[ES] Suplemento de la revista Free Radical Biology and Medicine: Effect of RONS in 6-NBDG/glucose uptake in C2C12 myotubes and single isolated skeletal muscle fibres

Fernández Puente, Escarlata

Martín-Prieto, Eva

Palomero Labajos, Jesús

Free Radical Biology and Medicine

English

Gestion del Repositorio Documental de la Universidad de Salamanca

Abstracts Free Radical Biology and Medicine 201 (2023) 1–64Espa~na; 4 Unidad de Investigacion, Hospital Universitario Santa Cristina, Instituto deInvestigacion Sanitaria Princesa (IIS-IP), Madrid, Espa~na* Corresponding authorFibrinogen is an abundant blood plasma glycoprotein formed by two hetero-trimers composed of the chains Aα, Bβ and γ. It plays an important role in bloodclotting (fibrin formation and fibrinolysis), cellular and matrix interactions, in-flammatory response, wound healing, and neoplasia1. These functions areregulated by interactive sites within the molecule, and when those sites arealtered functional impairments arise2. Nitroxidative posttranslational modifica-tions occur during nitroxidative distress, when the production of oxygen andnitrogen reactive species surplus the antioxidant capacity, with harmful effectson biomolecules as proteins, nucleic acids and lipids3,4. The addition of a nitrofunctional group in the ring of tyrosine residue producing 3nitrotyrosine is amarker of nitroxidative distress reported present in cardiovascular and inflam-matory diseases. The nitration of fibrinogen is present in thrombotic diseases asthrombosis, myocardial infarction and stroke5,6, and alterations in fibrin poly-merization, fibrinolysis and cellular interactions are observed when 3nitro-tyrosine is present7,8,9. The objective of the present work is to explore thepossible functional impact of nitration of fibrinogen as a diagnostic tool forcardiovascular diseases as ischemic stroke.Enriched fibrinogen from human plasma was in vitro nitrated with peroxynitriteand, after in-gel protein digestion, reverse phase-liquid chromatography tandemmass spectrometry (dynamic exclusion mode) was performed. The data pro-cessing was carried out with PEAKS Studio XPro software for protein identifi-cation and PTM characterization. Finally, fibrinogen visualization and additionof observed 3-nitrotyrosine residues was made in Molecular Operating Envi-ronment software.Sixteen of the 52 tyrosine residues were found to be nitrated within the fibrin-ogen molecule, being 3 in the Aα chain, 9 in the Bβ chain and 4 in the γ chain.Nine of the 3-nitrotyrosine positions found were within important sites whichplay roles in mediating fibrin assembly, interaction with thrombin, tissue plas-minogen activator, plasminogen and α2-plasmin inhibitor (all of them regulatorsof coagulation processes). They were in sites related with integrin binding tocells, including leukocytes, platelet spreading, fibroblast proliferation, endothe-lial cell spreading, proliferation and capillary tube formation, release of vonWillebrand factor and binding heparin. It results of interest to investigate theimpact of nitration of tyrosine residues on fibrinogen structure and properties inorder to shed light into the subjacent mechanisms of diseases with high incidenceand mortality rates as thrombotic ones.doi: https://doi.org/10.1016/j.freeradbiomed.2023.03.258PP XV_L2THE ROLE OF PEROXIPORINS IN TUNING THE TRANSMISSION OFINTRACELLULAR REDOX SIGNALINGIlaria Sorrentino 1, Angie Molina-Oviedo 1, Eduardo Arevalo-Nu~nez de Arenas 1,Mauro Galli 1, Stefano Bestetti 2, Roberto Sitia 2, Iria Medra~no-Fernandez 1.1 Redox Signaling in Regenerative Medicine lab, Tissue Engineering andRegenerative Medicine (TERMeG), Dept. of Bioengineering, University Carlos III ofMadrid, Madrid, Spain; 2 Protein transport and secretion Unit, Division of geneticand cell biology, Vita-Salute San Raffaele University, Milan, ItalyFor many years H2O2 has been considered a waste in cells, a relic of when oxygenappeared. Nowadays, this concept is completely reverted, and it is widelyaccepted that H2O2 acts as a signaling molecule capable of amplifying differentintracellular pathways1,2. Interestingly, H2O2-sources are localized behinddouble membranes. Hence, it is not surprising that specific