Titel
Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation
Autor*in
Nicolás Campolo
Departamento de Bioquímica and Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República
Mauricio Mastrogiovanni
Departamento de Bioquímica and Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República
Michele Mariotti
Department of Biomedical Sciences, Panum Institute, University of Copenhagen
... show all
Abstract
Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutamine from L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays a pivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highly expressed in astrocytes, where its activity sustains the glutamate-glutamine cycle at glutamatergic synapses and is thus essential for maintaining brain homeostasis. In fact, decreased GS levels or activity have been associated with neurodegenerative diseases, with these alterations attributed to oxidative post-translational modifications of the protein, in particular tyrosine nitration. In this study, we expressed and purified human GS (HsGS) and performed an in-depth analysis of its oxidative inactivation by peroxynitrite (ONOO−) in vitro. We found that ONOO− exposure led to a dose-dependent loss of HsGS activity, the oxidation of cysteine, methionine, and tyrosine residues and also the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MS through combined H216O/H218O trypsin digestion identified up to 10 tyrosine nitration sites and five types of dityrosine cross-links; these modifications were further scrutinized by structural analysis. Tyrosine residues 171, 185, 269, 283, and 336 were the main nitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that their sole nitration was not responsible for enzyme inactivation. In addition, we observed that ONOO− induced HsGS aggregation and activity loss. Thiol oxidation was a key modification to elicit aggregation, as it was also induced by hydrogen peroxide treatment. Taken together, our results indicate that multiple oxidative events at various sites are responsible for the inactivation and aggregation of human GS.
Stichwort
glutamine synthetaseoxidantsfree radicalsperoxynitritehydrogen peroxidenitrotyrosinedityrosinethiol oxidationaggregation
Objekt-Typ
Sprache
Englisch [eng]
Persistent identifier
phaidra.univie.ac.at/o:2064278
Erschienen in
Titel
Journal of Biological Chemistry
Band
299
Ausgabe
3
ISSN
0021-9258
Erscheinungsdatum
2023
Publication
Elsevier BV
Erscheinungsdatum
2023
Zugänglichkeit
Rechte
Rechteangabe
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