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Basal Glutathionylation of Na,K-ATPase α-Subunit Depends on Redox Status of Cells during the Enzyme Biosynthesis.

Mitkevich VA, Petrushanko IY, Poluektov YM, Burnysheva KM, Lakunina VA, Anashkina AA, Makarov AA - Oxid Med Cell Longev (2016)

Bottom Line: It was found that Na,K-ATPase α-subunit has a basal glutathionylation which is not abrogated by reducing agent.We have analyzed all X-ray structures of the Na,K-ATPase α-subunit from pig kidney and found that there are a number of isolated cavities with unresolved electron density close to the relevant cysteine residues.Here, we discuss the role of basal glutathionylation of Na,K-ATPase α-subunit and provide evidence supporting the view that this modification is cotranslational.

View Article: PubMed Central - PubMed

Affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, Moscow 119991, Russia.

ABSTRACT
Many viruses induce oxidative stress and cause S-glutathionylation of Cys residues of the host and viral proteins. Changes in cell functioning during viral infection may be associated with glutathionylation of a number of key proteins including Na,K-ATPase which creates a gradient of sodium and potassium ions. It was found that Na,K-ATPase α-subunit has a basal glutathionylation which is not abrogated by reducing agent. We have shown that acute hypoxia leads to increase of total glutathionylation level of Na,K-ATPase α-subunit; however, basal glutathionylation of α-subunit increases under prolonged hypoxia only. The role of basal glutathionylation in Na,K-ATPase function remains unclear. Understanding significance of basal glutathionylation is complicated by the fact that there are no X-ray structures of Na,K-ATPase with the identified glutathione molecules. We have analyzed all X-ray structures of the Na,K-ATPase α-subunit from pig kidney and found that there are a number of isolated cavities with unresolved electron density close to the relevant cysteine residues. Analysis of the structures showed that this unresolved density in the structure can be occupied by glutathione associated with cysteine residues. Here, we discuss the role of basal glutathionylation of Na,K-ATPase α-subunit and provide evidence supporting the view that this modification is cotranslational.

No MeSH data available.


Related in: MedlinePlus

Part of Na,K-ATPase α1-subunit near Cys452 and Cys456 (PDB code 3WGU). (a) Unresolved density in the isolated cavity. (b) GSH incorporated into the unresolved density.
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fig3: Part of Na,K-ATPase α1-subunit near Cys452 and Cys456 (PDB code 3WGU). (a) Unresolved density in the isolated cavity. (b) GSH incorporated into the unresolved density.

Mentions: Models of the Na,K-ATPase α-subunit with glutathione in isolated cavities were built manually using COOT to verify fitting of glutathione molecules to the cavities. For this purpose, two models of glutathione molecule were used (Figure 1), which were placed in isolated cavities avoiding overlap with the resolved parts of structure. Glutathione molecule was placed in isolated cavities to fit unresolved densities, and thiol group of glutathione has been directed towards the thiol group of a cysteine in the cavity. Areas of unresolved electron density, large enough to insert a glutathione molecule, were found in a number of isolated cavities in Na,K-ATPase near the cysteine residues listed in Table 4. It was found that the electron density capable of accommodating glutathione molecule by shape and size is located in cavities near the cysteine residues Cys 204, Cys 242 (Figure 2) (3WGU); Cys 452, Cys 456, and Cys 457 (Figure 3) (3WGU, 3KDP, and 3B8E); Cys 698, Cys 367 (Figure 4) (3WGU); and Cys 599 (Figure 5) (3WGU, 3KDP, 3B8E, and 4HYT) (Table 4). The role of these Cys residues in the functioning of the enzyme was evaluated previously with point mutagenesis [24]. Replacing one of Cys 367 and Cys 698 residues or all three Cys residues (452, 456, and 457) with alanine or serine resulted in reduced activity by more than 75%, while replacing Cys 204 and Cys 599 residues resulted in reduced activity by more than 50% (Table 4). This indicates that these Cys residues are not critical for the enzyme activity but their glutathionylation could play an important role in regulating the protein function. Substitution of Cys 242 to Ala or Ser residues was lethal to the cells, suggesting the central role of this residue for the functioning of Na,K-ATPase. According to our data, Cys 242 is one of the residues undergoing regulatory glutathionylation in oxidative stress [13].


