Background: Na,K-ATPase activity is extremely sensitive to changes in the redox state. Results: Binding of glutathione to the regulatory cysteine residues of the catalytic subunit completely inhibits the Na,K-ATPase by blocking the ATP-binding site. Conclusion: S-Glutathionylation of the catalytic subunit is revealed as a mechanism controlling the Na,K-ATPase function. Significance: Regulatory S-glutathionylation adjusts Na,K-ATPase activity to the changes in intracellular redox state and ATP levels.

By maintaining the Na+ and K+ transmembrane gradient mammalian Na,K-ATPase acts as a key regulator of neuronal electrotonic properties. Na,K-ATPase has an important role in synaptic transmission and memory formation. Accumulation of beta-amyloid (Aβ) at the early stages of Alzheimer’s disease is accompanied by reduction of Na,K-ATPase functional activity. The molecular mechanism behind this phenomenon is not known. Here we show that the monomeric Aβ(1-42) forms a tight (Kd of 3 μM), enthalpy-driven equimolar complex with α1β1 Na,K-ATPase. The complex formation results in dose-dependent inhibition of the enzyme hydrolytic activity. The binding site of Aβ(1-42) is localized in the “gap” between the alpha- and beta-subunits of Na,K-ATPase, disrupting the enzyme functionality by preventing the subunits from shifting towards each other. Interaction of Na,K-ATPase with exogenous Aβ(1-42) leads to a pronounced decrease of the enzyme transport and hydrolytic activity and Src-kinase activation in neuroblastoma cells SH-SY5Y. This interaction allows regulation of Na,K-ATPase activity by short-term increase of the Aβ(1-42) level. However prolonged increase of Aβ(1-42) level under pathological conditions could lead to chronical inhibition of Na,K-ATPase and disruption of neuronal function. Taken together, our data suggest the role of beta-amyloid as a novel physiological regulator of Na,K-ATPase.

Objective HIV-infected patients are at an increased risk of developing atherosclerosis, in part due to downmodulation and functional impairment of ATP-Binding Cassette A1 (ABCA1) cholesterol transporter by the HIV-1 protein Nef. The mechanism of this effect involves Nef interacting with an endoplasmic reticulum (ER) chaperone calnexin and disrupting calnexin binding to ABCA1, leading to ABCA1 retention in ER, its degradation and resulting suppression of cholesterol efflux. However, molecular details of Nef-calnexin interaction remained unknown, limiting translational impact of this finding. Approach and results Here, we used molecular modeling and mutagenesis to characterize Nef-calnexin interaction and to identify small molecule compounds that could block it. We demonstrated that interaction between Nef and calnexin is direct and can be reconstituted using recombinant proteins in vitro with a binding affinity of 89.1 nM measured by surface plasmon resonance. The cytoplasmic tail of calnexin is essential and sufficient for interaction with Nef, and binds Nef with affinity of 9.4 nM. Replacing lysine residues in positions 4 and 7 of Nef with alanines abrogates Nef-calnexin interaction, prevents ABCA1 downregulation by Nef, and preserves cholesterol efflux from HIV-infected cells. Through virtual screening of the NCI library of compounds, we identified a compound, 1[(7-Oxo-7H-benz[de]anthracene-3-yl)amino]anthraquinone, which blocked Nef-calnexin interaction, partially restored ABCA1 activity in HIV-infected cells, and reduced foam cell formation in a culture of HIV-infected macrophages. Conclusion This study identifies potential targets that can be exploited to block the pathogenic effect of HIV infection on cholesterol metabolism and prevent atherosclerosis in HIV-infected subjects.

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.

a b s t r a c tIon pump, Na,K-ATPase specifically binds cardiotonic steroids (CTS), which leads to inhibition of the enzyme activity and activation of signaling network in the cell. We have studied interaction of Na,K-ATPase with CTS of two different types -marinobufagenin and ouabain. We have shown that both CTS inhibit activity of Na,K-ATPase with the same K i values, but binding of ouabain is sensitive to the conformation of Na,K-ATPase while binding of marinobufagenin is not. Furthermore, binding of ouabain and marinobufagenin results in different structural changes in Na,K-ATPase. Our data allow to explain the diversity of effects on the receptor function of Na,K-ATPase caused by different types of CTS.

A hypothesis is proposed that the cytokine storm syndrome, which complicates COVID-19 in some patients, is a consequence of antibody-dependent enhancement of virus infection, which is in turn happens due to a change in dominant antigenic determinants of SARS-CoV-2 S-protein. The antibody-dependent enhancement of virus infection is a phenomenon in which virus-specific antibodies that are not neutralizing enhance the entry of infectious virus into immune cells causing their death. Antibody-dependent enhancement has been reported for different coronaviruses. This phenomenon happens due to a decrease in the binding strength of neutralizing antibodies to the virus, which converts these antibodies into suboptimalnot neutralizing ones. According to our hypothesis, such a decrease in affinity may be associated with a change in the conformation of the viral S-protein. We believe that this conformational change is the major factor in the switching of antibodies affinity, which triggers antibody-dependent enhancement. However, other factors that contribute to antigen drift and antigenic determinant changes may also play a role.

Cholinergic dysfunction in Alzheimer’s disease (AD) can be mediated by the neuronal α7 nicotinic acetylcholine receptor (α7nAChR). Beta-amyloid peptide (Aβ) binds to the α7nAChR, disrupting the receptor’s function and causing neurotoxicity. In vivo not only Aβ but also its modified forms can drive AD pathogenesis. One of these forms, iso-Aβ (containing an isomerized Asp7 residue), shows an increased neurotoxicity in vitro and stimulates amyloidogenesis in vivo. We suggested that such effects of iso-Aβ are α7nAChR-dependent. Here, using calcium imaging and electrophysiology, we found that iso-Aβ is a more potent inhibitor of the α7nAChR-mediated calcium current than unmodified Aβ. However, Asp7 isomerization eliminated the ability of Aβ to decrease the α7nAChR levels. These data indicate differences in the interaction of the peptides with the α7nAChR, which we demonstrated using computer modeling. Neither Aβ nor iso-Aβ competed with 125I-α-bungarotoxin for binding to the orthosteric site of the receptor, suggesting the allosteric binging mode of the peptides. Further we found that increased neurotoxicity of iso-Aβ was mediated by the α7nAChR. Thus, the isomerization of Asp7 enhances the inhibitory effect of Aβ on the functional activity of the α7nAChR, which may be an important factor in the disruption of the cholinergic system in AD.

Molecular mechanisms of human disease progression often have complex genetic underpinnings, and sophisticated sequencing approaches coupled with advanced analytics [...]