Mechanisms of Copper Incorporation into Human Ceruloplasmin

Abstract

Ceruloplasmin is a multicopper oxidase essential for normal iron homeostasis. To elucidate the mechanisms of copper incorporation into this protein, holoceruloplasmin biosynthesis was examined by immunoblot analysis and 64 Cu metabolic labeling of Chinese hamster ovary cells transfected with cDNAs encoding wild-type or mutant ceruloplasmin. This analysis reveals that the incorporation of copper into newly synthesized apoceruloplasmin in vivo results in a detectable conformational change in the protein. Strikingly, despite the unique functional role of each copper site within ceruloplasmin, metabolic studies indicate that achieving this final conformation-driven state requires the occupation of all six copper-binding sites with no apparent hierarchy for copper incorporation at any given site. Consistent with these findings a missense mutation (G631R), resulting in aceruloplasminemia and predicted to alter the interactions at a single type I copper-binding site, results in the synthesis and secretion only of apoceruloplasmin. Analysis of copper incorporation into apoceruloplasmin in vitro reveals that this process is cooperative and that the failure of copper incorporation into copper-binding site mutants observed in vivo is intrinsic to the mutant proteins. These findings reveal a precise and sensitive mechanism for the formation of holoceruloplasmin under the limiting conditions of copper availability within the cell that may be generally applicable to the biosynthesis of cuproproteins within the secretory pathway.

Cisplatin binds human copper chaperone Atox1 and promotes unfolding in vitro

“…In humans, cellular Cu homeostasis is maintained by the Cu chaperone Atox1 that obtains Cu from Ctr1 and then delivers it to metal-binding domains (MBDs) of ATP7A and ATP7B in the secretory pathway (11)(12)(13). These latter proteins couple ATP hydrolysis to Cu transport into the Golgi lumen, for metallation of cuproenzymes, such as apoceruloplasmin (14). Several diseases are related to imbalance in Cu homeostasis-for example, Menkes and Wilson diseases (15,16) and aceruloplasminemia (17,18).…”

Conserved residues modulate copper release in human copper chaperone Atox1

“…In humans, the Cu(I) chaperone Atox1 delivers Cu to metal-binding domains of ATP7A (i.e., Menkes disease protein) and ATP7B (i.e., Wilson disease protein) proteins, which are P 1B -type ATPases (6,7,10). In an ATP-dependent process, ATP7A or ATP7B (depending on the cell type) translocate copper from the cytoplasm into the Golgi lumen for insertion into enzymes in the secretory pathway (6), such as apo-ceruloplasmin (11).…”

In vitro unfolding of yeast multicopper oxidase Fet3p variants reveals unique role of each metal site

“…Studies have indicated that the apo form of hCp adopts an extended structure (like beads on a string) because of the lack of the Cu-ligand bonds, the TNC, that connect the first and the last domain (9). In support of all-ornone Cu binding to hCp (10), metabolic-labeling experiments indicated that achieving native hCp required occupation of all 6 Cu sites (11). However, there have been reports that endorse partially-metallated forms of hCp (12).…”