Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Harry Hines Blvd, Dallas, TX, United States of America.
PLoS Pathog. 2018 Oct 26;14(10):e1007404. doi: 10.1371/journal.ppat.1007404. eCollection 2018 Oct.
Polyamines are essential for cell growth of eukaryotes including the etiologic agent of human African trypanosomiasis (HAT), Trypanosoma brucei. In trypanosomatids, a key enzyme in the polyamine biosynthetic pathway, S-adenosylmethionine decarboxylase (TbAdoMetDC) heterodimerizes with a unique catalytically-dead paralog called prozyme to form the active enzyme complex. In higher eukaryotes, polyamine metabolism is subject to tight feedback regulation by spermidine-dependent mechanisms that are absent in trypanosomatids. Instead, in T. brucei an alternative regulatory strategy based on TbAdoMetDC prozyme has evolved. We previously demonstrated that prozyme protein levels increase in response to loss of TbAdoMetDC activity. Herein, we show that prozyme levels are under translational control by monitoring incorporation of deuterated leucine into nascent prozyme protein. We furthermore identify pathway factors that regulate prozyme mRNA translation. We find evidence for a regulatory feedback mechanism in which TbAdoMetDC protein and decarboxylated AdoMet (dcAdoMet) act as suppressors of prozyme translation. In TbAdoMetDC null cells expressing the human AdoMetDC enzyme, prozyme levels are constitutively upregulated. Wild-type prozyme levels are restored by complementation with either TbAdoMetDC or an active site mutant, suggesting that TbAdoMetDC possesses an enzyme activity-independent function that inhibits prozyme translation. Depletion of dcAdoMet pools by three independent strategies: inhibition/knockdown of TbAdoMetDC, knockdown of AdoMet synthase, or methionine starvation, each cause prozyme upregulation, providing independent evidence that dcAdoMet functions as a metabolic signal for regulation of the polyamine pathway in T. brucei. These findings highlight a potential regulatory paradigm employing enzymes and pseudoenzymes that may have broad implications in biology.
多胺是真核生物细胞生长所必需的，包括引起人类非洲锥虫病（HAT）的病原体布氏锥虫。在锥虫中，多胺生物合成途径中的关键酶 S-腺苷甲硫氨酸脱羧酶（TbAdoMetDC）与一种独特的无催化活性的同工酶 prozyme 异二聚化，形成活性酶复合物。在高等真核生物中，多胺代谢受到依赖于 spermidine 的严格反馈调节，而这种调节在锥虫中不存在。相反，在 T. brucei 中，已经进化出一种基于 TbAdoMetDC prozyme 的替代调节策略。我们之前证明，prozyme 蛋白水平会随着 TbAdoMetDC 活性的丧失而增加。在此，我们通过监测氘标记亮氨酸掺入新生 prozyme 蛋白来显示 prozyme 水平受翻译控制。此外，我们确定了调节 prozyme mRNA 翻译的途径因素。我们发现了一种调节反馈机制的证据，其中 TbAdoMetDC 蛋白和脱羧化的 AdoMet（dcAdoMet）作为 prozyme 翻译的抑制剂。在表达人 AdoMetDC 酶的 TbAdoMetDC 缺失细胞中，prozyme 水平持续上调。用 TbAdoMetDC 或活性位点突变体进行互补可恢复野生型 prozyme 水平，这表明 TbAdoMetDC 具有独立于酶活性的功能，可抑制 prozyme 翻译。通过三种独立策略耗尽 dcAdoMet 池：抑制/敲低 TbAdoMetDC、敲低 AdoMet 合酶或蛋氨酸饥饿，每种策略都会导致 prozyme 上调，这提供了独立的证据表明 dcAdoMet 作为调节 T. brucei 多胺途径的代谢信号。这些发现突出了一种可能的调节范例，该范例可能在生物学中具有广泛的意义。
