Studies on Regulatory Mechanisms of the Wilson Disease Protein, ATP7B

Das, Santanu (2022) Studies on Regulatory Mechanisms of the Wilson Disease Protein, ATP7B. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Santanu Das (18RS095)) 18RS095.pdf - Submitted Version Restricted to Repository staff only Download (12MB)

Abstract

Copper is an essential micronutrient for all life forms; however, in excess can be very toxic. In humans, the Wilson disease protein, ATP7B exports excess copper through biliary export and maintains copper levels in the liver. ATP7B traffics from trans-Golgi network (TGN) to endolysosomes to export excess copper. We investigated the recycling mechanism of ATP7B between TGN and the endolysosomal compartments. At high copper levels, ATP7B traffics primarily to active hydrolase positive endolysosomes and colocalizes with retromer subunit VPS35. Upon subsequent copper chelation, it returns to TGN. Knocking down VPS35 failed ATP7B to relocalize to the TGN, which was rescued by overexpressing wild-type VPS35. VPS35 and ATP7B juxtaposes on the lysosomal and Rab7 positive endolysosomes and interaction transiently. The interaction was detected by metabolic labelling using photo-amino acids and subsequent UV-crosslinking. We demonstrate that retromer regulates endolysosome to TGN trafficking of ATP7B in copper dependent manner. In Wilson disease, ATP7B gene is mutated resulting in impaired hepatic copper export leading copper accumulation in the liver. Also, in WD, many cuproenzymes are inactive as it requires copper from ATP7B for its maturation. Using cell biology and biochemical approach we studied the Wilson disease causing ATP7B mutations and tried to establish a genotype-phenotype correlationship of the disease. We have decoded the underpinning molecular pathology of fifteen prevalent and clinically significant disease variants of ATP7B by determining its (a) trafficking in response to high cooper and subsequent copper export capability and (b) copper transport to cuproenzyme, tyrosinase localized at the TGN. We could classify WD causing ATP7B mutations into 3 groups (A) Mutants that localizes at the TGN and are capable of copper-transport to cuproenzymes (B) mutants that are misfolded and are arrested in the endoplasmic reticulum (C) mutants that mislocalize in basal or copper treated conditions. Finally, we correlated the disease manifestations recorded in WD patients with prevalent mutations, R778L and H1069Q, with our in-cellulo and biochemical findings. Our study reflects the importance of different ATP7B protein domains and mutations harbored there within that contributes to the clinical heterogeneity observed among WD patients. The study provide insight into physiological consequences related to each mutant which dictates their degree of severity in the WD phenotype.

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