Investigating the Roles of Drosophila ppk20 in Cellular Iron Import
Creators: Wang, Qian
Publisher: name: University of Alberta Library
Year: 2022

<p>The ppk20 (pickpocket 20) gene was identified from a two-step screen that was aimed at identifying genes relevant to Drosophila iron and heme homeostasis. Initially, our lab collaborated with two other labs and carried out a prothoracic gland (PG)-specific genome-wide screen of 12,500 RNA interference (RNAi) lines seeking genes that might be relevant to ecdysone synthesis. Ecdysone is an insect steroid hormone that regulates Drosophila development. This initial screen identified 1,906 RNAi lines with developmental defects, including different larval stage arrest/delay and pupal lethality. The secondary screen was conducted in our lab, using ~800 lines that showed development defects, but survived to the third instar stage. This strategy identified 13 RNAi lines that showed enlargement of the PG (no red autofluorescence) and 21 RNAi lines that exhibited enlarged PGs together with a red autofluorescence signal. Due to the high iron demand in PG associated with heme and ecdysone synthesis, our lab has used the PG phenotype to study poorly characterized aspects of Drosophila iron and heme homeostasis.<br>
The heme production pathway is a multi-step biosynthesis process, and it requires the incorporation of iron at the last step. Several heme precursors contain the porphyrin ring structure, making them autofluoresce a red colour when exposed to ultraviolet (UV) light. The final product, heme, is a necessary cofactor for cytochrome P450 enzymes (CYPs) that are essential for Drosophila ecdysone biosynthesis. Ecdysone regulates all developmental transitions from embryonic to adult stages. Hence, an inappropriate drop in ecdysone concentration can lead to developmental defects. The PG maintains high iron levels to sustain heme production, thus supporting ecdysone biosynthesis. The red-fluorescing, protoporphyrin IX (PPIX), is the immediate precursor to heme, and without iron incorporation, it might halt and reduce heme production, causing PPIX to accumulate. In addition, interruption at other steps may result in the accumulation of other red-fluorescing precursors. Under UV light, the PG will fluoresce bright red with enough red-fluorescent precursor accumulation. Knocking down ppk20 in the PG causes developmental defects and red PG phenotype, possibly due to the lack of ecdysone and the accumulation of red-fluorescing precursors.<br>
PPK20 is a member of the epithelial sodium channel (ENaC) family, and some of its vertebrate homologs have been shown to exhibit sodium channel activity. No previous studies<br>
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have shown the relevance of ppk20 or any other ppk genes in iron and heme homeostasis. In this thesis, I explored how ppk20 this possible relevance.<br>
Iron might be imported into the cells through the iron-containing proteins, transferrin (Tsf) and ferritin, or through a non-protein-binding form. In humans, primary iron intake involves importing Tsf via Tsf receptor-mediated endocytosis. However, there is no identified fly Tsf receptor. Heme feeding and human transferrin receptor (hTfR) cDNA expression, but not iron feeding, rescued the loss-of-ppk20 animals. When ppk20 function was impaired in the larval intestine, free iron uptake from the midgut was interrupted. Moreover, ferritin injection could partially rescue the ppk20-deficient animals. Interestingly, the larval tracheal necrotic phenotype was present in the loss-of-ppk20 animals, which indicates its role in tracheal function.<br>
Taken together, this thesis presents evidence that ppk20 may play a role in cellular iron import via regulating the free iron and Tsf import pathways due to its possible function as the Tsf receptor-like protein or sodium channel. Additionally, ppk20 might play a role in larval tracheal development and water clearance, contributing to the porphyria-like PG phenotype and developmental defects observed in the loss-of-ppk20 animals.</p>