Basal Glutathionylation of Na,K-ATPase α-Subunit Depends on Redox Status of Cells during the Enzyme Biosynthesis.

Mitkevich VA, Petrushanko IY, Poluektov YM, Burnysheva KM, Lakunina VA, Anashkina AA, Makarov AA - Oxid Med Cell Longev (2016)

Part of Na,K-ATPase α1-subunit near Cys452 and Cys456 (PDB code 3WGU). (a) Unresolved density in the isolated cavity. (b) GSH incorporated into the unresolved density.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4863110&req=5

fig3: Part of Na,K-ATPase α1-subunit near Cys452 and Cys456 (PDB code 3WGU). (a) Unresolved density in the isolated cavity. (b) GSH incorporated into the unresolved density.
Mentions: Models of the Na,K-ATPase α-subunit with glutathione in isolated cavities were built manually using COOT to verify fitting of glutathione molecules to the cavities. For this purpose, two models of glutathione molecule were used (Figure 1), which were placed in isolated cavities avoiding overlap with the resolved parts of structure. Glutathione molecule was placed in isolated cavities to fit unresolved densities, and thiol group of glutathione has been directed towards the thiol group of a cysteine in the cavity. Areas of unresolved electron density, large enough to insert a glutathione molecule, were found in a number of isolated cavities in Na,K-ATPase near the cysteine residues listed in Table 4. It was found that the electron density capable of accommodating glutathione molecule by shape and size is located in cavities near the cysteine residues Cys 204, Cys 242 (Figure 2) (3WGU); Cys 452, Cys 456, and Cys 457 (Figure 3) (3WGU, 3KDP, and 3B8E); Cys 698, Cys 367 (Figure 4) (3WGU); and Cys 599 (Figure 5) (3WGU, 3KDP, 3B8E, and 4HYT) (Table 4). The role of these Cys residues in the functioning of the enzyme was evaluated previously with point mutagenesis [24]. Replacing one of Cys 367 and Cys 698 residues or all three Cys residues (452, 456, and 457) with alanine or serine resulted in reduced activity by more than 75%, while replacing Cys 204 and Cys 599 residues resulted in reduced activity by more than 50% (Table 4). This indicates that these Cys residues are not critical for the enzyme activity but their glutathionylation could play an important role in regulating the protein function. Substitution of Cys 242 to Ala or Ser residues was lethal to the cells, suggesting the central role of this residue for the functioning of Na,K-ATPase. According to our data, Cys 242 is one of the residues undergoing regulatory glutathionylation in oxidative stress [13].

Bottom Line: It was found that Na,K-ATPase α-subunit has a basal glutathionylation which is not abrogated by reducing agent.We have analyzed all X-ray structures of the Na,K-ATPase α-subunit from pig kidney and found that there are a number of isolated cavities with unresolved electron density close to the relevant cysteine residues.Here, we discuss the role of basal glutathionylation of Na,K-ATPase α-subunit and provide evidence supporting the view that this modification is cotranslational.

View Article: PubMed Central - PubMed

Affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, Moscow 119991, Russia.

ABSTRACT
Many viruses induce oxidative stress and cause S-glutathionylation of Cys residues of the host and viral proteins. Changes in cell functioning during viral infection may be associated with glutathionylation of a number of key proteins including Na,K-ATPase which creates a gradient of sodium and potassium ions. It was found that Na,K-ATPase α-subunit has a basal glutathionylation which is not abrogated by reducing agent. We have shown that acute hypoxia leads to increase of total glutathionylation level of Na,K-ATPase α-subunit; however, basal glutathionylation of α-subunit increases under prolonged hypoxia only. The role of basal glutathionylation in Na,K-ATPase function remains unclear. Understanding significance of basal glutathionylation is complicated by the fact that there are no X-ray structures of Na,K-ATPase with the identified glutathione molecules. We have analyzed all X-ray structures of the Na,K-ATPase α-subunit from pig kidney and found that there are a number of isolated cavities with unresolved electron density close to the relevant cysteine residues. Analysis of the structures showed that this unresolved density in the structure can be occupied by glutathione associated with cysteine residues. Here, we discuss the role of basal glutathionylation of Na,K-ATPase α-subunit and provide evidence supporting the view that this modification is cotranslational.

No MeSH data available.


Related in: MedlinePlus