Structural and biochemical insight into glycogenin inactivation by the glycogenosis-causing T82M mutation

AbstractThe X-ray structure of rabbit glycogenin containing the T82M (T83M according to previous authors amino acid numbering [1]) mutation causing glycogenosis showed the loss of Thr82 hydrogen bond to Asp162, the residue involved in the activation step of the glucose transfer reaction mechanism. Autoglucosylation, maltoside transglucosylation and UDP-glucose hydrolyzing activities were abolished even though affinity and interactions with UDP-glucose and positioning of Tyr194 acceptor were conserved. Substitution of Thr82 for serine but not for valine restored the maximum extent of autoglucosylation as well as transglucosylation and UDP-glucose hydrolysis rate. Results provided evidence sustaining the essential role of the lost single hydrogen bond for UDP-glucose activation leading to glycogenin-bound glycogen primer synthesis

3-Nitrotyrosine and related derivatives in proteins: precursors, radical intermediates and impact in function

Abstract Oxidative post-translational modification of proteins by molecular oxygen (O2)- and nitric oxide (•NO)-derived reactive species is a usual process that occurs in mammalian tissues under both physiological and pathological conditions and can exert either regulatory or cytotoxic effects. Although the side chain of several amino acids is prone to experience oxidative modifications, tyrosine residues are one of the preferred targets of one-electron oxidants, given the ability of their phenolic side chain to undergo reversible one-electron oxidation to the relatively stable tyrosyl radical. Naturally occurring as reversible catalytic intermediates at the active site of a variety of enzymes, tyrosyl radicals can also lead to the formation of several stable oxidative products through radical–radical reactions, as is the case of 3-nitrotyrosine (NO2Tyr). The formation of NO2Tyr mainly occurs through the fast reaction between the tyrosyl radical and nitrogen dioxide (•NO2). One of the key endogenous nitrating agents is peroxynitrite (ONOO−), the product of the reaction of superoxide radical (O2•−) with •NO, but ONOO−-independent mechanisms of nitration have been also disclosed. This chemical modification notably affects the physicochemical properties of tyrosine residues and because of this, it can have a remarkable impact on protein structure and function, both in vitro and in vivo. Although low amounts of NO2Tyr are detected under basal conditions, significantly increased levels are found at pathological states related with an overproduction of reactive species, such as cardiovascular and neurodegenerative diseases, inflammation and aging. While NO2Tyr is a well-established stable oxidative stress biomarker and a good predictor of disease progression, its role as a pathogenic mediator has been laboriously defined for just a small number of nitrated proteins and awaits further studies.</jats:p

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

Funding Information: This work was supported by grants of Universidad de la República (CSIC_2018 and EI_2020) to R. R., Universidad de la República (CSIC Iniciación_2017_ID_159) to N. C., Alexander von Humboldt Foundation (AvHF) to T. G. and R. R., the Novo Nordisk Foundation ( NNF13OC0004294 and NNF20SA0064214 ) to M. J. D., and PICT 2018-0795 from Agencia I+d+i Argentina , University of Buenos Aires (grant 20020120300025BA ), and CONICET (grant 11220150100303CO ) to D. E. N. C. was partially supported by a fellowship from Comisión Académica de Posgrado (CAP), Universidad de la República , Uruguay. Additional funding was obtained from Programa de Desarrollo de las Ciencias Básicas (PEDECIBA, Uruguay), Agencia Nacional de Investigación e Innovación (ANII-SNI, Uruguay), EU-LAC Health (EULACH16/T01-0131), and Programa de Alimentos y Salud Humana (PAyS, Uruguay). Funding Information: The authors would like to thank Dr Tobias Karlberg and Dr Susanne Gräslund from the Karolinska Institutet - Structural Genomics Consortium for providing the HsGS plasmid (Construct ID GLULA-c004). We also thank Dr Verónica Tórtora for her major aid within the initial steps of the transformation and HsGS expression experiments. N. C. D. E. P. H. T. G. M. J. D. S. B. and R. R. conceptualization; N. C. Mauricio Mastrogiovanni, Michele Mariotti, F. M. I. and P. H. methodology; N. C. Mauricio Mastrogiovanni, Michele Mariotti, and F. M. I. investigation; N. C. writing–original draft; M. J. D. S. B. and R. R. writing–review and editing; R. R. supervision. This work was supported by grants of Universidad de la República (CSIC_2018 and EI_2020) to R. R. Universidad de la República (CSIC Iniciación_2017_ID_159) to N. C. Alexander von Humboldt Foundation (AvHF) to T. G. and R. R. the Novo Nordisk Foundation (NNF13OC0004294 and NNF20SA0064214) to M. J. D. and PICT 2018-0795 from Agencia I+d+i Argentina, University of Buenos Aires (grant 20020120300025BA), and CONICET (grant 11220150100303CO) to D. E. N. C. was partially supported by a fellowship from Comisión Académica de Posgrado (CAP), Universidad de la República, Uruguay. Additional funding was obtained from Programa de Desarrollo de las Ciencias Básicas (PEDECIBA, Uruguay), Agencia Nacional de Investigación e Innovación (ANII-SNI, Uruguay), EU-LAC Health (EULACH16/T01-0131), and Programa de Alimentos y Salud Humana (PAyS, Uruguay). Publisher Copyright: © 2023 The AuthorsGlutamine 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.publishersversionpublishe

