Deciphering the Molecular Mechanism of Dysregulated Cholesterol Biosynthesis and Its Endosomal Trafficking in ATP7B Knockout Hepatocytes

Majumder, Avijit (2025) Deciphering the Molecular Mechanism of Dysregulated Cholesterol Biosynthesis and Its Endosomal Trafficking in ATP7B Knockout Hepatocytes. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (0MS Dissertation of Avijit Majumder (20MS088)) 20MS088_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (1MB)

Abstract

Copper (Cu) is an essential micronutrient for healthy physiology. It acts as a cofactor for several enzymes and hormones. However, it becomes toxic when its levels exceed the permissible limit. ATP7B, a Cu-ATPase exclusively present in hepatocytes, traffics from the trans-Golgi network to the lysosomal membrane when the cellular copper level rises and pumps copper from the cytosol to the lysosomal lumen. These lysosomes then fuse with the plasma membrane to export the excess copper outside the cell. In ATP7B Knock out HepG2 cell line (cellular model of Wilson disease), we found that cholesterol biosynthesis-related genes are severely downregulated. We also found that cholesterol distribution is drastically altered in those cells. In this thesis, I worked on two aspects: how cholesterol biosynthesis is downregulated in ATP7B KO cells and how endosomal cholesterol trafficking gets dysregulated inside the ATP7B KO hepatocytes. We found that transcription factor for cholesterol biosynthesis-related genes, N-term SREBP- 2 in higher amounts inside the nucleus of ATP7B KO cells. Here, we proposed a mTORC1- mediated pathway that inhibits SREBP-2 inside the nucleus of ATP7B KO cells, leading to cholesterol biosynthesis downregulation. Getting hints from our experimental data, next we investigated the role of ATP7B in endosomal cholesterol trafficking. We found NPC1, the key protein for endosomal cholesterol trafficking, binds with copper. We also showed that ATP7B and NPC1 interact directly with each other. We hypothesize that ATP7B might supply the Cu to NPC1 for effective cholesterol transfer from lysosome to ER. Thus, in ATP7B KO hepatocytes, cholesterol distribution is altered. This work shows that there is a link between copper and cholesterol metabolism and the broader functions of copper in cellular physiology.

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