Structural and functional fine mapping of cysteines in mammalian glutaredoxin reveal their differential oxidation susceptibility

Elizabeth M. Corteselli, Mona Sharafi, Robert Hondal, Maximilian MacPherson, Sheryl White, Ying-Wai Lam, Clarissa Gold, Allison M. Manuel, Albert Vliet, Severin T. Schneebeli, Vikas Anathy, Jianing Li () and Yvonne M. W. Janssen-Heininger ()
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Elizabeth M. Corteselli: University of Vermont Larner College of Medicine
Mona Sharafi: University of Vermont
Robert Hondal: University of Vermont
Maximilian MacPherson: University of Vermont Larner College of Medicine
Sheryl White: University of Vermont Larner College of Medicine
Ying-Wai Lam: University of Vermont
Clarissa Gold: University of Vermont
Allison M. Manuel: University of Vermont Larner College of Medicine
Albert Vliet: University of Vermont Larner College of Medicine
Severin T. Schneebeli: Purdue University
Vikas Anathy: University of Vermont Larner College of Medicine
Jianing Li: Purdue University
Yvonne M. W. Janssen-Heininger: University of Vermont Larner College of Medicine

Nature Communications, 2023, vol. 14, issue 1, 1-14

Abstract: Abstract Protein-S-glutathionylation is a post-translational modification involving the conjugation of glutathione to protein thiols, which can modulate the activity and structure of key cellular proteins. Glutaredoxins (GLRX) are oxidoreductases that regulate this process by performing deglutathionylation. However, GLRX has five cysteines that are potentially vulnerable to oxidative modification, which is associated with GLRX aggregation and loss of activity. To date, GLRX cysteines that are oxidatively modified and their relative susceptibilities remain unknown. We utilized molecular modeling approaches, activity assays using recombinant GLRX, coupled with site-directed mutagenesis of each cysteine both individually and in combination to address the oxidizibility of GLRX cysteines. These approaches reveal that C8 and C83 are targets for S-glutathionylation and oxidation by hydrogen peroxide in vitro. In silico modeling and experimental validation confirm a prominent role of C8 for dimer formation and aggregation. Lastly, combinatorial mutation of C8, C26, and C83 results in increased activity of GLRX and resistance to oxidative inactivation and aggregation. Results from these integrated computational and experimental studies provide insights into the relative oxidizability of GLRX’s cysteines and have implications for the use of GLRX as a therapeutic in settings of dysregulated protein glutathionylation.

Date: 2023
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DOI: 10.1038/s41467-023-39664-2

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