Our previous findings suggested that reversible thiol modifications of cysteine residues within the actuator (AD) and nucleotide binding domain (NBD) of the Na,K-ATPase may represent a powerful regulatory mechanism conveying redox- and oxygen-sensitivity of this multifunctional enzyme. S-glutathionylation of Cys244 in the AD and Cys 454-458-459 in the NBD inhibited the enzyme and protected cysteines' thiol groups from irreversible oxidation under hypoxic conditions. In this study mutagenesis approach was used to assess the role these cysteines play in regulation of the Na,K-ATPase hydrolytic and signaling functions. Several constructs of mouse α1 subunit of the Na,K-ATPase were produced in which Cys244, Cys 454-458-459 or Cys 244-454-458-459 were replaced by alanine. These constructs were expressed in human HEK293 cells. Non-transfected cells and those expressing murine α1 subunit were exposed to hypoxia or treated with oxidized glutathione (GSSG). Both conditions induced inhibition of the wild type Na,K-ATPase. Enzymes containing mutated mouse α1 lacking Cys244 or all four cysteines (Cys 244-454-458-459) were insensitive to hypoxia. Inhibitory effect of GSSG was observed for wild type murine Na,K-ATPase, but was less pronounced in Cys454-458-459Ala mutant and completely absent in the Cys244Ala and Cys 244-454-458-459Ala mutants. In cells, expressing wild type enzyme, ouabain induced activation of Src and Erk kinases under normoxic conditions, whereas under hypoxic conditions this effect was inversed. Cys454-458-459Ala substitution abolished Src kinase activation in response to ouabain treatment, uncoupled Src from Erk signaling, and interfered with O-sensitivity of Na,K-ATPase signaling function. Moreover, modeling predicted that S-glutathionylation of Cys 458 and 459 should prevent inhibitory binding of Src to NBD. Our data indicate for the first time that cysteine residues within the AD and NBD influence hydrolytic as well as receptor function of the Na,K-ATPase and alter responses of the enzyme to hypoxia or upon treatment with cardiotonic steroids.
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http://dx.doi.org/10.1016/j.redox.2017.05.021 | DOI Listing |
Sci Adv
January 2025
State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin 300353, People's Republic of China.
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View Article and Find Full Text PDFNat Cancer
January 2025
Department of Medical Oncology and Pneumology, University Hospital Tübingen, Tübingen, Germany.
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View Article and Find Full Text PDFJ Am Chem Soc
January 2025
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
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View Article and Find Full Text PDFPhys Chem Chem Phys
January 2025
School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China.
Globin X is a newly discovered member of the globin family, while its structure and function are not fully understood. In this study, we performed protein modelling studies using Alphafold3 and molecular dynamics simulations, which suggested that the protein adopts a typical globin fold, with the formation of a potential disulfide bond of Cys65 and Cys141. To elucidate the role of this unique disulfide in protein structure and stability, we constructed a double mutant of C65S/C141S by mutating the two cysteine residues to serine.
View Article and Find Full Text PDFOrg Biomol Chem
January 2025
Laboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical University, 3-2-1 Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, Japan.
The development of covalent drugs, particularly those utilizing Michael acceptors, has garnered significant attention in recent pharmaceutical research due to the ability of such molecules to irreversibly inhibit protein function. This study focusses on the synthesis and evaluation of ethynylsulfonamides, which are predicted to have superior covalent binding ability, metabolic stability, and water solubility compared to traditional amides. We developed a straightforward synthesis method for ethynylsulfonamides and comprehensively evaluated the covalent binding abilities of these compounds using NMR with various nucleophilic amino acids in different solvents.
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