SHORT COMMUNICATION: Functional consequences of RNA interference targeting COMMD1 in a canine hepatic cell line in relation to copper toxicosis

Abstract

A deletion in the copper metabolism (Murr1) domain containing 1 (COMMD1) gene is associated with hepatic copper toxicosis in dogs, yet evidence of copper retention in COMMD1-depleted hepatic cells has not been shown. In a dog hepatic cell line, we analysed the copper metabolic functions after an 80% (mRNA and protein) COMMD1 reduction with COMMD1-targeting siRNAs. Exposure to 64Cu resulted in a significant increase in copper retention in COMMD1-depleted cells. COMMD1-depleted cells were almost three times more sensitive to high extracellular copper concentrations. Copper-mediated regulation of metallothionein gene expression was enhanced in COMMD1-depleted cells. Based on the increased copper accumulation and enhanced cellular copper responses upon COMMD1 reduction, we conclude that COMMD1 has a major regulatory function for intracellular copper levels in hepatic cells.

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

“…The total cellular copper then represents the balance between copper sequestration and export. Because ATP7B is thought to be primarily involved in copper sequestration (6,57) this would explain the more significant increase in copper retention seen with COMMD1 knockdown in the dog hepatic cell line (56). These observations are consistent with both clusterin and COMMD1 levels influencing the turnover of the Cu-ATPases, which in turn impacts on cellular copper levels.…”

“…Alternatively, saturation of the localization pathways may affect normal targeting of the overexpressed proteins to the degradation pathways; or COMMD1 may have different binding efficiencies depending on whether the transiently overexpressed proteins are mislocalized to the endoplasmic reticulum or to vesicular/endocytic pathways. Both clusterin and COMMD1 were shown to affect intracellular copper levels in HEK293(T) cells (15,52), a dog hepatic cell line (56) and in a mouse hepatoma cell line (55). Knockdown of either clusterin or COMMD1 would lead to increased levels of one (hepatic cell line) or both (HEK293 cells) CuATPases, which in turn would lead to an increase in copper sequestration and efflux.…”

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

“…In addition, knockdown of COMMD1 by RNA interference in several cell lines results in increased cellular copper levels 25,28 . COMMD1 interacts with the copper transport protein ATP7B, which is mutated in WD, suggesting that these two proteins cooperate in the excretion of copper 13 .…”

Copper-Induced Translocation of the Wilson Disease Protein ATP7B Independent of Murr1/COMMD1 and Rab7

“…34 Depletion of Murr1/COMMD1 by RNA interference results in an intracellular copper accumulation. 35,36 The lack of exon 2 of the Murr1 gene leads to copper toxicosis in dogs, but the same deletion is embryonically lethal in mice. 37 Murr1 is part of a larger protein family sharing a C-terminal leucine-rich domain termed the copper metabolism Murr1 domain (COMMD), 34,38 and there is evidence that the other members of this family also bind to ATP7B.…”