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首页> 外文期刊>Biochemistry >Lysine Acetylation Activates Mitochondrial Aconitase in the Heart
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Lysine Acetylation Activates Mitochondrial Aconitase in the Heart

机译：赖氨酸乙酰化激活心脏中的线粒体乌头酸酶

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High-throughput proteomics studies have identified several thousand acetylation sites on more than 1000 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-coenzyme A resulted in increased aconitase activity that was reversed with SIRT3 treatment. Quantitative mass spectrometry was used to measure acetylation at 21 lysine residues and revealed significant increases with both in vitro treatments. A high-fat diet (60% of kilocalories 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 an increased level of 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.
机译：高通量蛋白质组学研究已经鉴定出1000多种蛋白质上的数千个乙酰化位点。线粒体乌头酸酶是一种将柠檬酸转化为异柠檬酸的克雷布斯循环酶，已在许多此类报告中得到了证实。乙酰化线粒体乌头酸酶也已被确定为瑟土因3（SIRT3）催化脱乙酰化的目标。但是，尚未确定线粒体乌头酸酶乙酰化的功能意义。使用体外策略，质谱分析和肥胖症的体内小鼠模型，我们发现了乌头酸酶的显着乙酰化依赖性激活。离体心脏线粒体经过乙酸酐或乙酰辅酶A的体外化学乙酰化处理后，乌头酸酶活性增加，而SIRT3处理则可以逆转。定量质谱法用于测量21个赖氨酸残基处的乙酰化程度，并显示两种体外处理均显着增加。高脂饮食（60卡路里的热量来自脂肪）被用作体内模型，还显示线粒体乌头酸酶活性显着增加，而蛋白质水平没有变化。高脂肪饮食还通过定量质谱分析测定了多个部位乌头酸乙酰化水平的提高。用SIRT3处理这些小鼠分离的线粒体，消除了高脂饮食诱导乌头酸酶的活化并减少了乙酰化。最后，动力学分析发现活性的增加是最大速度增加的结果，分子模型表明在K144发生乙酰化作用可能会扰乱酶的三级结构。这项研究的结果揭示了一种新的乙酰化激活线粒体乌头酸酶的方法。

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