Photochemically-induced protein tyrosine nitration in vitro and in cellula by 5-methyl-1,4-dinitro-1H-imidazole (DNI): synthesis and biochemical characterization

Natalia Ríos: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay -- Adrian Aicardo: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Departamento de Nutrición Clínica, Escuela de Nutrición, Universidad de la República, Montevideo, Uruguay -- Cecilia Chavarría: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay -- Rodrigo Ivagnes: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay -- Mauricio Mastrogiovanni: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay -- Rafael Radi: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay -- José M. Souza: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay; Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República, Montevideo, Uruguay. Contacto: [email protected] photochemical nitrating agent 5-methyl-1,4-dinitro-1H-imidazole (DNI) has been recently described as an effective tool for nitrating tyrosine residues in proteins under 390 nm irradiation (Long T. et al., 2021). Herein, we describe the one-step synthesis of DNI from the precursor 4-methyl-5-nitro-1H-imidazole with good yield (66%) and high purity (>99%). Spectral analysis of DNI reveals two maximum peaks (228 and 290 nm) with maximum nitration yields and kinetics occurring at 290 nm. Electron paramagnetic resonance (EPR)- and mass spectrometry (MS)- spin trapping analysis evidenced the formation of nitrogen dioxide (•NO2) upon irradiation of DNI, implying the homolysis of the N–N bond in the DNI molecule. Irradiation of DNI at 290, 390 nm, or UVA light (315–400 nm), produced tyrosine nitration, with yields approaching ca. 30% with respect to DNI at 290 nm exposure. Indeed, using alpha-synuclein as a model protein, the main protein post-translational modification triggered by DNI was the generation of 3-nitrotyrosine as shown by MS analysis. Additionally, the formation of di-tyrosine was also observed. Finally, intracellular •NO2 production upon DNI photolysis in bovine aortic endothelial cells was evidenced by the nitration of the tyrosine analog probe p-hydroxyphenylacetic acid (PHPA) and cellular protein tyrosine nitration

HPLC-MS/MS Oxylipin Analysis of Plasma from Amyotrophic Lateral Sclerosis Patients

Oxylipins play a critical role in regulating the onset and resolution phase of inflammation. Despite inflammation is a pathological hallmark in amyotrophic lateral sclerosis (ALS), the plasma oxylipin profile of ALS patients has not been assessed yet. Herein, we develop an oxylipin profile-targeted analysis of plasma from 74 ALS patients and controls. We found a significant decrease in linoleic acid-derived oxylipins in ALS patients, including 9-hydroxy-octadecadienoic acid (9-HODE) and 13-HODE. These derivatives have been reported as important regulators of inflammation on different cell systems. In addition, some 5-lipoxygenase metabolites, such as 5-hydroxy- eicosatetraenoic acid also showed a significant decrease in ALS plasma samples. Isoprostanes of the F2 alpha family were detected only in ALS patients but not in control samples, while the hydroxylated metabolite 11-HETE significantly decreased. Despite our effort to analyze specialized pro-resolving mediators, they were not detected in plasma samples. However, we found the levels of 14-hydroxy-docosahexaenoic acid, a marker pathway of the Maresin biosynthesis, were also reduced in ALS patients, suggesting a defective activation in the resolution programs of inflammation in ALS. We further analyze oxylipin concentration levels in plasma from ALS patients to detect correlations between these metabolites and some clinical parameters. Interestingly, we found that plasmatic levels of 13-HODE and 9-HODE positively correlate with disease duration, expressed as days since onset. In summary, we developed a method to analyze (oxy)lipidomics in ALS human plasma and found new profiles of metabolites and novel lipid derivatives with unknown biological activities as potential footprints of disease onset

Administration of Maresin-1 ameliorates the physiopathology of experimental autoimmune encephalomyelitis

