Clusterin (Apolipoprotein J), a Molecular Chaperone That Facilitates Degradation of the Copper-ATPases ATP7A and ATP7B

Abstract

The copper-transporting P 1B -type ATPases (Cu-ATPases) ATP7A and ATP7B are key regulators of physiological copper levels. They function to maintain intracellular copper homeostasis by delivering copper to secretory compartments and by trafficking toward the cell periphery to export excess copper. Mutations in the genes encoding ATP7A and ATP7B lead to copper deficiency and toxicity disorders, Menkes and Wilson diseases, respectively. This report describes the interaction between the Cu-ATPases and clusterin and demonstrates a chaperone-like role for clusterin in facilitating their degradation. Clusterin interacted with both ATP7A and ATP7B in mammalian cells. This interaction increased under conditions of oxidative stress and with mutations in ATP7B that led to its misfolding and mislocalization. A Wilson disease patient mutation (G85V) led to enhanced ATP7B turnover, which was further exacerbated when cells overexpressed clusterin. We demonstrated that clusterin-facilitated degradation of mutant ATP7B is likely to involve the lysosomal pathway. The knockdown and overexpression of clusterin increased and decreased, respectively, the Cu-ATPase-mediated copper export capacity of cells. These results highlight a new role for intracellular clusterin in mediating Cu-ATPase quality control and hence in the normal maintenance of copper homeostasis, and in promoting cell survival in the context of disease. Based on our findings, it is possible that variations in clusterin expression and function could contribute to the variable clinical expression of Menkes and Wilson diseases.

Clusterin and COMMD1 Independently Regulate Degradation of the Mammalian Copper ATPases ATP7A and ATP7B

“…2B). These results are in contrast to our previous data, which demonstrated that clusterin interacted to a greater extent with ATP7A and ATP7B when cells were treated with the same oxidizing agents (15). The data presented here suggests that COMMD1 interaction with the Cu-ATPases occurs in response to different cellular signals compared with clusterin.…”

“…1, A and B) verified that endogenous ATP7B interacts with both clusterin and COMMD1 in mammalian cells as previously reported (15)(16)(17). The anti-ATP7B antibody immunoprecipitated ATP7B, clusterin, and COMMD1, whereas preimmune serum did not precipitate these proteins (Fig.…”

“…Oxidative Stress Does Not Influence the Interaction between ATP7B and COMMD1-Previous observations revealed that oxidative stress increased the interaction between clusterin and ATP7B (15), and that both clusterin and COMMD1 act to facilitate the degradation of ATP7B (15,17). To determine whether the interaction between ATP7B and COMMD1 also can be modulated by oxidative stress, HEK293T cells were treated with CuCl 2 (200 M), bathocuproinedisulfonic acid/D-penicillamine (200 M), diamide (10 mM), or H 2 O 2 (2 mM).…”

“…To determine whether the interaction between ATP7B and COMMD1 also can be modulated by oxidative stress, HEK293T cells were treated with CuCl 2 (200 M), bathocuproinedisulfonic acid/D-penicillamine (200 M), diamide (10 mM), or H 2 O 2 (2 mM). We previously showed that these conditions effectively increase oxidative stress levels in HEK293T cells by demonstrating an increase in the levels of hemoxygenase-1, a commonly used marker of oxidative stress (15). Co-immunoprecipitation experiments were carried out using the anti-ATP7B antibody ( Fig.…”

“…Clusterin (apolipoprotein J) recently was shown to interact with both Cu-ATPases (15). The interaction between COMMD1 (copper metabolism MURR1 domain) and ATP7B has been the subject of other studies (16 -18).…”