proteinaceous64channels are responsible for tightly regulating the fluxes of H2O2 across bio-logical membranes. In the past, we characterized aquaporin- 8 (AQP8) as a mainH2O2 transporter at the plasma membrane, allowing for the transmission offluxes after Tyrosine Kinase Receptor (TKR) activation. Moreover, we found thatAQP8 can be regulated from an open to a closed conformation following cellularneeds3. During the last years, we have also demonstrated that the endoplasmicreticulum (ER) accommodates a specific H2O2 transporter, AQP11, that conductsa constitutive H2O2-flux from the ER to the cytosol that maintains cellularredoxstasis4. To depict the nature of this signal, we have used stable cell linesequipped with the H2O2-sensitive biosensor HyPer targeted to different sub-cellular compartments. We have discovered that AQP11 can also transmit anH2O2-flux that different strategies, we have determined that the silencing of thisprotein triggers the activation of ROS production by the mitochondrial complexIII5. Thus, we postulate that the existence and the architecture of these differentredox fluxes are fundamental to achieving redox homeostasis.doi: https://doi.org/10.1016/j.freeradbiomed.2023.03.259PP XIII_L4EFFECT OF RONS IN 6-NBDG/GLUCOSE UPTAKE IN C2C12 MYOTUBES ANDSINGLE ISOLATED SKELETAL MUSCLE FIBRESEva Martín-Prieto 1,2,*, Escarlata Fernandez-Puente 1,2,3, Jesús Palomero 1,2,3.1 Department of Physiology and Pharmacology, University of Salamanca, Spain;2 Institute of Neurosciences of Castilla y Leon (INCYL), Spain; 3 Institute ofBiomedical Research of Salamanca (IBSAL), Spain* Corresponding authorOxidative eustress, which refers to moderate intracellular level of Reactive Ox-ygen and Nitrogen Species (RONS), is essential to modulate redox signallingprocesses that maintain and govern cellular homeostasis and adaptation. Inskeletal muscle, glucose uptake is essential to provide to the cell the substrate toproduce energy through ATP, which is needed as the energy molecule by thisorgan and others to maintain vital functions. It has been reported that the ho-meostasis and level of RONS may modulate glucose uptake in skeletal muscle.Hydrogen peroxide (H2O2) and nitric oxide (NO) are two specific RONS that areresponsible for oxidative eustress, and they act as signalling molecules thatinteract with components of the cellular signalling pathways that modulate andregulate glucose uptake in skeletal muscle. We have adapted, developed, opti-mized, and validated a methodology based on the fluorescence glucose analogue6-NBDG, combined with quantitative fluorescence microscopy image analysis, todetermine the glucose uptake in two models of skeletal muscle cells: C2C12myotubes and single fibres isolated from muscle. We have assayed differentexperimental conditions that affect redox homeostasis, such as exposure of cellsto different concentrations of H2O2, pro-oxidant environment, impairment, andreplenishment of the main antioxidant -glutathione-, and exposure of cells to NOdonors. The results showed that pro-oxidative intracellular redox environmentevoked by RONS, such as H2O2 and NO, and which might be associated withoxidative eustress, improved 6-NBDG/glucose uptake in myotubes and skeletalmuscle fibres. However, when oxidation was excessive (i.e., oxidative distress),cells appeared damaged and cellular viability was compromised. Moreover, inthis circumstance, glucose uptake appeared to be elevated. In conclusion, thedetermination of 6-NBDG/glucose uptake in myotubes and skeletal muscle cellsis feasible and was validated. Furthermore, a pro-oxidative environment mayfavour glucose uptake in C2C12 myotubes and skeletal muscle fibres.doi: https://doi.org/10.1016/j.freeradbiomed.2023.03.260

Effect of RONS in 6-NBDG/glucose uptake in C2C12 myotubes and single isolated skeletal muscle fibres

https://gredos.usal.es/bitstream/10366/154091/1/Effect%20of%20RONS%20in%206-NBDG_glucose%20uptake%20in%20C2C12%20myotubes%20and%20single%20isolated%20skeletal%20muscle%20fibres.pdf

Effect of RONS in 6-NBDG/glucose uptake in C2C12 myotubes and single isolated skeletal muscle fibres

Abstract

Similar works

Full text

Available Versions

Gestion del Repositorio Documental de la Universidad de Salamanca