Studies on Localisation and Function of Wilson Disease Protein ATP7B at the Lysosome

Maji, Saptarshi (2024) Studies on Localisation and Function of Wilson Disease Protein ATP7B at the Lysosome. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Saptarshi Maji (18RS020)) 18RS020.pdf - Submitted Version Restricted to Repository staff only Download (17MB)

Abstract

Copper is an essential yet toxic micronutrient for our body. Golgi localized Cu-ATPase ATP7A and ATP7B transports copper to the biosynthetic pathway as well as pumps-out excess copper from the cell. In copper stress, the liver-specific Cu-ATPase ATP7B, traffics to the lysosome, and pumps excess copper to the lysosomal lumen. Upon reestablishment of copper homeostasis, ATP7B recycles back to the TGN. I discovered a liver-specific copper-insensitive trafficking of ATP7B from TGN to lysosome. We also found a novel TGN-lysosome contact site that plays a crucial role in this copper-insensitive trafficking of ATP7B. Modulating lysosomal positioning affected TGNlysosome proximity and lysosomal localization of ATP7B. Upon copper chelation, contrary to the previous findings, we found that ATP7B stays localized on lysosomes and recycles to a TGN-proximal lysosome but does not translocate to TGN entirely, indicating an incomplete recycling of ATP7B. Liver being the primary metal-detoxifying tissue, the presence of a lysosomal pool of ATP7B can be a part of combating metal stress via a quick response of the export mechanism. The cell utilizes lysosomal exocytosis to export excess copper. In ATP7B⁻/⁻ cells, we found a higher copper accumulation, significant reduction in lysosomal activity, increased autophagic induction, reduction in mTOR activity and perturbation in cholesterol biosynthesis. mTOR, being a master regulator of lysosomal biogenesis, autophagy, and cholesterol biosynthesis, can significantly affect all these processes. Interestingly, knocking in wt-ATP7B in the ATP7B⁻/⁻ cells can rescue lysosomal functions suggesting excess copper accumulation and the absence of a copper-export-mechanism in ATP7B⁻/⁻ cells influence cellular physiology to a greater extent. However, the exact mechanism of this perturbation is not clear. To summarise, we discovered a novel TGN-lysosome contact site that plays a vital role in the copper-insensitive trafficking of ATP7B in hepatocytes. The absence of ATP7B leads to excess copper accumulation that alters major physiological processes.

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