Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation

CAMPOLO, NICOLÁS; MASTROGIOVANNI, MAURICIO; MARIOTTI, MICHELE; ISSOGLIO, FEDERICO M.; ESTRIN, DARÍO; HÄGGLUND, PER; GRUNE, TILMAN; DAVIES, MICHAEL J.; BARTESAGHI, SILVINA; RADI, RAFAEL

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Año: 2023

0021-9258

Glutamine synthetase (GS), which catalyzes the ATP-dependent synthesis of L-glutaminefrom L-glutamate and ammonia, is a ubiquitous and conserved enzyme that plays apivotal role in nitrogen metabolism across all life domains. In vertebrates, GS is highlyexpressed in astrocytes, where its activity sustains the glutamate-glutamine cycle atglutamatergic 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 theprotein, in particular tyrosine nitration. In this study, we expressed and purified humanGS (HsGS) and performed an in-depth analysis of its oxidative inactivation byperoxynitrite (ONOO - ) in vitro. We found that ONOO - exposure led to a dose-dependentloss of HsGS activity, the oxidation of cysteine, methionine and tyrosine residues andalso the nitration of tryptophan and tyrosine residues. Peptide mapping by LC-MS/MSthrough combined H 216 O/H 218 O trypsin digestion identified up to 10 tyrosine nitrationsites and five types of dityrosine cross-links; these modifications were further scrutinizedby structural analysis. Tyrosine residues 171, 185, 269, 283 and 336 were the mainnitration targets; however, tyrosine-to-phenylalanine HsGS mutants revealed that theirsole nitration was not responsible for enzyme inactivation. In addition, we observed thatONOO - induced HsGS aggregation and activity loss. Thiol oxidation was a keymodification to elicit aggregation, as it was also induced by hydrogen peroxide treatment.Taken together, our results indicate that multiple oxidative events at various sites areresponsible for the inactivation and aggregation of human GS.