The transition metal copper is an essential cofactor for many redox-active enzymes, but excessive copper can generate toxic reactive oxygen species. Copper homeostasis is maintained by highly conserved proteins, to balance copper uptake, distribution and export on the systemic and cellular level. The research in this thesis entitled “Posttranslational regulation of copper transporters” aims to unravel the mechanisms that regulate cellular copper uptake and export in relation to the molecular pathogenesis of Wilson disease (WD), a hereditary copper overload disorder. To allow quantitative, sensitive and high-throughput assessment of changes in cellular copper homeostasis, we developed the MRE-Luciferase reporter that was able to monitor bio-available copper in a copper-concentration-dependent manner. The human high-affinity copper transporter 1 (hCTR1) is essential for cellular copper uptake, but several observations have indicated the existence of hCTR1-independent copper uptake pathways. We characterized the homologous human CTR2 (hCTR2) as a bona fide copper uptake protein with low affinity for copper compared to hCTR1 using our MRE-Luciferase reporter. hCTR1 was localized at the plasma membrane and in intracellular organelles, whereas hCTR2 was exclusively localized in late endosomes and lysosomes. Hence, we speculate that hCTR2 enables mobilization of copper from lysosomal copper pools in analogy with its function in yeast. Electron microscopic crystallography of recombinant hCTR1 had revealed that hCTR1 subunits assemble in oligomeric complexes comprising three hCTR1 subunits. Here, we assessed the necessity of hCTR1 oligomerization in functional copper uptake. hCTR1-dependent copper uptake was completely abolished by conversion of two highly conserved methionine residues in the second transmembrane helix of hCTR1 into isoleucines (hCTR1 M150I-M154I). Co-expression of hCTR1 M150I-M154I with wild type (WT) hCTR1 did not affect oligomerization, expression or cellular localization of WT hCTR1, but WT hCTR1-dependent copper uptake was inhibited in a dominant fashion. Similar results were obtained for hCTR2, suggesting that oligomerization of hCTR1 and hCTR2 is required for copper transport activity by permitting the formation of a copper permeable channel. Hepatic copper excretion is mediated by ATP7B, and mutations in ATP7B result in WD. We investigated the possibility that defects arising from some WD mutations are ameliorated by drug treatment to improve protein folding and function. Homology modeling of distinct ATP7B missense mutations suggested that most mutations result in misfolded ATP7B proteins. Indeed, almost all tested mutations resulted in reduced ATP7B protein expression. Furthermore, physiological localization of ATP7B to the trans-Golgi network was abrogated, and mutant ATP7B was retained in the endoplasmic reticulum instead. The increased expression and normalization of localization after culturing cells at 30C suggested that these proteins were indeed misfolded. Four distinct mutations exhibited residual copper export capacity, which suggests that improved expression of these mutants could restore ATP7B expression in these patients. Interestingly, treatment with pharmacological chaperones curcumin and 4-phenylbutyrate, a clinically approved compound, partially restored expression and localization of most ATP7B mutants. The surprisingly large number of WD mutations that result in protein misfolding indicates that pharmacological chaperone-treatment to improve protein folding, localization and function might actually improve clinical management of a significant proportion of WD-patients in the future
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