SFRR-E OP 2.1:
Erythrocyte catalase S-nitrosation as a sensor of chronic subclinical oxidative stress and metabolic complications associated with childhood obesity
Prof. Dr. Rosa María Mateos | Dept. of Biomedicine, Biotechnology and Public Health, University of Cadiz, Spain. | Spain
Chronic subclinical oxidative stress suffered by obese children is characterized by an alteration of redox homeostasis and a depleted antioxidant capacity of their erythrocytes. We previously observed that obese children with metabolic complications present difficulties in activating erythroid antioxidant responses when facing compromised metabolic situations such as an acute glucose ingestion. Among others, erythroid catalase was postulated as a good sensor of such metabolic dystress in children with evidence of insulin resistance (prediabetes), in contrast to eustress in metabolically healthy obese children. Our aim was to study the nitrosation of the enzyme as possible cause of the enzyme inhibition. The study included 95 prepubescent children divided into 3 groups according to their clinical and anthropometric profile: metabolically healthy and unhealthy obese children (Ob.IR- and Ob. IR+, respectively), versus a lean children group. Fasting blood was collected in all three groups, and at different points after an acute glucose intake only in obese children. Catalase levels and activity were analyzed by western blotting and spectrophotometry, respectively. Catalase nitrosation was studied by the biotin-switch method and combined by a final analysis of the erythrocyte nitrosylated protein profile by LCS-MS/MS. Catalase levels were similar among groups, but the baseline activity of the enzyme was partially inhibited in the obese ones. Nevertheless, an induction in catalase activity was showed after glucose intake in healthy obese children, but not in those with metabolic disorders, in which the enzyme activity remained unchanged. The enzyme activity inhibition was proportional to the increased level of its nitrosation in the obese IR+, and incubation of erythrocytes with a nitric oxide donor had detrimental effects over catalase activity in every group. We have showed that erythrocyte catalase nitrosation levels is a good indicator of metabolic complications associated with chronic stress in obese prepubescent children. [Funding codes: PI18/01316 ISCIII; PI-0209-2019; LII19/16IN-CO24].
SFRR-E OP 2.2:
Role of the Thioredoxin-1 in heart failure
Tania Medali | Sorbonne Université, CNRS, INSERM, Institut de Biologie Paris Seine, Biological Adaptation and Ageing (B2A-IBPS), F-75005, Paris, France. | France
Thioredoxin-1 (Trx-1) is a 12 kDa protein with antioxidant and anti-inflammatory properties. It is a highly protective protein against myocardial infarction in murine model. Whether this protective phenomenon can be attributed to cardiomyocyte regeneration, reduced levels of ROS, inflammatory cytokines, protein oxidation or another phenomenon is not known. Nevertheless, its cleavage leading to the truncated pro-inflammatory and pro-oxidant isoform, Trx-80, could compromise its therapeutic use. To circumvent this problem, Trx-mimetic peptide called CB3 has been developed.
The aim of the study is to evaluate the impact of Trx-1 and CB3 on heart failure in mice model and to identify the mechanisms involved in their effects.
For this purpose, we analyzed the impact of Trx-1, Trx-80 and CB3, on cultured neonatal mice cardiomyocytes and in vivo on adult mice with experimental myocardial infarction. To overexpress Trx-1 and Trx-80, we generated different AAV with a TnT chicken cardiac promoter which is specific to the cardiomyocytes.
Our results showed that Trx-1 and CB3 improved cardiac functions such as cardiac contractility and dilatation. In contrast, Trx-80 aggravated such parameters. Also, Trx-1 and CB3 reduced the infarct size. These results suggest that both Trx-1 and CB3 have beneficial effects and in all cases the effect of CB3 is greater than Trx-1 indicating that the effect of Trx-1 is probably attenuated by the cleavage process. In vitro studies showed that CB3 decreases intracellular level of ROS which could be linked to the observed reduced protein carbonylation. As a consequence, an increase of cell proliferation was found.
In conclusion, Trx-1 plays an important role against heart failure through probably a reduction of protein oxidation involved in cell cycling. Nevertheless, such beneficial effect is attenuated by the cleavage process. In this context, the use of CB3 for therapeutic purposes could constitute a way to overcome this obstacle.
