Elucidating the Role of the N-terminus of Human Copper Transporter-1 in Physiological Copper Uptake

Sen, Samarpita (2021) Elucidating the Role of the N-terminus of Human Copper Transporter-1 in Physiological Copper Uptake. Masters thesis, Indian Institute of Science, Education and Research Kolkata.

Text (MS dissertation of Samarpita Sen (15MS020)) 15MS020_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (10MB)

Abstract

Copper is an essential micro-nutrient for all life forms and hCTR1 is the only high affinity plasma membrane copper importer that has been known to date in mammalian cells. The highly variable and unstructured extracellular N-term of this importer is what is thought to be predominantly important for copper acquisition from the basolateral side of polarized epithelia. Cu(II), though is the most abundant state in the environment, it is the lower oxidation state of copper, Cu(I) which has physiological relevance and we hypothesize that the N-terminal domain of hCTR1 might have some role to play in this crucial reduction process. Our in-silico and in-vivo studies have led us to postulate that while the amino-terminal histidine residues play an important role as a concentrator for Cu(II) under copper limiting conditions, methionines and aspartates facilitates Cu(II) to Cu(I) reduction preceding its import. We have also tried to purify the hCTR1 N-term for our copper-binding assays in order to have a better understanding about the differential interaction of Cu(II) and Cu(I) with the residues. The transient binding of both Cu(II) and Cu(I) during the reduction process facilitated by the aspartates, and subsequent coordination of Cu(I) by the N-term methionine residues are crucial determinants of hCTR1 endocytosis to common recycling endosomes. Mutating ⁷Met-Gly-Met⁹ and Asp¹³ abrogates copper uptake and copper-induced endocytosis, that was corrected by providing the cells with a non-reoxidizable source of Cu(I). We also show that the two N-terminal His-Met-Asp clusters exhibit functional complementarity in regulating Cu(I)-induced hCTR1 endocytosis. Finally, this study proposes a model where the His-Met-Asp residues of hCTR1 amino-terminal not only bind copper, but also maintain its reduced state which is crucial for its intracellular uptake and utilization.

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