Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation
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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.
In: Journal of Biological Chemistry, Vol. 299, No. 3, 102941, 2023.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Multiple oxidative post-translational modifications of human glutamine synthetase mediate peroxynitrite-dependent enzyme inactivation and aggregation
AU - Campolo, Nicolás
AU - Mastrogiovanni, Mauricio
AU - Mariotti, Michele
AU - Issoglio, Federico M.
AU - Estrin, Darío
AU - Hägglund, Per
AU - Grune, Tilman
AU - Davies, Michael J.
AU - Bartesaghi, Silvina
AU - Radi, Rafael
N1 - Publisher Copyright: © 2023 The Authors
PY - 2023
Y1 - 2023
N2 - 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.
AB - 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.
KW - aggregation
KW - dityrosine
KW - free radicals
KW - glutamine synthetase
KW - hydrogen peroxide
KW - nitrotyrosine
KW - oxidants
KW - peroxynitrite
KW - thiol oxidation
UR - http://www.scopus.com/inward/record.url?scp=85149804682&partnerID=8YFLogxK
U2 - 10.1016/j.jbc.2023.102941
DO - 10.1016/j.jbc.2023.102941
M3 - Journal article
C2 - 36702251
AN - SCOPUS:85149804682
VL - 299
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 3
M1 - 102941
ER -
ID: 340406100