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

该建议的中心思想是，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，类固醇生成酶和它们的铁辅助因子在PG中产生的水平非常高，以维持类固醇的产生。因此，像PG这样的甾体腺对铁的需求远远超过大多数其他组织。大多数类固醇激素以脉冲形式释放，有明确的开始、幅度和结束。这就要求来自细胞外或细胞内来源的铁动员与类固醇生成酶的合成同步。我们的中心假设是，类固醇的产生和铁的动员必须通过迄今尚未确定的调节机制来协调，以确保铁辅助因子的及时产生，以应对即将到来的类固醇脉冲。我们最近发现AGBE基因是果蝇PG正常铁代谢所必需的新参与者。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。通过在饮食中补充铁或类固醇前体7-脱氢胆固醇（7DC），可以挽救这种致死率。AGBE是一种具有良好特征的基因，具有单一的记录作用，即向糖原添加侧分支，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与已知的铁调节蛋白IRP1（果蝇中的IRP1A）发生物理相互作用。在这个提案中，我们讨论实验来探索AGBE和IRP1A的功能。我们的方法包括为AGBE以及IRP1A及其同类IRP1B制作一系列突变等位基因和转基因构建物。这将允许我们有条件地去除基因，给产生的蛋白质添加标签，并产生经典的突变体。我们将使用这些转基因细胞系进行一系列实验，以分子表征AGBE和两种果蝇IRP蛋白。我们还提出了实验来检验为什么7DC在拯救AGBE功能丧失动物方面与铁一样有效。我们怀疑7DC介导铁动员或铁摄取进入线粒体，我们正在提出实验来测试不同可能的机制。我们期望在两个主要领域做出重要贡献，即a)铁稳态与类固醇产生和释放的协调，确保发育的正常进展；b)铁运输、进口和储存在类固醇激素产生组织中的控制机制。

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.