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Intracellular copper (Cu) in eukaryotic organisms is regulated by homeostatic systems, which rely on the activities of soluble metallochaperones that participate in Cu exchange through highly tuned protein-protein interactions. Recently, the human enzyme glutaredoxin-1 (hGrx1) has been shown to possess Cu metallochaperone activity. The aim of this study was to ascertain whether hGrx1 can act in Cu delivery to the metal binding domains (MBDs) of the P1B-type ATPase ATP7B and to determine the thermodynamic factors that underpin this activity. hGrx1 can transfer Cu to the metallochaperone Atox1 and to the MBDs 5-6 of ATP7B (WLN5-6). This exchange is irreversible. In a mixture of the three proteins, Cu is delivered to the WLN5-6 preferentially, despite the presence of Atox1. This preferential Cu exchange appears to be driven by both the thermodynamics of the interactions between the proteins pairs and of the proteins with Cu(I). Crucially, protein-protein interactions between hGrx1, Atox1 and WLN5-6 were detected by NMR spectroscopy both in the presence and absence of Cu at a common interface. This study augments the possible activities of hGrx1 in intracellular Cu homeostasis and suggests a potential redundancy in this system, where hGrx1 has the potential to act under cellular conditions where the activity of Atox1 in Cu regulation is attenuated.

Intracellular copper (Cu) in eukaryotic organisms is regulated by homeostatic systems, which rely on the activities of soluble metallochaperones that participate in Cu exchange through highly tuned protein-protein interactions. Recently, the human enzyme glutaredoxin-1 (hGrx1) has been shown to possess Cu metallochaperone activity. The aim of this study was to ascertain whether hGrx1 can act in Cu delivery to the metal binding domains (MBDs) of the P1B-type ATPase ATP7B and to determine the thermodynamic factors that underpin this activity. hGrx1 can transfer Cu to the metallochaperone Atox1 and to the MBDs 5-6 of ATP7B (WLN5-6). This exchange is irreversible. In a mixture of the three proteins, Cu is delivered to the WLN5-6 preferentially, despite the presence of Atox1. This preferential Cu exchange appears to be driven by both the thermodynamics of the interactions between the proteins pairs and of the proteins with Cu(I). Crucially, protein-protein interactions between hGrx1, Atox1 and WLN5-6 were detected by NMR spectroscopy both in the presence and absence of Cu at a common interface. This study augments the possible activities of hGrx1 in intracellular Cu homeostasis and suggests a potential redundancy in this system, where hGrx1 has the potential to act under cellular conditions where the activity of Atox1 in Cu regulation is attenuated.

Ubiquitous expression of mutant Cu/Zn-superoxide dismutase (SOD1) selectively affects motor neurons in the central nervous system (CNS), causing the adult-onset degenerative disease amyotrophic lateral sclerosis (ALS). The CNS-specific impact of ubiquitous mutant SOD1 expression is recapitulated in transgenic mouse models of the disease. Here we present outcomes for the metallo-complex CuII(atsm) tested for therapeutic efficacy in mice expressing SOD1G93A on a mixed genetic background. Oral administration of CuII(atsm) delayed the onset of neurological symptoms, improved locomotive capacity and extended overall survival. Although the ALS-like phenotype of SOD1G93A mice is instigated by expression of the mutant SOD1, we show the improved phenotype of the CuII(atsm)-treated animals involves an increase in mature mutant SOD1 protein in the disease-affected spinal cord, where concomitant increases in copper and SOD1 activity are also evident. In contrast to these effects in the spinal cord, treating with CuII(atsm) had no effect in liver on either mutant SOD1 protein levels or its activity, indicating a CNS-selective SOD1 response to the drug. These data provide support for CuII(atsm) as a treatment option for ALS as well as insight to the CNS-selective effects of mutant SOD1.

Ubiquitous expression of mutant Cu/Zn-superoxide dismutase (SOD1) selectively affects motor neurons in the central nervous system (CNS), causing the adult-onset degenerative disease amyotrophic lateral sclerosis (ALS). The CNS-specific impact of ubiquitous mutant SOD1 expression is recapitulated in transgenic mouse models of the disease. Here we present outcomes for the metallo-complex CuII(atsm) tested for therapeutic efficacy in mice expressing SOD1G93A on a mixed genetic background. Oral administration of CuII(atsm) delayed the onset of neurological symptoms, improved locomotive capacity and extended overall survival. Although the ALS-like phenotype of SOD1G93A mice is instigated by expression of the mutant SOD1, we show the improved phenotype of the CuII(atsm)-treated animals involves an increase in mature mutant SOD1 protein in the disease-affected spinal cord, where concomitant increases in copper and SOD1 activity are also evident. In contrast to these effects in the spinal cord, treating with CuII(atsm) had no effect in liver on either mutant SOD1 protein levels or its activity, indicating a CNS-selective SOD1 response to the drug. These data provide support for CuII(atsm) as a treatment option for ALS as well as insight to the CNS-selective effects of mutant SOD1.