Role of Glutaredoxin1 and Glutathione in Regulating the Activity of the Copper-transporting P-type ATPases, ATP7A and ATP7B

Abstract

The copper-transporting P-type ATPases (Cu-ATPases), ATP7A and ATP7B, are essential for the regulation of intracellular copper homeostasis. In this report we describe new roles for glutathione (GSH) and glutaredoxin1 (GRX1) in Cu homeostasis through their regulation of Cu-ATPase activity. GRX1 is a thiol oxidoreductase that catalyzes the reversible reduction of GSH-mixed disulfides to their respective sulfhydryls (deglutathionylation). Here, we demonstrated that glutathionylation of the Cu-ATPases and their interaction with GRX1 were affected by alterations in Cu levels. The data support our hypothesis that the Cu-ATPases serve as substrates for Cu-dependent GRX1-mediated deglutathionylation. This in turn liberates the CuATPase cysteinyl thiol groups for Cu binding and transport. GSH depletion experiments led to reversible inhibition of the Cu-ATPases that correlated with effects on intracellular Cu levels and GRX1 activity. Finally, knockdown of GRX1 expression resulted in an increase in intracellular Cu accumulation. Together, these data directly implicate GSH and GRX1 with important new roles in redox regulation of the Cu-ATPases, through modulation of Cu binding by the Cu-ATPase cysteine motifs.

An Expanding Range of Functions for the Copper Chaperone/Antioxidant Protein Atox1

“…Evidence exists for both models (25,39,64,95 The role of the cellular redox-maintaining machinery in the copper transfer/transport mechanism is an important and only partially characterized issue. Recent study suggests that the MBDs of Cu-ATPases can be glutathionylated (89). It has been proposed that upon copper elevation glutaredoxin(s) remove the GSH units from the CxxC motif making it available for copper binding (89).…”

“…Recent study suggests that the MBDs of Cu-ATPases can be glutathionylated (89). It has been proposed that upon copper elevation glutaredoxin(s) remove the GSH units from the CxxC motif making it available for copper binding (89). The model is logical, and the dependence of copper efflux on the presence of glutaredoxins (Grx1 and Grx2) in a cell has been clearly demonstrated.…”

Copper induces Cu-ATPase ATP7A mRNA in a fish cell line, SAF1

“…Murine GR is regulated by the oxidative stress-activated transcription factor Nrf2 (Harvey et al, 2009), and although similar oxidative mechanisms may also play a role in MT induction (Ohtsuji et al, 2008), the lack of change in ATP7A mRNA after Cd treatment suggests that Cu is not affecting ATP7A mRNA expression via a mechanism dependent on oxidative stress. Recently Singleton et al (2010) have provided evidence that the reversible glutaredoxin1-catalysed glutathionylation of the Cu-binding Cys residues of ATP7A is required for normal copper transport. Since depletion of glutathione inhibits the Cu-transporting activity of ATP7A, the induction of GR shown in our study may be part of the cellular response to excess Cu, working to maintain glutathione levels in order to facilitate Cu excretion by ATP7A.…”

Functional Partnership of the Copper Export Machinery and Glutathione Balance in Human Cells

“…It was assumed that in the cytosol, the CXXC sites of Atox1 and Cu-ATPases were constitutively reduced and available for copper. However, recent studies suggest that CuATPases are glutathionylated and that glutaredoxins are required to reverse modification and enable copper binding (11).…”