Understanding the Extended Role of Zinc as a Therapeutic Agent for Wilson’s Disease

Bhattacharya, Sharanya (2025) Understanding the Extended Role of Zinc as a Therapeutic Agent for Wilson’s Disease. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Sharanya Bhattacharya (23IP002)) 23IP002_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (3MB)

Abstract

Wilson’s Disease (WD) is a rare genetic disorder of copper metabolism, caused by mutations in the copper-exporting ATPase ATP7B, leading to toxic copper accumulation in tissues, especially the liver. Although zinc acetate has been FDA- approved for WD therapy since 1997—primarily due to its induction of metallothioneins that sequester labile copper—the complete mechanism behind its therapeutic effect remains poorly understood. In this study, I explored the extended role of zinc in WD using ATP7B-knockout HepG2 (ATP7B⁻/⁻) cells as an in-cellulo model. I found that ATP7B⁻/⁻ cells were more resistant to zinc stress and exhibited dynamic, zinc- dependent changes in the expression of metal-responsive and oxidative stress-related genes, including SOD1, NRF2, and CCS. Although lysosomal biogenesis and ATP7B localization were unaffected by zinc treatment, I observed that ATP7B⁻/⁻ cells had elevated basal levels of TFEB and LC3B-II, indicating a pre-activated autophagy machinery likely compensating for copper-induced oxidative damage. Upon zinc supplementation, both TFEB and LC3B-II levels decreased, suggesting zinc may alleviate oxidative stress and reduce the need for active autophagy. I also found that SOD1 levels increased upon zinc treatment, though the protein remained extranuclear, suggesting potential non-canonical antioxidant roles. Separately, I performed an evolutionary and structural analysis of zinc transporters (ZNTs) and identified the HXXXD motif in transmembrane domains 2 and 5 as essential for zinc specificity. By introducing a point mutation (H70N) in ZNT7, I demonstrated altered subcellular localization, highlighting the motif’s functional significance. Together, my findings broaden our understanding of zinc’s multifaceted role in WD therapy and provide new insights into the structure-function relationship of metal transporters.

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