Metabolic and Structural Insights into Hydrogen Sulfide Mis-Regulation in Enterococcus faecalis

Hydrogen sulfide (H2S) is implicated as a cytoprotective agent that bacteria employ in response to host-induced stressors, such as oxidative stress and antibiotics. The physiological benefits often attributed to H2S, however, are likely a result of downstream, more oxidized forms of sulfur, collectively termed reactive sulfur species (RSS) and including the organic persulfide (RSSH). Here, we investigated the metabolic response of the commensal gut microorganism Enterococcus faecalis to exogenous Na2S as a proxy for H2S/RSS toxicity. We found that exogenous sulfide increases protein abundance for enzymes responsible for the biosynthesis of coenzyme A (CoA). Proteome S-sulfuration (persulfidation), a posttranslational modification implicated in H2S signal transduction, is also widespread in this organism and is significantly elevated by exogenous sulfide in CstR, the RSS sensor, coenzyme A persulfide (CoASSH) reductase (CoAPR) and enzymes associated with de novo fatty acid biosynthesis and acetyl-CoA synthesis. Exogenous sulfide significantly impacts the speciation of fatty acids as well as cellular concentrations of acetyl-CoA, suggesting that protein persulfidation may impact flux through these pathways. Indeed, CoASSH is an inhibitor of E. faecalis phosphotransacetylase (Pta), suggesting that an important metabolic consequence of increased levels of H2S/RSS may be over-persulfidation of this key metabolite, which, in turn, inhibits CoA and acyl-CoA-utilizing enzymes. Our 2.05 &Aring; crystallographic structure of CoA-bound CoAPR provides new structural insights into CoASSH clearance in E. faecalis

Exploring the Catalytic Mechanism of Human Glutamine Synthetase by Computer Simulations

Glutamine synthetase is an important enzyme that catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. In mammals, it plays a key role in preventing excitotoxicity in the brain and detoxifying ammonia in the liver. In plants and bacteria, it is fundamental for nitrogen metabolism, being critical for the survival of the organism. In this work, we show how the use of classical molecular dynamics simulations and multiscale quantum mechanics/molecular mechanics simulations allowed us to examine the structural properties and dynamics of human glutamine synthetase (HsGS), as well as the reaction mechanisms involved in the catalytic process with atomic level detail. Our results suggest that glutamine formation proceeds through a two-step mechanism that includes a first step in which the γ-glutamyl phosphate intermediate forms, with a 5 kcal/mol free energy barrier and a −8 kcal/mol reaction free energy, and then a second rate-limiting step involving the ammonia nucleophilic attack, with a free energy barrier of 19 kcal/mol and a reaction free energy of almost zero. A detailed analysis of structural features within each step exposed the relevance of the acid–base equilibrium related to protein residues and substrates in the thermodynamics and kinetics of the reactions. These results provide a comprehensive study of HsGS dynamics and establish the groundwork for further analysis regarding changes in HsGS activity, as occur in natural variants and post-translational modifications

SARS-CoV2 Nsp1 is a metal-dependent DNA and RNA endonuclease

Over recent years, we have been living under a pandemic, caused by the rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). One of the major virulence factors of Coronaviruses is the Non-structural protein 1 (Nsp1), known to suppress the host cells protein translation machinery, allowing the virus to produce its own proteins, propagate and invade new cells. To unveil the molecular mechanisms of SARS-CoV2 Nsp1, we have addressed its biochemical and biophysical properties in the presence of calcium, magnesium and manganese. Our findings indicate that the protein in solution is a monomer and binds to both manganese and calcium, with high affinity. Surprisingly, our results show that SARS-CoV2 Nsp1 alone displays metal-dependent endonucleolytic activity towards both RNA and DNA, regardless of the presence of host ribosome. These results show Nsp1 as new nuclease within the coronavirus family. Furthermore, the Nsp1 double variant R124A/K125A presents no nuclease activity for RNA, although it retains activity for DNA, suggesting distinct binding sites for DNA and RNA. Thus, we present for the frst time, evidence that the activities of Nsp1 are modulated by the presence of different metals, which are proposed to play an important role during viral infection. This research contributes significantly to our understanding of the mechanisms of action of Coronaviruses