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 20min of ischemia and 90-min 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.

中文翻译：

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心脏琥珀酸组的特征及其在缺血再灌注损伤中的作用。

琥珀酰化是指用琥珀酰辅酶A提供的琥珀酰基团修饰赖氨酸残基。Sirtuin5 (Sirt5) 是一种线粒体 NAD(+) 依赖性脱酰基酶，可催化从蛋白质中去除琥珀酰基。Sirt5 和蛋白质琥珀酰化在物种间是保守的，表明修饰的功能重要性。Sirt5 丢失会影响肝脏代谢，但尚未探索琥珀酰化在心脏中的作用。我们将亲和富集与蛋白质组学和质谱法相结合，分析从 WT 和 Sirt5(-/-) 小鼠心脏中分离的线粒体的总琥珀酰赖氨酸含量。我们在 184 种蛋白质中鉴定了 887 个琥珀酰化赖氨酸残基。44 种肽（5 种蛋白质）在 WT 样品中独特出现，289 种（46 种蛋白质）在 Sirt5(-/-) 样品中出现，554 种（133 种蛋白质）是两组共有的。Sirt5(-/-) 心脏中的 46 种独特蛋白质参与代谢过程，如脂肪酸 β-氧化 (Eci2) 和支链氨基酸分解代谢，包括呼吸链蛋白质（Ndufa7、12、13、Dhsa）。我们对 WT 和 Sirt5(-/-) 心脏常见的肽进行了无标记分析。来自 9 种蛋白质的 16 种肽在 Sirt5(-/-) 中显着增加了至少 30%。腺嘌呤核苷酸转运蛋白 1 在 Sirt5(-/-) 中显示出最高的琥珀酰化增加（108.4 倍）。数据表明琥珀酰化在心脏中广泛存在并在代谢途径中丰富。我们检查了 Sirt5 的缺失是否会影响缺血再灌注 (I/R) 损伤，并且我们发现与 WT 同窝仔 (68.5(+)/-1.1% Sirt5(-) 相比，Sirt5(-/-) 心脏的梗死面积增加/-) 与 39.6(+)/(-) 6. 8% WT) 在缺血 20 分钟和再灌注 90 分钟后。我们进一步证明，通过用丙二酸二甲酯（一种琥珀酸脱氢酶 (SDH) 的竞争性抑制剂）预处理，Sirt5(-/-) 心脏中的 I/R 损伤恢复到 WT 水平，这表明 SDH 活性的改变是损伤的原因。