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Investigating the function of novel players of iron homeostasis, heme metabolism and steroid hormone biosynthesis in Drosophila melanogaster
- Author / Creator
- Sattar Soltani
Iron is a transition metal that is essential for all organisms in trace amounts. It participates in biological reactions in the form of different enzymatic cofactors such as a single atom to an iron-sulfur cluster and heme. Therefore, iron functions in numerous cellular processes such as oxygen transport, electron transfer, steroid hormone biosynthesis and DNA synthesis. Unbalanced cellular iron levels result in divergent disorders, including iron deficiency anemia, iron overload hemochromatosis, porphyrias and neurodegenerative disorders. While extensive efforts have focused on general aspects of systemic and cellular iron regulation, little is known about iron homeostasis in tissues with dynamic iron demands. The Drosophila prothoracic gland (PG) is an excellent model for investigating dynamic iron regulation and characterizing novel genes or genes with hitherto unknown links to iron. The PG is a part of the endocrine gland known as the ring gland (RG) and is the primary tissue responsible for the biosynthesis of the steroid hormone ecdysone in insects. The PG has been demonstrated to require substantial amounts of iron. Indeed, the PG releases pulses of ecdysone throughout the Drosophila life cycle to govern developmental transitions such as the molts and puparium formation. Multiple cytochrome P450 enzymes (P450s) with dynamic and remarkably high expression in the PG cells produce ecdysone. These P450 enzymes require iron in the form of heme cofactors. Therefore, the PG has to import a high amount of iron or release stored iron to keep up with the ecdysone biosynthesis.
My thesis focuses on three distinct projects aiming at understanding the regulatory mechanisms of heme synthesis, iron homeostasis and ecdysone biosynthesis. First, I investigated the roles of a gene referred to as Ecotropic viral integration site 5 (Evi5) in the PG cells’ iron trafficking and heme production. Evi5 is a GTPase-activating protein (GAP) that regulates the small GTPase Rab11. Depletion of Evi5 function in the PG cells causes larval developmental retardation, pupal lethality and cellular accumulation of heme precursors (known as the porphyria-like phenotype). These phenotypes are due to impaired vesicular trafficking and reduced cellular iron level that can be rescued with dietary heme administration. Further investigation suggested a hitherto unknown role for Evi5 in iron homeostasis and trafficking. I found that Evi5 interacts with the iron protein ferritin and autophagy components such as dynamin, Clathrin and Hsc70-4. These results led to the hypothesis that Evi5 regulates iron release from ferritin via autophagy-lysosomal degradation.
The second project was to evaluate the transcriptional responses to dietary iron manipulation. I carried out an RNA sequencing (RNA-Seq) analysis using the larval brain ring gland complex (BRGC), larval gut, and whole larval body (WB) to identify differentially expressed genes (DEGs). This study aimed at identifying novel iron homeostasis genes. My approach determined that in PG cells, the heat shock proteins Hsp22 and Hsp70 are iron-responsive proteins. Both proteins appear to participate in cellular iron homeostasis by binding to NFS1, a component of iron-sulfur (Fe-S) cluster assembly machinery, Fe-S cluster recipient proteins, and ferritin. Further analysis of all significant DEGs indicated decreased food iron levels boost the immune responses and chitin metabolism. Given these findings, I propose that chitin acts as a novel iron deficiency immune responsive molecule that creates a physical barrier against pathogens. Furthermore, this work demonstrated that Dietary and metabolic glutamate transporter (dmGlut) and modifier of mdg4 (mod(mdg4)) have significant homology to unidentified ferroportin (FPN1) and ATF2 transcription factor in Drosophila, respectively.
I also studied novel functions of the Spen protein in ecdysone biosynthesis. Spen is a corepressor transcription factor that governs gene expression by binding to regulatory sequences in the nucleus or to mRNA in the cytosol. PG-specific loss of Spen alters the RG morphology of the larvae and reduces the numbers of PG cells. This results in a larval developmental arrest due to ecdysone deficiency that can be rescued with ecdysone or precursors of the ecdysone pathway. I further found evidence that Spen is a regulatory protein downstream of the MAPK/ERK signaling pathway in PG cells. The exact regulatory mechanism of Spen is unknown, but expressing a constitutively active form of Ras in the PG cells also rescues the arrested animals and improves the RG morphology. These preliminary data suggest a novel role for Spen in ecdysone biosynthesis.
- Graduation date
- Fall 2021
- Type of Item
- Doctor of Philosophy
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