Interaction of the copper chaperone HAH1 with the Wilson disease protein is essential for copper homeostasis

Abstract

The delivery of copper to specific sites within the cell is mediated by distinct intracellular carrier proteins termed copper chaperones. Previous studies in Saccharomyces cerevisiae suggested that the human copper chaperone HAH1 may play a role in copper trafficking to the secretory pathway of the cell. In this current study, HAH1 was detected in lysates from multiple human cell lines and tissues as a single-chain protein distributed throughout the cytoplasm and nucleus. Studies with a glutathione S-transferase-HAH1 fusion protein demonstrated direct protein-protein interaction between HAH1 and the Wilson disease protein, which required the cysteine copper ligands in the amino terminus of HAH1. Consistent with these in vitro observations, coimmunoprecipitation experiments revealed that HAH1 interacts with both the Wilson and Menkes proteins in vivo and that this interaction depends on available copper. When these studies were repeated utilizing three diseaseassociated mutations in the amino terminus of the Wilson protein, a marked diminution in HAH1 interaction was observed, suggesting that impaired copper delivery by HAH1 constitutes the molecular basis of Wilson disease in patients harboring these mutations. Taken together, these data provide a mechanism for the function of HAH1 as a copper chaperone in mammalian cells and demonstrate that this protein is essential for copper homeostasis.

Distinct Wilson’s Disease Mutations in ATP7B Are Associated With Enhanced Binding to COMMD1 and Reduced Stability of ATP7B

“…ATP7B plays a key role in hepatic copper excretion, by virtue of its ability to transport copper across cellular membranes at the cost of ATP hydrolysis. Within its amino terminal region, ATP7B contains six copper binding sequences, through which it receives copper by a transient copper-dependent interaction with the copper chaperone ATOX1 7,8 . Under basal conditions, ATP7B resides in the trans Golgi network (TGN), where it facilitates the incorporation of copper in cuproenzymes such as ceruloplasmin.…”

Unresolved questions in human copper pump mechanisms

“…Subsequent modeling studies indicated that this kinked helix could be a docking site for Atox1 (Gourdon et al 2012) as well as for the 6th metalbinding domain (Arumugam & Crouzy, 2012) making the question of where Atox1 delivers the Cu ion still unresolved. Regardless, the importance of the metal-binding domains in vivo is clear: at least three disease-causing point mutations are found in the metal-binding domains of ATP7B (Hamza et al 1999).…”

“…Subsequently, this motif was found in the promotor regions of several other genes (Muller & Klomp, 2009). In support of playing a role in the nucleus, the sequence of Atox1 contains an apparent nuclear localization signal KKTGK within its C-terminal part and, although not discussed, in the initial discovery paper of Atox1 from 1999 (Hamza et al 1999), immunofluorescence of HeLa cells indicated that Atox1 was distributed throughout the cell, including the nucleus. In our work, we have also detected Atox1 in the nuclei of HeLa cells (Fig.…”

Posttranslational regulation of copper transporters

“…Copper binding to ATP7A and ATP7B forms a key event in the catalytic cycle. An essential step in ATP7A-and ATP7B-dependent copper transport is copper delivery by the copper chaperone ATOX1 [90][91][92][93]. The importance of ATOX1 in mammalian biology is clearly illustrated by the Atox1 knockout mice that displayed a copper deficiency phenotype similar to Menkes disease [90].…”