Resolution of inflammation is an active and regulated process that leads to the clearance of cell debris and immune cells from the challenged tissue, facilitating the recovery of homeostasis. This physiological response is coordinated by endogenous bioactive lipids known as specialized pro-resolving mediators (SPMs). When resolution fails, inflammation becomes uncontrolled leading chronic inflammation and tissue damage, as occurs in multiple sclerosis (MS). SPMs and the key biosynthetic enzymes involved in SPM production were analysed by metabololipidomics and qPCR in active brain lesions, serum and peripheral blood mononuclear cells (PBMC) of MS patients as well as in the spinal cord of mice with experimental autoimmune encephalomyelitis (EAE). We also tested the therapeutic actions of the SPM coined Maresin-1 (MaR1) in EAE mice and studied its impact on inflammation by doing luminex and flow cytometry analysis. We show that levels of MaR1 and other SPMs were below the limit of detection or not increased in the spinal cord of EAE mice, whereas the production of pro-inflammatory eicosanoids was induced during disease progression. Similarly, we reveal that SPMs were undetected in serum and active brain lesion samples of MS patients, which was linked to impaired expression of the enzymes involved in the biosynthetic pathways of SPMs. We demonstrate that exogenous administration of MaR1 in EAE mice suppressed the protein levels of various pro-inflammatory cytokines and reduced immune cells counts in the spinal cord and blood. MaR1 also decreased the numbers of Th1 cells but increased the accumulation of regulatory T cells and drove macrophage polarization towards an anti-inflammatory phenotype. Importantly, we provide clear evidence that administration of MaR1 in mice with clinical signs of EAE enhanced neurological outcomes and protected from demyelination. This study reveals that there is an imbalance in the production of SPMs in MS patients and in EAE mice, and that increasing the bioavailability of SPMs, such as MaR1, minimizes inflammation and mediates therapeutic actions. Thus, these data suggest that immunoresolvent therapies, such as MaR1, could be a novel avenue for the treatment of MS. The online version contains supplementary material available at 10.1186/s12974-022-02386-1

Acidity and nucleophilic reactivity of glutathione persulfide

Persulfides (RSSH/RSS2) participate in sulfur trafficking and metabolic processes, and are proposed to mediate the signaling effects of hydrogen sulfide (H2S). Despite their growing relevance, their chemical properties are poorly understood. Herein, we studied experimentally and computationally the formation, acidity, and nucleophilicity of glutathione persulfide (GSSH/ GSS2), the derivative of the abundant cellular thiol glutathione (GSH). We characterized the kinetics and equilibrium of GSSH formation from glutathione disulfide and H2S. A pKa of 5.45 for GSSH was determined, which is 3.49 units below that of GSH. The reactions of GSSH with the physiologically relevant electrophiles peroxynitrite and hydrogen peroxide, and with the probe monobromobimane, were studied and compared with those of thiols. These reactions occurred through SN2 mechanisms. At neutral pH, GSSH reacted faster than GSH because of increased availability of the anion and, depending on the electrophile, increased reactivity. In addition, GSS2 presented higher nucleophilicity with respect to a thiolate with similar basicity. This can be interpreted in terms of the so-called a effect, i.e. the increased reactivity of a nucleophile when the atom adjacent to the nucleophilic atom has high electron density. The magnitude of the a effect correlated with the Brønsted nucleophilic factor, bnuc, for the reactions with thiolates and with the ability of the leaving group. Our study constitutes the first determination of the pKa of a biological persulfide and the first examination of the a effect in sulfur nucleophiles, and sheds light on the chemical basis of the biological properties of persulfides.Fil: Benchoam, Dayana. Universidad de la República; UruguayFil: Semelak, Jonathan Alexis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Cuevasanta, Ernesto. Universidad de la República; UruguayFil: Mastrogiovanni, Mauricio. Universidad de la Republica; UruguayFil: Grassano, Juan S.. Universidad de Buenos Aires; ArgentinaFil: Ferrer-Sueta, Gerardo. Universidad de la Republica; UruguayFil: Zeida Camacho, Ari Fernando. Universidad de la Republica; UruguayFil: Trujillo, Madia. Universidad de la Republica; UruguayFil: Möller, Matías N.. Universidad de la Republica; UruguayFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Alvarez, Beatriz. Universidad de la Republica; Urugua

Detección, cuantificación y análisis de oxilipinas en pacientes con Esclerosis Lateral Amiotrófica

