Elucidating the dynamic links between iron, heme and steroid hormone biosynthesis in Drosophila.

阐明果蝇铁、血红素和类固醇激素生物合成之间的动态联系。

• 批准号: RGPIN-2018-04357
• 负责人: KingJones, Kirst
• 金额: $ 3.06万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=712954
• 关键词: Elucidating; dynamic; links; between; iron

The central idea of this proposal is that we can use the Drosophila prothoracic gland (PG) as a model to study unknown aspects of iron and steroid biology. This is because the synthesis of steroids relies on enzymes that require iron-containing cofactors such as heme and iron-sulfur (Fe-S) clusters. Importantly, both the steroidogenic enzymes and their iron cofactors are produced at very high levels in the PG to sustain steroid production. Thus, iron demand of steroidal glands such as the PG far exceeds that of most other tissues. Most steroid hormones are released as pulses that have a defined beginning, amplitude and end. This necessitates that iron mobilization from extra- or intracellular sources is in sync with the synthesis of steroid-producing enzymes. Our central hypothesis is that the production of steroids and the mobilization of iron must be coordinated by hitherto unidentified regulatory mechanisms to ensure that iron cofactors are produced in a timely fashion for an upcoming steroid pulse. We have recently identified the AGBE gene as a novel player required for normal iron metabolism in the Drosophila PG. Upon PG-specific disruption of AGBE function, heme precursors accumulate in the affected cells, resulting in a larval lethality. The lethality can be rescued by supplementing the diet with either iron or, surprisingly, the steroid precursor 7-dehydrocholesterol (7DC). AGBE is a well-characterized gene that has a single documented role, which is to add side branches to glycogen, suggesting that this protein has a “moonlighting” function in cellular iron homeostasis. Indeed, we were able to show that AGBE interacts physically with a known iron-regulatory protein called IRP1 (IRP1A in Drosophila). In this proposal, we discuss experiments to explore the functions of AGBE and IRP1A. Our approach includes making a series of mutant alleles for and transgenic constructs for AGBE, as well for IRP1A and its paralog IRP1B. These will allow us to conditionally remove the genes, add tags to the resulting proteins and generate classic mutants. We will use these genetically altered lines to conduct a range of experiments to molecularly characterize both AGBE and the two Drosophila IRP proteins. We also propose experiments to examine why 7DC appears to be equally effective in rescuing AGBE loss-of-function animals as iron. We suspect that 7DC mediates either iron mobilization or iron uptake into the mitochondria, and we are proposing experiments that test different possible mechanisms. We expect to make important contributions in two main areas, namely a) the coordination of iron homeostasis with steroid production and release, ensuring the normal progression of development and b) the mechanisms by which iron transport, import, and storage are controlled in a steroid hormone-producing tissue.

这项建议的中心思想是，我们可以使用果蝇前胸腺(PG)作为模型来研究铁和类固醇生物学的未知方面。这是因为类固醇的合成依赖于需要含铁辅助因子的酶，如血红素和铁-硫(Fe-S)簇。重要的是，类固醇生成酶及其铁辅助因子在PG中以非常高的水平产生，以维持类固醇的产生。因此，类固醇腺体，如PG，对铁的需求远远超过大多数其他组织。大多数类固醇激素是以脉冲的形式释放的，这些脉冲有明确的开始、幅度和结束。这就需要细胞外或细胞内来源的铁动员与类固醇产生酶的合成同步。我们的中心假设是，类固醇的产生和铁的动员必须由迄今未知的调节机制来协调，以确保铁辅助因子在即将到来的类固醇脉冲中以及时的方式产生。 我们最近发现AGBE基因是果蝇PG体内正常铁代谢所必需的一个新的成员。当PG特异性破坏AGBE功能时，血红素前体在受影响的细胞中积累，导致幼虫死亡。这种致命性可以通过补充铁或令人惊讶的类固醇前体7-脱氢胆固醇(7DC)来挽救。AGBE是一个特征明确的基因，只有一个已知的作用，那就是增加糖原的侧枝，这表明该蛋白在细胞铁稳态中具有“兼职”功能。事实上，我们能够证明AGBE与一种已知的铁调节蛋白IRP1(果蝇中的IRP1A)在物理上相互作用。 在这个建议中，我们讨论了探索AGBE和IRP1A功能的实验。我们的方法包括为AGBE以及IRP1A和其类似的IRP1B制造一系列突变等位基因和转基因构建体。这些将允许我们有条件地移除基因，为所产生的蛋白质添加标签，并产生经典的突变。我们将使用这些基因改变的品系来进行一系列实验，以确定AGBE和两个果蝇IRP蛋白的分子特征。我们还建议进行实验，以检验为什么7DC在拯救AGBE功能丧失的动物方面似乎与铁一样有效。我们怀疑7DC介导铁动员或铁摄取进入线粒体，我们正在提议进行实验，以测试不同的可能机制。 我们希望在两个主要领域做出重要贡献，即a)铁的动态平衡与类固醇的产生和释放的协调，确保发育的正常进行，以及b)在类固醇激素产生组织中铁的运输、输入和储存的控制机制。