Proteomics

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Structural and functional fine mapping of cysteines in mammalian glutaredoxin reveal a hierarchy of susceptibility to oxidative inactivation


ABSTRACT: Protein-S-glutathionylation (PSSG) is a post-translational modification involving the conjugation of glutathione to reactive protein thiols, which can modulate the activity and structure of key cellular proteins. Glutaredoxins (GLRX) are oxidoreductases that regulate this process by performing the reversible deglutathionylation reaction. Mammalian GLRX only requires the N-terminal cysteine in the thioredoxin domain to carry out deglutathionylation. However, GLRX has five cysteines that are potentially vulnerable to oxidative modification, and oxidation of GLRX itself has been reported in close association with aggregation and loss of activity. Further, the specific cysteines within GLRX that are vulnerable to oxidative modification and their relative reactivities with oxidants remain unknown. Herein, we utilized various molecular modeling approaches coupled with site-directed mutagenesis of each cysteine both individually and in combination to address how the five cysteines present in GLRX influence the enzyme’s biological activity and susceptibility to dimerization and inactivation. We report that mutation of cysteine 26 (C26) to serine of murine GLRX1 confers a higher rate of deglutathionylation. We further demonstrate that C8 and C83 are targets for PSSG in vitro while C79 is less vulnerable due to steric hindrance. Hydrogen peroxide also caused inactivation of GLRX that was only protected against with mutation of C8 or C83. Both experimental and modeling evidence indicates C8 contributes to dimer formation. Lastly, combinatorial mutation of C8, C26, and C83 results in increased biological activity and resistance to aggregation and oxidative inactivation. Overall, these results from our corroborated experimental and computational studies have valuable implications for the use of GLRX as a therapeutic in settings of dysregulated protein glutathionylation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Ying Wai Lam  

LAB HEAD: Yvonne Janssen-Heininger

PROVIDER: PXD026486 | Pride | 2023-10-24

REPOSITORIES: Pride

Dataset's files

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Action DRS
Allison_QE__0077-_1_.msf Msf
Allison_QE__0077-_1_.mzid.gz Mzid
Allison_QE__0077-_1_.mzid_Allison_QE__0077-_1_.MGF Mzid
Allison_QE__0077_C8S.raw Raw
Allison_QE__0078-_1_.msf Msf
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Structural and functional fine mapping of cysteines in mammalian glutaredoxin reveal their differential oxidation susceptibility.

Corteselli Elizabeth M EM   Sharafi Mona M   Hondal Robert R   MacPherson Maximilian M   White Sheryl S   Lam Ying-Wai YW   Gold Clarissa C   Manuel Allison M AM   van der Vliet Albert A   Schneebeli Severin T ST   Anathy Vikas V   Li Jianing J   Janssen-Heininger Yvonne M W YMW  

Nature communications 20230728 1


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  ...[more]

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