Lysine Acetylation Activates Mitochondrial Aconitase in the Heart.
Lysine Acetylation Activates Mitochondrial Aconitase in the Heart.
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DOI：
10.1021/acs.biochem.5b00375
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发表时间：
2015-06-30
期刊：
影响因子：
2.9
通讯作者：
Kinter M
中科院分区：
文献类型：
--
作者：
Fernandes J;Weddle A;Kinter CS;Humphries KM;Mather T;Szweda LI;Kinter M
高通量蛋白质组学研究已经在超过一千种蛋白质上鉴定了几千个乙酰化位点。线粒体顺乌头酸酶是将柠檬酸盐转化为异柠檬酸盐的克雷布斯循环酶，已在许多这些报告中鉴定。乙酰化线粒体顺乌头酸酶也已被鉴定为沉默调节蛋白3（SIRT3）催化的脱乙酰化的靶标。然而，线粒体乌头酸酶乙酰化的功能意义尚未确定。使用体外策略、质谱分析和体内肥胖小鼠模型，我们发现顺乌头酸酶具有显着的乙酰化依赖性激活。用乙酸酐或乙酰辅酶A进行体外化学乙酰化的分离的心脏线粒体导致乌头酸酶活性增加，而SIRT3治疗则逆转了这一点。使用定量质谱法测量21个赖氨酸残基的乙酰化，发现两种体外处理均显著增加。高脂饮食（60%千卡来自脂肪）被用作体内模型，也显示出线粒体顺乌头酸酶活性显著增加，而蛋白质水平没有变化。高脂饮食也产生了增加乌头酸酶乙酰化在多个网站，如通过定量质谱测定。用SIRT3处理从这些小鼠分离的线粒体，消除了高脂饮食诱导的乌头酸酶活化和乙酰化减少。最后，动力学分析发现，活性的增加是最大速度增加的结果，分子模拟表明K144处的乙酰化可能会扰乱酶的三级结构。本研究的结果揭示了一种新的激活线粒体乌头酸酶的乙酰化。
High throughput proteomics studies have identified several thousand acetylation sites on over one thousand proteins. Mitochondrial aconitase, the Krebs cycle enzyme that converts citrate to isocitrate, has been identified in many of these reports. Acetylated mitochondrial aconitase has also been identified as a target for sirtuin 3 (SIRT3) catalyzed deacetylation. However, the functional significance of mitochondrial aconitase acetylation has not been determined. Using in vitro strategies, mass spectrometric analyses, and an in vivo mouse model of obesity, we found a significant acetylation-dependent activation of aconitase. Isolated heart mitochondria subjected to in vitro chemical acetylation with either acetic anhydride or acetyl-CoA resulted in increased aconitase activity that was reversed with SIRT3 treatment. Quantitative mass spectrometry was used to measure acetylation at 21 lysine residues and found significant increases with both in vitro treatments. A high fat diet (60% kcal from fat) was used as an in vivo model and also showed significantly increased mitochondrial aconitase activity without changes in protein level. The high fat diet also produced increased aconitase acetylation at multiple sites as measured by the quantitative mass spectrometry assays. Treatment of isolated mitochondria from these mice with SIRT3 abolished the high fat diet-induced activation of aconitase and reduced acetylation. Finally, kinetic analyses found that the increase in activity was a result of increased maximal velocity and molecular modeling suggests the potential for acetylation at K144 to perturb the tertiary structure of the enzyme. The results of this study reveal a novel activation of mitochondrial aconitase by acetylation.