Cathepsin Protease Controls Copper and Cisplatin Accumulation via Cleavage of the Ctr1 Metal-binding Ectodomain

Abstract

Copper is an essential metal ion for embryonic development, iron acquisition, cardiac function, neuropeptide biogenesis, and other critical physiological processes. Ctr1 is a high affinity Cu ؉ transporter on the plasma membrane and endosomes that exists as a full-length protein and a truncated form of Ctr1 lacking the methionine-and histidine-rich metal-binding ectodomain, and it exhibits reduced Cu ؉ transport activity. Here, we identify the cathepsin L/B endolysosomal proteases functioning in a direct and rate-limiting step in the Ctr1 ectodomain cleavage. Cells and mice lacking cathepsin L accumulate full-length Ctr1 and hyper-accumulate copper. As Ctr1 also transports the chemotherapeutic drug cisplatin via direct binding to the ectodomain, we demonstrate that the combination of cisplatin with a cathepsin L/B inhibitor enhances cisplatin uptake and cell killing. These studies identify a new processing event and the key protease that cleaves the Ctr1 metal-binding ectodomain, which functions to regulate cellular Cu ؉ and cisplatin acquisition.Copper is essential for key biological processes, including electron transfer, iron acquisition, dopamine hydrolysis, and superoxide disproportionation, and defects in copper metabolism are associated with cardiomyopathy, anemia, peripheral neuropathy, and neutropenia (1-6). Although many proteins involved in the acquisition and intracellular distribution of copper have been identified, little is known about the regulation of copper import. The transport of Cu ϩ from the extracellular environment is accomplished by the evolutionarily conserved homotrimeric integral membrane protein, copper transporter 1 (Ctr1) (7-13), which resides on the plasma membrane and in endosomal compartments (14 -18). High affinity Cu ϩ import via Ctr1 requires a methionine-and histidine-rich metal-binding extracellular domain (ectodomain) that is thought to concentrate extracellular Cu ϩ near the ion trans-membrane pore (19 -23). Additionally, Ctr1 binds the chemotherapeutic agent cisplatin via the methionine ligands in the ectodomain and imports cisplatin and other platinum-based chemotherapeutic agents via an endocytic mechanism (24 -28). Despite a critical role for the Ctr1 ectodomain in both Cu ϩ and cisplatin import, both a full-length form and a truncated form of Ctr1 (tCtr1) 4 are present in cultured cells and tissues (18,29,30). The latter lacks the ectodomain and drives ϳ50% of the Cu ϩ uptake as compared with full-length Ctr1 (31).Previously, an integral membrane protein similar to Ctr1, denoted Ctr2, was shown to both interact with and regulate the ratio of full-length Ctr1 to tCtr1 in mouse embryonic fibroblasts (MEFs) and in specific tissues in Ctr2 knock-out mice (32). In the absence of Ctr2, MEFs possess dramatically lower levels of tCtr1, while simultaneously expressing high levels of full-length Ctr1 and accumulating Cu ϩ and cisplatin. A large fraction of the Cu ϩ accumulated in Ctr2 Ϫ/Ϫ MEFs and mouse tissues is found in endosomal compartments (32). The mechanism by which Ct...

Core fucosylation of copper transporter 1 plays a crucial role in cisplatin‐resistance of epithelial ovarian cancer by regulating drug uptake

“…The removal of Asn 15 ‐linked glycan by mutation (N 15 Q) does not affect trafficking of the transporter to the cell surface . Several studies on EOC cells reported that the expression levels of CTR1 are not correlated to the amount of cDDP accumulation.17, Conversely, it has been reported that the elevation of CTR1 expression in the tumor cells increased their sensitivity to platinum drugs and the survival of cancer patients . We found that there is no significant difference in CTR1 expressions of A2780S and A2780CP cells (Figure C), the CTR1 expression is not changed in A2780S and A2780CP cells after cDDP‐treatment.…”

“…CTR1 on cell membrane is the most important cDDP‐transporting protein with the N‐linked glycosylation on its metal‐binding ecto‐domain . The colocalization of CTR1 with core fucosylation of N‐glycans on the cell membrane of ovarian carcinoma implied that the function of CTR1 may be correlated with core fucosylation in cDDP‐treated EOC (Figure D).…”

“…CTR1 transports cDDP via direct binding it by the Met‐rich motifs within the ecto‐domain . Thus the Met‐rich motifs within the CTR1 ecto‐domain are especially critical for cDDP acquisition of cells . Contrast to the CTR1 −/−/WT cells, the Pt content of CTR1 −/−/M ( 40 MXXXM 45 was deleted) and CTR1 −/−/Truncated (the first N‐terminal 45 amino acids were deleted) variants were increased by 2.5‐ and 2.3‐fold, respectively .…”

“…These findings indicate that core fucosylation is necessary for many molecular interactions on cell membrane. Since the drug uptake of cancer cell is based on the molecular interaction between cell surface proteins and drugs, the relationship between core fucosylation and the membrane protein‐dependent drug uptake should be a novel point for the studies on the drug‐resistance of EOC.…”

“…Over‐expression of CTR1 in human ovarian tumor cells increases their sensitivity to platinum drugs . CTR1‐mediated cDDP‐uptake was dependant on its metal‐binding ecto‐domain, which is linked with the N‐glycan (Asn 15 ) and the O‐glycan (Thr 27 ) . Blocking CTR1 ecto‐domain O‐linked glycosylation by mutating Thr 27 , increased the abundance of truncated CTR1 .…”