Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Biochemistry. 2010 Jan 19;49(2):304-11. doi: 10.1021/bi901627u.
A member of the sirtuin family of NAD(+)-dependent deacetylases, SIRT3, is identified as one of the major mitochondrial deacetylases located in mammalian mitochondria responsible for deacetylation of several metabolic enzymes and components of oxidative phosphorylation. Regulation of protein deacetylation by SIRT3 is important for mitochondrial metabolism, cell survival, and longevity. In this study, we identified one of the Complex II subunits, succinate dehydrogenase flavoprotein (SdhA) subunit, as a novel SIRT3 substrate in SIRT3 knockout mice. Several acetylated Lys residues were mapped by tandem mass spectrometry, and we determined the role of acetylation in Complex II activity in SIRT3 knockout mice. In agreement with SIRT3-dependent activation of Complex I, we observed that deacetylation of the SdhA subunit increased the Complex II activity in wild-type mice. In addition, we treated K562 cell lines with nicotinamide and kaempferol to inhibit deacetylase activity of SIRT3 and stimulate SIRT3 expression, respectively. Stimulation of SIRT3 expression decreased the level of acetylation of the SdhA subunit and increased Complex II activity in kaempherol-treated cells compared to control and nicotinamide-treated cells. Evaluation of acetylated residues in the SdhA crystal structure from porcine and chicken suggests that acetylation of the hydrophilic surface of SdhA may control the entry of the substrate into the active site of the protein and regulate the enzyme activity. Our findings constitute the first evidence of the regulation of Complex II activity by the reversible acetylation of the SdhA subunit as a novel substrate of the NAD(+)-dependent deacetylase, SIRT3.
Sirtuin 家族是 NAD(+)依赖的去乙酰化酶家族的一员，SIRT3 是哺乳动物线粒体中主要的线粒体去乙酰化酶之一，负责对几种代谢酶和氧化磷酸化成分进行去乙酰化。SIRT3 对蛋白质去乙酰化的调节对于线粒体代谢、细胞存活和寿命至关重要。在这项研究中，我们在 SIRT3 敲除小鼠中鉴定出复合物 II 的一个亚基，琥珀酸脱氢酶黄素蛋白（SdhA）亚基，为 SIRT3 的一个新的底物。通过串联质谱分析鉴定了几个乙酰化赖氨酸残基，并确定了 SIRT3 敲除小鼠中复合物 II 活性的乙酰化作用。与 SIRT3 依赖性的复合物 I 激活一致，我们观察到 SdhA 亚基的去乙酰化增加了野生型小鼠中的复合物 II 活性。此外，我们用烟酰胺和山奈酚处理 K562 细胞系，以抑制 SIRT3 的去乙酰化酶活性和分别刺激 SIRT3 的表达。与对照和烟酰胺处理的细胞相比，刺激 SIRT3 的表达降低了 SdhA 亚基的乙酰化水平，并增加了山奈酚处理细胞中的复合物 II 活性。评估来自猪和鸡的 SdhA 晶体结构中的乙酰化残基表明，SdhA 的亲水性表面的乙酰化可能控制底物进入蛋白质的活性部位，并调节酶活性。我们的发现构成了第一个证据，表明复合物 II 活性受到 SdhA 亚基的可逆乙酰化调节，SdhA 亚基是 NAD(+)依赖的去乙酰化酶 SIRT3 的一个新底物。
