Knyphausen Philipp, de Boor Susanne, Kuhlmann Nora, Scislowski Lukas, Extra Antje, Baldus Linda, Schacherl Magdalena, Baumann Ulrich, Neundorf Ines, Lammers Michael
From the Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), Joseph-Stelzmann-Strasse 26, University of Cologne, 50931 Cologne and.
the Institute for Biochemistry, Zülpicher Strasse 47b, University of Cologne, 50674 Cologne, Germany.
J Biol Chem. 2016 Jul 8;291(28):14677-94. doi: 10.1074/jbc.M116.726307. Epub 2016 May 18.
Sirtuins are NAD(+)-dependent lysine deacylases, regulating a variety of cellular processes. The nuclear Sirt1, the cytosolic Sirt2, and the mitochondrial Sirt3 are robust deacetylases, whereas the other sirtuins have preferences for longer acyl chains. Most previous studies investigated sirtuin-catalyzed deacylation on peptide substrates only. We used the genetic code expansion concept to produce natively folded, site-specific, and lysine-acetylated Sirt1-3 substrate proteins, namely Ras-related nuclear, p53, PEPCK1, superoxide dismutase, cyclophilin D, and Hsp10, and analyzed the deacetylation reaction. Some acetylated proteins such as Ras-related nuclear, p53, and Hsp10 were robustly deacetylated by Sirt1-3. However, other reported sirtuin substrate proteins such as cyclophilin D, superoxide dismutase, and PEPCK1 were not deacetylated. Using a structural and functional approach, we describe the ability of Sirt1-3 to deacetylate two adjacent acetylated lysine residues. The dynamics of this process have implications for the lifetime of acetyl modifications on di-lysine acetylation sites and thus constitute a new mechanism for the regulation of proteins by acetylation. Our studies support that, besides the primary sequence context, the protein structure is a major determinant of sirtuin substrate specificity.
沉默调节蛋白是依赖烟酰胺腺嘌呤二核苷酸（NAD⁺）的赖氨酸脱酰基酶，可调节多种细胞过程。细胞核中的Sirt1、细胞质中的Sirt2和线粒体中的Sirt3是强大的脱乙酰酶，而其他沉默调节蛋白则更倾向于作用于更长的酰基链。此前大多数研究仅调查了沉默调节蛋白对肽底物的催化脱酰基作用。我们利用遗传密码扩展概念来生产天然折叠、位点特异性且赖氨酸乙酰化的Sirt1 - 3底物蛋白，即Ras相关核蛋白、p53、磷酸烯醇式丙酮酸羧激酶1（PEPCK1）、超氧化物歧化酶、亲环蛋白D和热休克蛋白10（Hsp10），并分析了脱乙酰化反应。一些乙酰化蛋白，如Ras相关核蛋白、p53和Hsp10，能被Sirt1 - 3有效地脱乙酰化。然而，其他已报道的沉默调节蛋白底物蛋白，如亲环蛋白D、超氧化物歧化酶和PEPCK1，并未发生脱乙酰化。通过结构和功能研究方法，我们描述了Sirt1 - 3对两个相邻乙酰化赖氨酸残基进行脱乙酰化的能力。这一过程的动力学对双赖氨酸乙酰化位点上乙酰化修饰的寿命有影响，因此构成了一种通过乙酰化调节蛋白质的新机制。我们的研究支持，除了一级序列背景外，蛋白质结构是沉默调节蛋白底物特异性的主要决定因素。
