Department of Pathology and Laboratory of Medicine, University of Vermont Larner College of Medicine, Burlington, VT, 05405, USA.
Department of Chemistry, University of Vermont, Burlington, VT, 05405, USA.
Nat Commun. 2023 Jul 28;14(1):4550. doi: 10.1038/s41467-023-39664-2.
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.
蛋白质 S-谷胱甘肽化是一种涉及谷胱甘肽与蛋白质巯基结合的翻译后修饰，可调节关键细胞蛋白的活性和结构。谷氧还蛋白（GLRX）是通过进行去谷胱甘肽化来调节此过程的氧化还原酶。然而，GLRX 有五个半胱氨酸，容易受到氧化修饰的影响，这与 GLRX 聚集和失活有关。迄今为止，GLRX 中被氧化修饰的半胱氨酸及其相对易感性仍不清楚。我们利用分子建模方法、使用重组 GLRX 的活性测定，以及对每个半胱氨酸的定点突变（单独和组合），来研究 GLRX 半胱氨酸的氧化性。这些方法表明，C8 和 C83 是体外 S-谷胱甘肽化和过氧化氢氧化的靶标。计算机模拟和实验验证证实了 C8 对半胱氨酸二聚体形成和聚集的重要作用。最后，C8、C26 和 C83 的组合突变导致 GLRX 活性增加，对氧化失活和聚集的抵抗力增强。这些综合计算和实验研究的结果提供了对 GLRX 半胱氨酸相对氧化性的深入了解，并对 GLRX 在蛋白质谷胱甘肽化失调的情况下作为治疗药物的应用具有重要意义。
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