Correlating Copper Transport Activity and Intracellular Trafficking of Wilson Disease Protein

Mandal, Taniya (2021) Correlating Copper Transport Activity and Intracellular Trafficking of Wilson Disease Protein. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Taniya Mandal (16MS075)) 16MS075_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (25MB)

Abstract

Copper is a heavy metal essential to aerobic organisms. In excess it is toxic. Wilson disease is a monogenic metabolic disorder which leads to impaired Cu homeostasis. The key feature of this disease is over accumulation of the metal in liver, brain and kidney. WD is caused by mutations in Cu-ATPase ATP7B. A diverse array of mutations can lead to development of this disease. The clinical features associated with this disease also vary largely. ATP7B, a multi-pass transmembrane protein is expressed majorly in hepatic and neural tissues. It exports Cu from cytosolic to the trans-golgi compartment using ATP hydrolysis. There, Cu is incorporated into enzymes. ATP7B also plays a vital role in excretion of Cu through bile. WD mutations fail to deliver Cu to TGN either (1) by affecting the Cu-transport ability of the protein or (2) due to defects in trafficking or (3) both. This study analyzes the mechanism underlying development of WD in 5 widely found pathogenic mutations across the world. Initially 15 pathogenic point mutations were selected for this study based on disease phenotype, their location in the protein and their frequency. Tyrosinase assay was performed on the variants to identify the mutations which lead to a complete loss of Cu-delivery to TGN in mammlian cells. The transporter activity of 5 WD variants was further quantified utilizing yeast complementation assay. Immunofluorescence studies were done to investigate whether these variants are transported to TGN in polarised cells, under different Cu-concentrations. We asked whether WD variants can be categorized using these features. This study gives an understanding of the correlation between genotype and phenotype in WD. In parallel it puts forward a framework for comprehending and categorizing complex metabolic disorders.

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