation sites were quantified. The data identified multiple pathways that are affected by SIRT1. From this wealth of new information on the SIRT1-modulated Lys acetylome, we discovered consensus motifs for SIRT1-mediated Lys acetylation sites and identified extensive correlation between Lys acetylation and phosphorylation, SUMOylation, and mutations associated with disease,

Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

“…However, Ran has recently been shown to be lysine acetylated at five distinct sites in human (K37, K60, K71, K99, and K159) (22). The lysine acetylation sites were found independently by several studies in different species using highly sensitive quantitative MS (22)(23)(24)(25)(26). K37 is located within switch I, K60 in the β3-strand preceding switch II, K71 in switch II, K99 in α-helix three (α3), and K159 in α5 C-terminal to the 150-SAK-152 motif interacting with the nucleotide base (Fig.…”

Succinylome Analysis Reveals the Involvement of Lysine Succinylation in Metabolism in Pathogenic Mycobacterium tuberculosis*

“…Among the hundreds of different PTMs, acylations at lysine residues, such as acetylation (3)(4)(5)(6), malonylation (7,8), crotonylation (9,10), propionylation (11)(12)(13), butyrylation (11,13), and succinylation (7, 14 -16) are crucial for functional regulations of many prokaryotic and eukaryotic proteins. Because these lysine PTMs depend on the acyl-CoA metabolic intermediates, such as acetyl-CoA (Ac-CoA), succinyl-CoA, and malonylCoA, lysine acylation could provide a mechanism to respond to changes in the energy status of the cell and regulate energy metabolism and the key metabolic pathways in diverse organisms (17,18).…”

Regulation of S-Adenosylhomocysteine Hydrolase by Lysine Acetylation

“…Protein lysine acetylation is now understood to be a very abundant post-translational modification in the cell, with thousands of proteins and specific sites being recently identified using mass spectrometry (29,43,44). A large number of these acetylation events have been linked to metabolic enzymes inside and outside of the mitochondria.…”