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

这个建议的中心思想是，我们可以使用果蝇前胸腺（PG）作为一个模型来研究未知方面的铁和类固醇生物学。这是因为类固醇的合成依赖于需要含铁辅因子如血红素和铁硫（Fe-S）簇的酶。重要的是，类固醇生成酶及其铁辅因子在PG中以非常高的水平产生，以维持类固醇的产生。因此，类固醇腺如PG的铁需求远远超过大多数其他组织。大多数类固醇激素作为脉冲释放，具有定义的开始，幅度和结束。这就要求铁从细胞外或细胞内来源的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通过迄今尚未确定的调节机制来协调，以确保铁辅因子在即将到来的类固醇脉冲中及时产生。我们最近已经确定了AGBE基因作为一个新的球员所需的正常铁代谢的果蝇PG。PG特异性中断AGBE功能后，血红素前体积累在受影响的细胞，导致幼虫致死。死亡率可以通过补充铁或类固醇前体7-脱氢胆固醇（7 DC）来挽救。AGBE是一种特征明确的基因，具有单一记录的作用，即为糖原添加侧支，这表明该蛋白质在细胞铁稳态中具有“兼职”功能。事实上，我们能够证明AGBE与一种称为IRP 1（果蝇中的IRP 1A）的已知铁调节蛋白发生物理相互作用。在这个提议中，我们讨论了探索AGBE和IRP 1A功能的实验。我们的方法包括制作一系列AGBE的突变等位基因和转基因构建体，以及IRP 1A和它的副基因IRP 1B。这将使我们能够有条件地去除基因，在产生的蛋白质上添加标签，并产生经典的突变体。我们将使用这些基因改变的品系进行一系列实验，从分子上表征AGBE和两种果蝇IRP蛋白。我们还提出了实验，以研究为什么7 DC似乎是同样有效地挽救AGBE功能丧失的动物铁。我们怀疑7 DC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的可能机制。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和B）铁在产生类固醇的组织中的运输、输入和储存的控制机制。

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.