Protein lipoylation, a vital lysine posttranslational modification (PTM), plays a crucial role in the function of key mitochondrial TCA cycle enzymatic complexes. In eukaryotes, lipoyl PTM synthesis occurs exclusively through de novo pathways, relying on lipoyl synthesis/transfer enzymes, dependent upon mitochondrial fatty acid and Fe-S cluster biosynthesis. Dysregulation in any of these pathways leads to diminished cellular lipoylation. Efficient restoration of lipoylation in lipoylation deficiency cell states using either chemical or genetic approaches has been challenging due to pathway complexity and multiple upstream regulators. To address this challenge, we explored the possibility that a bacterial lipoate protein ligase (lplA) enzyme, that can salvage free lipoic acid bypassing the dependency on de novo synthesis, could be engineered to be functional in human cells. Overexpression of the engineered lplA in lipoylation null cells restored lipoylation levels, cellular respiration, and growth in low glucose conditions. Engineered lplA restored lipoylation in all tested lipoylation null cell models, mimicking defects in mitochondrial fatty acid synthesis (MECR KO), Fe-S cluster biosynthesis (BOLA3 KO), and specific lipoylation regulating enzymes (FDX1, LIAS and LIPT1 KOs). Furthermore, we describe a patient with a homozygous c.212C>T variant LIPT1 with a previously uncharacterized syndromic congenital sideroblastic anemia. K562 erythroleukemia cells engineered to harbor this missense LIPT1 allele recapitulate the lipoylation deficient phenotype and exhibit impaired proliferation in low glucose that is completely restored by engineered lplA. This synthetic approach offers a potential therapeutic strategy for treating lipoylation disorders.

中文翻译：

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工程细菌硫辛酸蛋白连接酶 A （lplA） 可恢复硫辛酰化缺陷细胞模型中的硫辛酰化。


蛋白质硫辛酰化是一种重要的赖氨酸翻译后修饰 （PTM），在关键线粒体 TCA 循环酶复合物的功能中起着至关重要的作用。在真核生物中，硫辛酰 PTM 合成仅通过从头途径发生，依赖于硫辛酰合成/转移酶，依赖于线粒体脂肪酸和 Fe-S 簇生物合成。这些途径中的任何一个失调都会导致细胞硫辛酰化减少。由于通路复杂性和多个上游调节因子，使用化学或遗传方法有效恢复硫辛酰化缺陷细胞状态下的硫辛酰化一直具有挑战性。为了应对这一挑战，我们探索了一种细菌硫辛酸蛋白连接酶 （lplA） 酶的可能性，该酶可以绕过对从头合成的依赖性来挽救游离硫辛酸，从而在人类细胞中发挥作用。在硫辛酰化无效细胞中过表达工程化 lplA 可恢复硫辛酰化水平、细胞呼吸和低葡萄糖条件下的生长。工程化 lplA 在所有测试的硫辛酰化无效细胞模型中恢复了硫辛酰化，模拟了线粒体脂肪酸合成 （MECR KO）、Fe-S 簇生物合成 （BOLA3 KO） 和特异性硫辛酰化调节酶（FDX1、LIAS 和 LIPT1 KOs）中的缺陷。此外，我们描述了一名患有纯合子 c.212C>T 变体 LIPT1 的患者，该患者患有先前未表征的综合征性先天性铁粒幼细胞性贫血。K562 红白血病细胞被工程改造为携带这种错义 LIPT1 等位基因，概括了硫辛酰化缺陷表型，并在低葡萄糖中表现出增殖受损，而工程改造的 lplA 完全恢复了这种细胞增殖。这种合成方法为治疗硫辛酰化障碍提供了一种潜在的治疗策略。