An Investigation of the Differential Transport of P-Type ATPases, ATP7A and ATP7B

Nayak, Ankita (2021) An Investigation of the Differential Transport of P-Type ATPases, ATP7A and ATP7B. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Ankita Nayak (16MS135)) 16MS135_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (5MB)

Abstract

Copper is an essential micronutrient due to its role as a cofactor for enzymes that play a critical role in biochemical processes such as superoxide dismutase and ceruloplasmin. thus, it is extremely important to tightly regulate copper homeostasis. While copper intake is regulated by hCTR1, copper efflux in cells is regulated by ATP7A and ATP7B. At basal copper levels, ATP7A and ATP7B reside within the Trans-Golgi Network (TGN) where they receive copper frommetallochaperones and transfer it to cuproenzymes. However, as copper accumulates in the cell, these proteins traffic out of the TGN towards intracellular vesicles or the plasma membrane in order to expel copper. As their names suggest, these proteins share a high sequence homology. However, the difference in their trafficking behaviour becomes apparent in polarised cells where ATP7A traffics to the basolateral membrane and ATP7B traffics to the apical membrane. In this project,we have used two approaches to better understand this phenomenon. the first involves identifying the different substations in the trafficking itinerary of both these proteins in polarised MDCK cells through the use of Immunostaining and Confocal Microscopy. MDCK cells that stably express mKO2-ATP7A have been used to identify these substations for ATP7A and GFP-ATP7B has been transfected in MDCK cells for the same for ATP7B. through these experiments, it has been found that ATP7A is indeed localised at the TGN under copper chelated conditions, at the basolateral membrane under elevated copper conditions and that Clathrin may be involved in the endocytosis of ATP7A. Additionally, the localisation of ATP7B at the TGN under copper chelated conditions and at the apical membrane under elevated copper conditions has also been seen. ATP7B has been found to colocalise with Apical Recycling Endosome markers as well as Common Recycling Endosome/ Basolateral Sorting Endosome markers. the former is likely to be the final substation in the trafficking itinerary before the Apical membrane while the latter corroborates an earlier result in a different cell line that ATP7B sorting occurs via a Basolateral intermediate. The second approach involves the identification of proteins that interact with ATP7A and ATP7B to regulate their trafficking behaviour through a novel technique known as Proximity Biotinylation. This technique involves the tagging of proteins within a small radius of the protein of interest with a biotin tag, allowing the proteins to be isolated via Streptavidin and analysed by Mass spectrometry. While the constructs necessary for this approach could not be prepared, PCR cycling parameters have been standardised. Keywords: ATP7A, ATP7B, Differential Trafficking

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