CSIC: Grupos_2018 ID: 53

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

Profile of Arachidonic Acid-Derived Inflammatory Markers and Its Modulation by Nitro-Oleic Acid in an Inherited Model of Amyotrophic Lateral Sclerosis

The lack of current treatments for amyotrophic lateral sclerosis (ALS) highlights the need of a comprehensive understanding of the biological mechanisms of the disease. A consistent neuropathological feature of ALS is the extensive inflammation around motor neurons and axonal degeneration, evidenced by accumulation of reactive astrocytes and activated microglia. Final products of inflammatory processes may be detected as a screening tool to identify treatment response. Herein, we focus on (a) detection of arachidonic acid (AA) metabolization products by lipoxygenase (LOX) and prostaglandin endoperoxide H synthase in SOD1G93A mice and (b) evaluate its response to the electrophilic nitro-oleic acid (NO2-OA). Regarding LOX-derived products, a significant increase in 12-hydroxyeicosatetraenoic acid (12-HETE) levels was detected in SOD1G93A mice both in plasma and brain whereas no changes were observed in age-matched non-Tg mice at the onset of motor symptoms (90 days-old). In addition, 15-hydroxyeicosatetraenoic acid (15-HETE) levels were greater in SOD1G93A brains compared to non-Tg. Prostaglandin levels were also increased at day 90 in plasma from SOD1G93A compared to non-Tg being similar in both types of animals at later stages of the disease. Administration of NO2-OA 16 mg/kg, subcutaneously (s/c) three times a week to SOD1G93A female mice, lowered the observed increase in brain 12-HETE levels compared to the non-nitrated fatty acid condition, and modified many others inflammatory markers. In addition, NO2-OA significantly improved grip strength and rotarod performance compared to vehicle or OA treated animals. These beneficial effects were associated with increased hemeoxygenase 1 (HO-1) expression in the spinal cord of treated mice co-localized with reactive astrocytes. Furthermore, significant levels of NO2-OA were detected in brain and spinal cord from NO2-OA -treated mice indicating that nitro-fatty acids (NFA) cross brain–blood barrier and reach the central nervous system to induce neuroprotective actions. In summary, we demonstrate that LOX-derived oxidation products correlate with disease progression. Overall, we are proposing that key inflammatory mediators of AA-derived pathways may be useful as novel footprints of ALS onset and progression as well as NO2-OA as a promising therapeutic compound

Mitochondrial Peroxiredoxin 3 Is Rapidly Oxidized and Hyperoxidized by Fatty Acid Hydroperoxides

Human peroxiredoxin 3 (HsPrx3) is a thiol-based peroxidase responsible for the reduction of most hydrogen peroxide and peroxynitrite formed in mitochondria. Mitochondrial disfunction can lead to membrane lipoperoxidation, resulting in the formation of lipid-bound fatty acid hydroperoxides (LFA-OOHs) which can be released to become free fatty acid hydroperoxides (fFA-OOHs). Herein, we report that HsPrx3 is oxidized and hyperoxidized by fFA-OOHs including those derived from arachidonic acid and eicosapentaenoic acid peroxidation at position 15 with remarkably high rate constants of oxidation (>3.5 × 107 M−1s−1) and hyperoxidation (~2 × 107 M−1s−1). The endoperoxide-hydroperoxide PGG2, an intermediate in prostanoid synthesis, oxidized HsPrx3 with a similar rate constant, but was less effective in causing hyperoxidation. Biophysical methodologies suggest that HsPrx3 can bind hydrophobic structures. Indeed, molecular dynamic simulations allowed the identification of a hydrophobic patch near the enzyme active site that can allocate the hydroperoxide group of fFA-OOHs in close proximity to the thiolate in the peroxidatic cysteine. Simulations performed using available and herein reported kinetic data indicate that HsPrx3 should be considered a main target for mitochondrial fFA-OOHs. Finally, kinetic simulation analysis support that mitochondrial fFA-OOHs formation fluxes in the range of nM/s are expected to contribute to HsPrx3 hyperoxidation, a modification that has been detected in vivo under physiological and pathological conditions