A Directed Evolution System for Lysine Deacetylases
Lysine acetylation is a ubiquitous modification permeating the proteomes of organisms from all domains of life. Lysine deacetylases (KDACs) reverse this modification by following two fundamentally different enzymatic mechanisms, which differ mainly by the
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Introduction Lysine acetylation was first discovered on histone proteins and linked to transcriptional regulation by neutralizing the positive charge of lysine, thereby loosening the DNA–histone interaction [1]. The reversible nature of this process was first suggested in 1977 due to a spike in histone acetylation observed upon n-butyrate treatment [2]. Twenty years later, the responsible enzyme, histone deacetylase 1 (HDAC1), was isolated by affinity chromatography, and HDAC2 was independently identified by sequence homology to yeast Rpd3 as a transcription factor [3, 4]. This elicited interest in this enzyme family and led to the discovery of 16 further human KDACs [5]. KDACs are divided into two families (histone deacetylases and sirtuins) and four classes (I–IV) based on their reaction mechanism and sequence homology. Classes I, II, and IV consist of histone
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247, https://doi.org/10.1007/978-1-0716-1126-5_18, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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deacetylases that use a catalytic zinc ion to hydrolyze the peptide bond, while class III enzymes, the sirtuins, use NAD+ in a unique mechanism for cleavage [6, 7]. While histones were the first reported substrates of KDACs, thousands of other proteins and several new modifications have now been identified in mammals [8], plants [9, 10], fungi [11] and bacteria [12]. Among the identified proteins are important cellular components, for example, p53 as substrate of Sirt1 [13] and HDAC1 [14] and α-tubulin of HDAC6 [15]. In addition to the large number of protein substrates, it was found that various lysine modifications (butyryl, crotonyl, malonyl, etc.) can be reversed by KDACs [8]. While the function of many of these modifications is still unclear, it has been shown that they possess their own set of effector proteins such as readers [16], writers [17], and eraser [18]. Due to the promiscuous activity of KDACs to various substrates, regulation plays an important role, which in the case of KDACs often means that they are part of larger protein complexes, for example the transcriptional regulators CoREST [19], mSIN3 [20], NuRD [21], or NCoR/SMRT [22]. While most substrate proteins have long been known, the role of individual modifications and their connection to particular KDACs are still unclear. Problems arise from the high substrate overlap of the different KDACs, the vast number of modified proteins and the questionable significance of many acylations, since the modification reaction can also take place non-enzymatically on proteins [23]. Here we describe a powerful bacterial selection system to isolate KDACs by their activity and selectivity. Using genetic code expansion, we create an acylation-responsive bifunctional auxotrophic marker, Ura3 K93ac, to couple cell growth to KDAC activity. Applying the same general concept, we develop a straightforward luciferase assay to detect various deacylas
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