Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Proc Natl Acad Sci U S A. 2012 Apr 17;109(16):E916-25. doi: 10.1073/pnas.1121471109. Epub 2012 Apr 2.
Although the biological roles of many members of the sirtuin family of lysine deacetylases have been well characterized, a broader understanding of their role in biology is limited by the challenges in identifying new substrates. We present here an in vitro method that combines biotinylation and mass spectrometry (MS) to identify substrates deacetylated by sirtuins. The method permits labeling of deacetylated residues with amine-reactive biotin on the ε-nitrogen of lysine. The biotin can be utilized to purify the substrate and identify the deacetylated lysine by MS. The biotinyl-lysine method was used to compare deacetylation of chemically acetylated histones by the yeast sirtuins, Sir2 and Hst2. Intriguingly, Sir2 preferentially deacetylates histone H3 lysine 79 as compared to Hst2. Although acetylation of K79 was not previously reported in Saccharomyces cerevisiae, we demonstrate that a minor population of this residue is indeed acetylated in vivo and show that Sir2, and not Hst2, regulates the acetylation state of H3 lysine 79. The in vitro biotinyl-lysine method combined with chemical acetylation made it possible to identify this previously unknown, low-abundance histone acetyl modification in vivo. This method has further potential to identify novel sirtuin deacetylation substrates in whole cell extracts, enabling large-scale screens for new deacetylase substrates.
尽管赖氨酸去乙酰化酶 sirtuin 家族的许多成员的生物学作用已经得到了很好的描述，但由于鉴定新底物的挑战，人们对其在生物学中的作用的理解仍然有限。我们在此提出了一种体外方法，该方法结合了生物素化和质谱 (MS) 技术，用于鉴定 sirtuins 去乙酰化的底物。该方法允许赖氨酸 ε-氮上的反应性胺基将去乙酰化残基标记上生物素。生物素可以用于通过 MS 纯化底物并鉴定去乙酰化的赖氨酸。我们使用生物素化赖氨酸方法比较了酵母 sirtuins Sir2 和 Hst2 对化学乙酰化组蛋白的去乙酰化作用。有趣的是，与 Hst2 相比，Sir2 优先去乙酰化组蛋白 H3 赖氨酸 79。尽管此前在酿酒酵母中未报道过 K79 的乙酰化，但我们证明该残基的一小部分确实在体内被乙酰化，并且表明 Sir2 而不是 Hst2 调节 H3 赖氨酸 79 的乙酰化状态。体外生物素化赖氨酸方法与化学乙酰化相结合，使得鉴定这种以前未知的低丰度组蛋白乙酰化修饰成为可能。该方法还有进一步的潜力，可以在全细胞提取物中鉴定新的 sirtuin 去乙酰化底物，从而实现新去乙酰化酶底物的大规模筛选。
