Somatic mutations that perturb Parkin ubiquitin ligase activity and the misregulation of iron homeostasis have both been linked to Parkinson’s disease. Lactotransferrin (LTF) is a member of the family of transferrin iron binding proteins that regulate iron homeostasis, and increased levels of LTF and its receptor have been observed in neurodegenerative disorders like Parkinson’s disease. Here, we report that Parkin binds to LTF and ubiquitylates LTF to influence iron homeostasis. Parkin-dependent ubiquitylation of LTF occurred most often on lysines (K) 182 and 649. Substitution of K182 or K649 with alanine (K182A or K649A, respectively) led to a decrease in the level of LTF ubiquitylation, and substitution at both sites led to a major decrease in the level of LTF ubiquitylation. Importantly, Parkin-mediated ubiquitylation of LTF was critical for regulating intracellular iron levels as overexpression of LTF ubiquitylation site point mutants (K649A or K182A/K649A) led to an increase in intracellular iron levels measured by ICP-MS/MS. Consistently, RNAi-mediated depletion of Parkin led to an increase in intracellular iron levels in contrast to overexpression of Parkin that led to a decrease in intracellular iron levels. Together, these results indicate that Parkin binds to and ubiquitylates LTF to regulate intracellular iron levels. These results expand our understanding of the cellular processes that are perturbed when Parkin activity is disrupted and more broadly the mechanisms that contribute to Parkinson’s disease.

中文翻译：

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通过 Parkin 介导的乳铁蛋白泛素化调节铁稳态。

扰乱帕金森泛素连接酶活性的体细胞突变和铁稳态的失调都与帕金森病有关。乳转铁蛋白 (LTF) 是转铁蛋白铁结合蛋白家族的成员，可调节铁稳态，在帕金森病等神经退行性疾病中观察到 LTF 及其受体水平升高。在这里，我们报告 Parkin 与 LTF 结合并泛素化 LTF 以影响铁稳态。LTF 的 Parkin 依赖性泛素化最常发生在赖氨酸 (K) 182 和 649 上。用丙氨酸取代 K182 或 K649（分别为 K182A 或 K649A）导致 LTF 泛素化水平降低，两个位点的取代导致LTF泛素化水平显着下降。重要的，Parkin 介导的 LTF 泛素化对于调节细胞内铁水平至关重要，因为 LTF 泛素化位点突变体（K649A 或 K182A/K649A）的过表达导致通过 ICP-MS/MS 测量的细胞内铁水平增加。一致地，与导致细胞内铁水平降低的Parkin过表达相比，RNAi介导的Parkin耗竭导致细胞内铁水平增加。总之，这些结果表明 Parkin 与 LTF 结合并泛素化以调节细胞内铁水平。这些结果扩展了我们对帕金森活动被破坏时受到干扰的细胞过程以及更广泛地导致帕金森病的机制的理解。