Iron-Sulfur Center Regulation and Crosstalk of two Radical SAM Modifiers by one Electron Transfer Protein in Yeast?

自由基SAM酶中的铁硫（FeS）中心使用电子进行还原SAM切割和自由基形成，这些化学反应对所有细胞（包括我们自己的细胞）都至关重要。因此，自由基SAM酶的缺陷可导致人类疾病。例如，卵巢癌与用于EF 2的二苯二甲酰胺修饰的自由基SAM酶（Dph 1-Dph 2）的突变有关，EF 2是真核生物中的一种必需mRNA翻译因子，而Elongator（一种tRNA反密码子修饰复合物，具有自由基SAM催化亚基（Elp 3））的缺陷与严重的神经病变有关。为了确保EF 2和tRNA的正常功能，这两种修饰剂应该始终处于活性状态。然而，数据显示修改确实发生了变化，表明情况并非如此。有趣的是，这两种酶共享一个电子转移蛋白：Kti 11（又名Dph 3）。Dph 3/Kti 11向Dph 1-Dph 2提供电子，并与Kti 13复合，参与向Elp 3的电子流。这表明两个自由基SAM酶的调节通过电子流到（和从）他们的FeS中心。原则上，Kti 13-Kti 11/Dph 3复合物可以将电子馈送到两种修饰途径中或将它们限制在Elongator（Elp 3）中。虽然有证据支持任一选项，但Kti 13在联苯二酰胺途径中的相关性是没有实际意义的，并且还不能排除两种模型的组合，其中Kti 13支持电子流到两种自由基SAM酶，但对每种酶具有不同的贡献。我们的目的是研究Kti 13的确切作用及其在Dph 1-Dph 2或Elongator（Elp 3）络合物中引导电子从Kti 11/Dph 3流向FeS中心的潜力。使用酵母作为真核生物的模型，这将提供新的见解，适当调节自由基SAM催化和防止非生物电子流到随机的，潜在的有害受体。此外，与tRNA和EF 2修饰剂被连接到mRNA翻译，阐明电子流的模式（和从）他们的FeS中心可以告知两个自由基SAM酶之间的功能性串扰。为了实现这些目标，我们将回答以下问题：-Kti 13可以引导电子从Kti 11/Dph 3到不同酶（Elp 3和/或Dph 1-Dph 2）中的FeS中心吗？Kti 11/Dph 3（和Kti 13）功能突变的分离是否揭示了不同的FeS中心调控？两种自由基SAM酶通过Kti 11/Dph 3或Kti 13在mRNA翻译中是否存在串扰？由于钼辅因子（MOCO）合成的S-转移和与自由基SAM化学密切相关的延伸子依赖性tRNA的硫醇化以及与人类健康状况相关的MOCO和修饰的tRNA的不适当水平，我们的项目在长期内具有生物医学意义。因此，它完全符合SPP 1927的重点铁硫生命，并与参与mRNA翻译的自由基SAM酶的功能和适当调节有关，这是所有细胞（包括我们自己的细胞）蛋白质合成的重要步骤。

Iron-sulfur (FeS) centers in radical SAM enzymes use electrons for reductive SAM cleavage and radical formation in numerous biochemical reactions that are crucial for all cells (including our own). Accordingly, defects in radical SAM enzymes can cause disease in humans. For instance, ovarian cancer is linked to mutations in a radical SAM enzyme (Dph1-Dph2) for diphthamide modification of EF2, an essential mRNA translation factor in eukaryotes, and defects in Elongator, a tRNA anticodon modifying complex with a radical SAM catalytic subunit (Elp3), are associated with severe neuropathies. To ensure proper EF2 and tRNA functioning, both modifiers ought to be active at all times. However, data showing the modifications do change, suggest otherwise. Intriguingly, both enzymes share one electron transfer protein: Kti11 (aka Dph3). Dph3/Kti11 donates electrons to Dph1-Dph2, and in complex with Kti13, is implicated in electron flow to Elp3. This suggests regulation of two radical SAM enzymes via electron flow to (and from) their FeS centers. In principle, the Kti13-Kti11/Dph3 complex may feed electrons into both modification pathways or restrict them to Elongator (Elp3). Although there is evidence to support either option, the relevance of Kti13 in the diphthamide pathway is moot, and a combination of both models, where Kti13 supports electron flow to both radical SAM enzymes but with a differential contribution to each, cannot be excluded yet.Hence, we aim to study the precise role of Kti13 and its potential to guide electron flow from Kti11/Dph3 to FeS centers in the Dph1-Dph2 or Elongator (Elp3) complexes. Using yeast as a model eukaryote, this will provide novel insights into proper regulation of radical SAM catalysis and prevention of non-biological electron flow to random, potentially harmful acceptors. Moreover, with the tRNA and EF2 modifiers being linked to mRNA translation, elucidation of the mode of electron flow to (and from) their FeS centers may inform about functional cross-talk among both radical SAM enzymes. To achieve these aims we will answer the following questions:- Can Kti13 guide electrons from Kti11/Dph3 to FeS centers in different enzymes (Elp3 and/or Dph1-Dph2)?- Do separation of Kti11/Dph3 (and Kti13) function mutations reveal differential FeS center regulation?- Is there cross-talk among both radical SAM enzymes in mRNA translation via Kti11/Dph3 or Kti13?With S-transfer for molybdenum cofactor (MOCO) synthesis and thiolation of Elongator dependent tRNAs intimately linked to radical SAM chemistry and inappropriate levels of MOCO and modified tRNAs related to human health conditions, our project is biomedically significant in the long term. So it neatly fits the SPP1927 focus Iron-Sulfur for Life and is relevant with regards to function and proper regulation of radical SAM enzymes involved in mRNA translation, an essential step in protein synthesis by all cells (including our own).