Investigating Drosophila midgut copper homeostasis and novel regulation mechanisms
thesisposted on 08.02.2017 by Binks, Tim Paul
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All animals require the essential biometal copper as an enzymatic cofactor for processes as diverse as energy production, free radical detoxification and pigmentation. The human body has an absolute need for copper but also must provide protection against toxicity. Therefore each cell produces a set of copper homeostasis proteins which are required for copper to enter the cell, be utilized and then be removed. In mammalian cells, hCtr1 and the Cu-ATPases have been identified as the primary transport proteins for import and export of copper respectively. Whilst the basic role of each copper transporter has been identified, many questions remain in regard to their regulation, localization and tissue specific function. In recent years Drosophila melanogaster (referred to as Drosophila) has emerged as an alternative model used to further investigate these questions due to the high conservation of the key copper transporters. As the major source of copper exposure to Drosophila (and humans) is ingestion of the nutrient, the first aim of this project was to explore the mechanisms of midgut copper absorption and its importance to distribution of the nutrient throughout the organism. The second and third aims explored regulation of copper homeostasis by investigating protein localization and the role of novel interacting proteins. Ctr1A and Ctr1B, orthologues of the human Ctr1, are the primary Drosophila copper import proteins and were suspected to be crucial for midgut absorption. However, midgut specific suppression of both in combination does not impact upon viability implying that reduced copper entry may be sufficient for survival, Drosophila as a whole can compensate for low intestinal copper import or that there are other sources of uptake. Ectopic expression of Ctr1B causes lethality suggesting that this protein is the more efficient importer and results in toxicity of both the midgut and lymph. Localization of both Ctr1A/B was predominantly at the apical membrane yet some was at the basolateral membrane implying that copper import back from the lymph was also possible. DmATP7, the sole orthologue of the mammalian Cu-ATPases, localizes predominantly to the basolateral membrane of enterocytes, with some protein likely at the trans-Golgi network. Midgut specific suppression of DmATP7 does not impact upon viability yet increases enterocyte copper content and is detrimental when copper import is increased. It appears likely that loss of DmATP7 expression results in midgut copper toxicity but the effect of limited copper delivery to the lymph appears negligible. Ectopic expression of DmATP7 results in lethality likely caused by copper toxicosis and in combination with suppression data further implies that Drosophila are more capable surviving decreased enterocyte copper import rather than increased. Using the midgut model (and other Drosophila tissues such as the eye and midline) this project was also able to demonstrate putative roles for dRab5 and HipK in copper homeostasis. This project shows that copper levels may influence dRab5 stability, whilst at the same time dRab5 expression is crucial in determining the localization of DmATP7. As dRab5 is a key component in the maturation of the early endosome, this suggests a link between endocytosis and the regulation of copper transporters. HipK is a homeodomain serine/threonine kinase that inhibits the activity of Slimb, an E3 ubiquitin ligase and therefore can stabilize targets proteins by blocking degradation. This project demonstrates that increased expression of HipK exacerbates Drosophila midline and midgut phenotypes caused by ectopic expression of DmATP7 implying that degradation of import protein is inhibited. At the same time, midline phenotypes caused by ectopic expression of HipK may be partially as a result of copper deficiency as the phenotypes are rescued by increased cellular copper levels. Work on both novel interacting proteins clearly demonstrated their regulation of copper homeostasis and will stimulate further use of Drosophila to explore this area of research.