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

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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.