Regulation of Retromer mediated trafficking of ATP7B; From perspective of mutational study

Ghosh, Archita (2019) Regulation of Retromer mediated trafficking of ATP7B; From perspective of mutational study. Masters thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

Copper is an indeed important micronutrient of our cell. Copper works as an essential co factor for the activity of a large number of enzymes for example Ceruloplasmin, Cu, ZN dependant Superoxide Dismutase, dopamine-β-hydroxylase, peptidyl-α-mono-oxygenase etc. Copper overload can cause both morphological and metabolic changes in tissues and if untreated, even this can lead to death. So a tight regulation of Copper is indeed important. Copper enters within the cell through hCTR1 and excess Cu is exported out through P- type Cu ATPase ATP7B. At basal Cu level ATP7B resides in TGN membrane but whenever the Cu concentration increases it gets vesicularised and leaves TGN and travels upto cell membrane to excrete out excess Cu out of the cell. When Cu concentration reaches the normal level ATP7B again traffics back to TGN. Bigger aim of our lab is to understand the trafficking mechanism of ATP7B from Golgi to Cell membrane and again back to Golgi. We have found that a heteropentameric protein complex known as Retromer regulates the retrograde trafficking (PM to Golgi) of ATP7B. But exactly which motif or specific amino acid is mediating the interaction between ATP7B and Retromer is not known. I have chosen two conserved motif YXXФ and NPXY in the c term and N term of ATP7B respectively for my study. To check their role I have generated both deletion and Substitution mutation in these two region. Our recent study has shown that due to the deletion mutatons (YXXФ motif in the C term, NPXY in the N term) ATP7B trafficking pathways is being highly affected. On the other hand Y1376D phosphomimetic mutation in the C term restore the wild type trafficking pattern to a great extent. But in the N term Y44D phosphoimetic mutation failed to mimic the wild type property. We have labelled the cells with LAMP1 to check upon Cu treatment whether the ATP7B is going to Lysosome or not. Our previous data are suggesting that there might be an indirect or transient interaction between VPS35 and N term of ATP7B. So we labelled the cell with VPS35 marker.

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