SFRR-E OP 2.3:
A chemoproteomic approach to identify redox-active methionines in secreted proteins of saprophytic fungi during biomass degradation
Lise Molinelli | Aix-Marseille University | France
Saprophytic fungi degrade lignocellulosic polymers present in plant cell walls using dedicated enzymes called ‘Carbohydrate Active enZYmes’ (CAZYmes). These enzymes are secreted in the extracellular environment together with reactive oxygen species (ROS), shown to participate actively in vegetal biomass degradation. Numerous CAZYmes are routinely used in biotechnology approaches and the understanding of their functioning is of crucial importance to improve and create new ways of biomass valorization. ROS can oxidize proteins on sensitive amino acids leading to oxidative post-translational modifications, the consequences of which range from damaging effects to fine-tuned regulation of protein functions and fates. For example, the reversible oxidation of methionine into methionine sulfoxide (MetO) can act as redox switch to regulate proteins involved in several cell functions. If the effects of ROS on numerous proteins of bacteria, plants or animals have been studied, almost nothing is known about fungal proteins and specifically during biomass degradation. Our study aims to identify and characterize proteins carrying redox-sensitive Met, that are secreted during biomass degradation in fungi. We chose Pycnoporus cinnabarinus as a model fungus for its capacity of producing a large arsenal of lignocellulosic enzymes. We developed a chemoproteomic approach using an oxaziridine probe targeting redox-sensitive Met. The probe has been synthesized and validated in a model protein, for which increased concentration of oxaziridine allowed ranking of the different Met according to their redox-sensibility. Thanks to coupled mass spectrometry analysis, and along with the use of 18O labelled hydrogen peroxide, this probe will allow us to identify proteins carrying redox-active Met in fungi. The effect of redox modification of Met will then be elucidated on recombinant proteins. Additionally, compared analysis of intracellular fungal proteins could give insight into mechanisms set up to protect extracellular proteins against oxidative stress. This approach should help to uncover redox regulated CAZYmes.
SFRR-E OP 2.4 [YIA]:
Identification of 11'-α-tocomonoenol in the microalgae Monodopsis subterranea and effect of nitrogen depletion on its tocomonoenol content
Alexander Montoya-Arroyo | University of Hohenheim | Germany
Introduction. α-Tocomonoenols (αT1) are tocochromanols differing form α-tocopherol (αT) and α-tocotrienol due to the presence of a single double bond in the side chain. Two αT1 isomers have been reported, 11'-αT1, mostly reported in land plants, and 12'-αT1, mainly found in marine organisms. Recently, it has been reported that 11'-αT1 and not 12'-αT1 is the major αT1 congener in microalgae and that it is metabolized in human liver cells similar to αT. The aim of the present study was to evaluate the effect of nitrogen depletion in Monodopsis subterranea on tocochromanol content with particular emphasis on αT1 isomers. Methods. M. subterranea was obtained under nitrogen-repleted and -depleted conditions for determination of the tocochromanol profile. Presence and identity of αT1 was determined by LC-MSn and GC-MS and quantification of tocochromanols was done by HPLC-FLD. Results. 11'-αT1 is the predominant αT1 congener in M. subterranea. Nitrogen depletion increased tocochromanol content with no significant effect on absolute αT1 concentration, but with a significant relative increase when normalized to chlorophyll content. Conclusions. 11'-αT1 is the major αT1 congener in M. subterranea in agreement with previous reports. Nitrogen depletion does not affect absolute αT1, despite significant increases in total tocochromanols.
SFRR-E OP 2.5:
Characterization of the copper chaperone PcuC as substrate of the periplasmic methionine sulfoxide reductase MsrP in Cereibacter sphaeroides.
Dr. Lionel Tarrago | INRAE, Aix-Marseille University | France
In proteins, methionine (Met) residues can be oxidized by various oxidants and converted to methionine sulfoxide (MetO). Depending on the affected protein, the consequences of Met oxidation range from uncontrolled damage to fine regulation of function or activity. Almost all characterized organisms possess methionine sulfoxide reductase (Msr) enzymes to reduce MetO to Met. Msrs play an important role in cellular functions by protecting against oxidative stress: they reduce oxidized proteins and scavenge reactive oxygen species through cyclic oxidation and reduction of Met. Moreover, reversible Met oxidation can act as a post-translational modification responsible for the activation of enzymes and transcription factors or the regulation of protein-protein interactions. Bacteria have specific molybdenum-containing Msrs, such as the periplasmic MsrP. We previously characterized the substrate specificity of MsrP from the purple bacterium Cereibacter (Rhodobacter) sphaeroides and identified potential protein targets by proteomics. Among these, we found the copper chaperone PcuC, involved in copper loading of cytochrome oxidase. PcuC has an unusually high Met content (5.8% instead of 2.4% for most proteins) and 5 Met are potentially involved in copper binding. In this study, we determined that an oxidized PcuC is efficiently reduced by MsrP. In particular, we mapped the sensitivity of each Met to oxidation using 18O-labeled hydrogen peroxide and oxaziridine probes coupled to proteomics. Solving the three-dimensional structure of PcuC helped to understand the sensitivity of Met to oxidation. We showed that PcuC can bind two copper ions and that, apparently, oxidation barely affects its ability to bind the metal. Experiments are underway to evaluate the effect of oxidation on the ability of PcuC to deliver copper to the cytochrome oxidase and to determine if MsrP can affect this process. The results should help in understanding the role of MsrP in the protection of bacteria against oxidative stress.