Cellular copper levels determine the phenotype of the Arg ⁸⁷⁵ variant of ATP7B/Wilson disease protein

Abstract

In human disorders, the genotype-phenotype relationships are often complex and influenced by genetic and/or environmental factors. Wilson disease (WD) is a monogenic disorder caused by mutations in the copper-transporting P-type ATPase ATP7B. WD shows significant phenotypic diversity even in patients carrying identical mutations; the basis for such diverse manifestations is unknown. We demonstrate that the 2623A/G polymorphism (producing the Gly 875 →Arg substitution in the A-domain of ATP7B) drastically alters the intracellular properties of ATP7B, whereas copper reverses the effects. Under basal conditions, the common Gly 875 variant of ATP7B is targeted to the trans-Golgi network (TGN) and transports copper into the TGN lumen. In contrast, the Arg 875 variant is located in the endoplasmic reticulum (ER) and does not deliver copper to the TGN. Elevated copper corrects the ATP7B-Arg 875 phenotype. Addition of only 0.5-5 μM copper triggers the exit of ATP7B-Arg 875 from the ER and restores copper delivery to the TGN. Analysis of the recombinant A-domains by NMR suggests that the ER retention of ATP7B-Arg 875 is attributable to increased unfolding of the Arg 875 -containing A-domain. Copper is not required for the folding of ATP7B-Arg 875 during biosynthesis, but it stabilizes protein and stimulates its activity. A chemotherapeutical drug, cisplatin, that mimics a copper-bound state of ATP7B also corrects the "disease-like" phenotype of ATP7B-Arg 875 and promotes its TGN targeting and transport function. We conclude that in populations harboring the Arg 875 polymorphism, the levels of bioavailable copper may play a vital role in the manifestations of WD.trafficking | cisplatin | phenotypic variability

Distinct phenotype of a Wilson disease mutation reveals a novel trafficking determinant in the copper transporter ATP7B

“…7 D and D′). Interestingly, the presence of copper appears to promote ATP7B S653Y/858TGE860>AAA exit from the ER, as has been reported for the ATP7B G875R variant (30). We conclude that the S653Y was not dominant in the context of the TGE>AAA mutation.…”

“…In addition to the cellular components required for copperdependent ATP7B trafficking, studies have shown that molecular signals within the protein, as well as intramolecular interactions, play important roles (30,40). Two well-characterized trafficking signals encoded within the primary sequences of both copper-ATPases have been identified: (i) F 37 -E 45 in ATP7B, which is required for retention of ATP7B in the TGN (−Cu) and targeting to the apical region in polarized hepatic cells (+Cu, anterograde trafficking) (11); and (ii) di-and trileucines in ATP7A and ATP7B's C-termini, which are needed for retrograde trafficking to the TGN (−Cu) (12,41,42).…”

“…How could small changes in the properties of TM1 result in such a significant effect on trafficking of ATP7B? Previous biochemical studies with recombinant peptides showed that ATP7B's N-terminal cytosolic domain independently interacted with two other cytosolic domains, the nucleotide binding (N) domain in low Cu conditions (29) and the actuator (A) domain in elevated Cu (30) (Fig. 1A).…”

Molecular Events Initiating Exit of a Copper-transporting ATPase ATP7B from the Trans-Golgi Network

“…Site-directed Mutagenesis in ATP7B-The previously generated construct of N-terminally Flag-tagged ATP7B in pcDNA 5.1 FRT/TO vector was used as a template (26). The Ser3 Ala, Ser3 Gly, Ser3 Thr, and Ser3 Asp mutations were introduced by PCR using the Quickchange XL mutagenesis kit (Stratagene).…”

“…To test this hypothesis, we took advantage of a naturally occurring ATP7B variant ATP7B R875 , which has a low apparent affinity to copper compared with a common ATP7B G875 variant and a very distinct cellular behavior. In basal or low copper, ATP7B R875 localizes in the endoplasmic reticulum (ER), but it traffics to the TGN and then to vesicles when copper is increased in a stepwise fashion (26). Stabilizing the "copper-bound" state for the ATP7B R875 variant is expected to change its localization from ER to TGN and/or vesicles under basal conditions.…”