hCTR1 and ATP7B: A Tale of Two Transporters

Maji, Saptarshi (2018) hCTR1 and ATP7B: A Tale of Two Transporters. Masters thesis, Indian Institute of Science Education and Research Kolkata.

PDF (MS dissertatiion of Saptarshi Maji (13MS018)) 13MS018.pdf - Submitted Version Restricted to Repository staff only Download (2MB)

Abstract

Copper is an essential micronutrient for cell. Diverse processes like energy generation, neurotransmitter function, maturation of extracellular matrix is controlled by copper containing enzymes. But increased level of intracellular copper leads to ROS generation and cell death. So, maintaining a tight copper homeostasis is a necessary process for the cell. Copper is imported into the cell by human copper transporter (hCTR1). The membrane ATPases, ATP7A and ATP7B export copper out of the cell. Proper functioning of these proteins regulates copper homeostasis inside the cell. The major aim of my project is to understand the regulation of hCTR1 and ATP7B in response to copper. hCTR1, a 190-amino acid long membrane channel protein, has three transmembrane domains and trimerizes to form a channel. Upon increased intracellular copper concentration, it gets internalized (endocytosed). The first aim of the project is to identify the amino acids (motifs) in CTR1 that are responsible for copper induced endocytosis. I have found that a well-studied HCH motif on the cytosolic C-terminal is linked to endocytosis. ¹⁸⁸HCH¹⁹⁰ motif, when mutated to ¹⁸⁸AAA¹⁹⁰ is found to be localized on the plasma membrane even in higher intracellular copper concentration. The copper pump, ATP7B, a P-type ATPase, resides in Golgi, but upon increased intracellular copper concentration, it traffics to the plasma membrane. The second aim of the project is to determine the role of retromer proteins in the regulation of copper induced localization of ATP7B. Previous unpublished data has shown that retromer complex proteins are more abundant in ATP7B containing vesicles in increased intracellular copper condition than normal physiological condition. Our recent study shows that ATP7B increases colocalization with VPS26 and VPS35 (core retromer proteins) in high intracellular coper concentration than copper deprived condition. Using colocalization studies, the vesicles were identified as Rab11+ vesicles. As a continuation of the study, we have also identified a possible retromer binding site on the C-terminal of ATP7B and generated both deletion and site directed mutations for them.

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