Characterization of the cardiac succinylome and its role in ischemia-reperfusion injury.
Characterization of the cardiac succinylome and its role in ischemia-reperfusion injury.
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DOI：
10.1016/j.yjmcc.2015.09.005
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发表时间：
2015-11
影响因子：
5
通讯作者：
Murphy E
中科院分区：
文献类型：
--
作者：
Boylston JA;Sun J;Chen Y;Gucek M;Sack MN;Murphy E
琥珀酰化是指用琥珀酰辅酶A提供的琥珀酰修饰赖氨酸残基。Sirtuin 5（Sirt 5）是一种线粒体NAD+依赖性脱酰酶，催化从蛋白质中去除琥珀酰基。Sirt 5和蛋白琥珀酰化在物种间是保守的，表明修饰的功能重要性。Sirt 5缺失影响肝脏代谢，但琥珀酰化在心脏中的作用尚未探索。我们将亲和富集与蛋白质组学和质谱法相结合，分析了从WT和Sirt 5 −/−小鼠心脏分离的线粒体的总琥珀酰赖氨酸含量。我们确定了887琥珀酰赖氨酸残基在184个蛋白质。44种肽（5种蛋白质）在WT样品中唯一出现，289种（46种蛋白质）在Sirt 5 −/−样品中出现，554种（133种蛋白质）在两组中共有。Sirt 5 −/−心脏中的46种独特蛋白质参与代谢过程，如脂肪酸β-氧化（Eci 2）和支链氨基酸催化剂，并包括呼吸链蛋白（Ndufa 7，12，13，Dhsa）。我们对WT和Sirt 5 −/−心脏共有的肽进行了无标记分析。Sirt 5-/-中来自9种蛋白质的16种肽显着增加了至少30%。腺嘌呤核苷酸转运蛋白1在Sirt 5 −/−中表现出最高的琥珀酰化增加（108.4倍）。这些数据表明，琥珀酰化在心脏中广泛存在，并在代谢途径中富集。我们检查了Sirt 5的缺失是否会影响缺血再灌注（I/R）损伤，我们发现在缺血20分钟和再灌注90分钟后，与WT同窝仔相比，Sirt 5 −/−心脏的梗死面积增加（68.5+/− 1.1%Sirt5 −/− vs 39.6+/− 6.8%WT）。我们进一步证明，在Sirt 5 −/−心脏的I/R损伤恢复到WT水平的预处理与丙二酸二甲酯，琥珀酸脱氢酶（SDH）的竞争性抑制剂，暗示SDH活性的改变作为损伤的原因。
Succinylation refers to modification of lysine residues with succinyl groups donated by succinyl-CoA. Sirtuin5 (Sirt5) is a mitochondrial NAD+-dependent deacylase that catalyzes the removal of succinyl groups from proteins. Sirt5 and protein succinylation are conserved across species, suggesting functional importance of the modification. Sirt5 loss impacts liver metabolism but the role of succinylation in the heart has not been explored. We combined affinity enrichment with proteomics and mass spectrometry to analyze total succinylated lysine content of mitochondria isolated from WT and Sirt5−/− mouse hearts. We identified 887 succinylated lysine residues in 184 proteins. 44 peptides (5 proteins) occurred uniquely in WT samples, 289 (46 proteins) in Sirt5−/− samples, and 554 (133 proteins) were common to both groups. The 46 unique proteins in Sirt5−/− heart participate in metabolic processes such as fatty acid β-oxidation (Eci2) and branched chain amino acid catabolism, and include respiratory chain proteins (Ndufa7, 12, 13, Dhsa). We performed label-free analysis of the peptides common to WT and Sirt5−/− hearts. 16 peptides from 9 proteins were significantly increased in Sirt5−/− by at least 30%. The adenine nucleotide transporter 1 showed the highest increase in succinylation in Sirt5−/− (108.4 fold). The data indicate that succinylation is widespread in the heart and enriched in metabolic pathways. We examined whether the loss of Sirt5 would impact ischemia-reperfusion (I/R) injury and we found an increase in infarct size in Sirt5−/− hearts compared to WT littermates (68.5+/− 1.1% Sirt5−/− vs 39.6+/− 6.8% WT) following 20 minutes of ischemia and 90 minutes reperfusion. We further demonstrate that I/R injury in Sirt5−/− heart is restored to WT levels by pretreatment with dimethyl malonate, a competitive inhibitor of succinate dehydrogenase (SDH), implicating alteration in SDH activity as causative of the injury.