Most mitochondrial proteins are encoded by nuclear DNA, and synthesised in the cytosol, then imported into the different mitochondrial subcompartments. To reach their destination, mitochondrial inner membrane proteins require import across the outer mitochondrial membrane, and through the intermembrane space. This passage through the IMS is assisted by the small Tim proteins. This family is characterised by conserved cysteine residues arranged in a twin CX3C motif. They can form Tim9-Tim10 and Tim8-Tim13 complexes, while Tim12 appears to form part of a Tim9-Tim10-Tim12 complex that is associated with the inner membrane translocase TIM22 complex. Current models suggest that the biogenesis of small Tim proteins and their assembly into complexes is dependent on the redox states of the proteins. However, the role of the conserved cysteine residues, and the disulphide bonds formed by them, in small Tim biogenesis and complex formation is not clear. As there is no research about the structural characterisation of Tim12 and double cysteine mutants of Tim9, purification of these proteins was attempted using different methods. To investigate how cysteine mutants affect complex formation, the purified double cysteine mutants of Tim9 were studied using in vitro methods. It showed that the double cysteine mutants were partially folded, and they can form complexes with Tim10 with low affinities, suggesting disulphide bonds are important for the structures and complex formation of small Tim proteins. The effect of cysteine mutants on mitochondrial function was addressed using in vivo methods. It showed that cysteines of small Tim proteins were not equally essential for cell viability, and growth defect of the lethal cysteine mutant was caused by low level of protein. Thus, the conclusion of this study is that disulphide bond formation is highly important for correct Tim9- Tim10 complex formation, and yeast can survive with low levels of complex, but it results in instability of the individual proteins.

Este estudo teve como objetivo avaliar o efeito da N-acetil-L-cisteína (NAC), adicionada ao diluente composto de leite em pó desnatado, sobre a viabilidade espermática e o estresse oxidativo do sêmen equino resfriado a 5°C. Ejaculados de 8 pôneis da raça Brasileira foram coletados em triplicata resultando em 24 ejaculados. O sêmen foi distribuído em 4 grupos: Equidil® + 0,00 mM (controle), 0,5 mM, 1,0 mM ou 2,5 mM de NAC. As amostras foram armazenadas em tubos de 15mL e mantidas em caixas de transporte de sêmen BotuFLEX® (Botupharma, Botucatu-SP, Brasil). Parâmetros como motilidade total (MT), motilidade progressiva (MP), vigor, pH, resposta ao teste hiposmótico (HOST) e atividade mitocondrial (MTT) foram avaliados nas 24 e 48 h, bem como no sêmen fresco, após a diluição. O vigor, MT e MP foram também avaliados após teste de termorresistência (TTR), na ausência e presença de peróxido. A MT, a MP, o vigor espermático e a MTT foram similares (P > 0.05) entre as concentrações de NAC, nas 24 e 48 h. A resposta ao HOST foi semelhante entre as concentrações de NAC (P > 0,05) nas 24 h de resfriamento, porém nas 48 h ocorreu diminuição da funcionalidade da membrana no grupo NAC 2,5 mM em comparação ao grupo EQUIDIL, sem adição de NAC. A MT, a MP e vigor, das amostras resfriadas por 24 h e submetidas ao TTR, diferiu entre sem e com peróxido (P < 0,05) nos grupos Equidil, 0,5 mM e 1,0 mM, mas foi similar na concentração de 2,5 mM de NAC. Quando o sêmen foi resfriado por 48 h, houve diferença no vigor e MT entre amostras com e sem peróxido (P < 0,05), em todos os grupos testados, mas a MP foi similar entre amostras com e sem peróxido, na concentração 2,5 mM. O pH do diluente Equidil® foi o maior e os grupos Equidil + 0,5 mM e 1,0 mM tiveram valores intermediários, enquanto a concentração de 2,5 mM de NAC gerou valores mais baixos, nos três momentos avaliados (P < 0,05). Não houve variação significativa de pH entre os momentos 0 e 24 h (P= 0,7075) e entre 0 e 48 h (P= 0,4617), em todos os grupos testados. As concentrações de NAC testadas não melhoraram a motilidade, integridade da membrana plasmática, atividade mitocondrial e resposta ao HOST de espermatozoides equinos resfriados a 5°C e armazenados por 48 h. Após TTR, as concentrações de NAC testadas não evitaram a diminuição da motilidade e vigor espermático, na presença de peróxido. / The aim of this study was to evaluate the effect of N-acetyl-L-cysteine (NAC) based on skim milk powder extender on sperm viability and oxidative stress of equine semen cooled to 5°C. Ejaculate of 8 ponies of the Brazilian breed were collected in triplicate, resulting in 24 ejaculates, distributed in 4 groups: Equidil® extender + 0.00mm (control), 0.5mM, 1.0mM and 2.5mM of NAC with the semen samples were The samples were stored in 15mL tubes and kept in BotuFLEX® semen transport boxes (Botupharm, Botucatu/SP/Brazil). Parameters, such as motility, vigor, pH, osmolarity, HOST, and MTT were evaluated at 24 and 48 h of cooling also in fresh semen. Vigor, total (TM) and progressive motility (PM) were also evaluated after thermoresistance test (TTR), in the absence and presence of peroxide. TM, PM, sperm vigor and MTT were similar (P > 0.05) between NAC concentrations at 24 and 48 h. The response to HOST was similar between NAC concentrations (P > 0.05) at 24 h cooling, but at 48 h there was a decreased in membrane functionality in 2.5mM NAC group compared to the EQUIDIL group. TM, PM, and vigor of the samples cooled by 24 h and submitted to TTR differed between without and with peroxide (P< 0.05) in the EQUIDIL, 0.5mM and 1.0mM groups, but was similar in 2.5mM NAC. After cooling for 48 h, there was difference in vigor and TM between samples with and without peroxide (P < 0.05) in all groups tested, but the PM was similar between samples with and without peroxide at concentration 2.5mM of NAC. The pH of the EQUIDIL extender was higher and the EQUIDIL + 0.5mM and 1.0mM groups had intermediate values, while the 2.5mM NAC concentration generated lower values in the three evaluated periods (P < 0.05). There was no significant variation of pH between 0 and 24 h (P=0.7075) and between 0 and 48 h (P=0.4617) in all groups tested. The concentrations of NAC tested did not improve motility, plasma membrane integrity, mitochondrial activity and response to HOST equine spermatozoa cooled to 5°C and stored for 48 h. After TTR, the concentrations of NAC tested did not prevent the decrease of motility and sperm vigor in the presence of peroxide.

IN ENGLISH Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Supervisors: prof. Dr. Michael Gütschow RNDr. Klára Konečná, Ph.D. Candidate: Michaela Hympánová Title of the diploma thesis: Enzyme kinetic evaluation of several potential inhibitors of certain human cysteine and serine proteases Background Cysteine and serine proteases are enzymes involved in many physiological processes. The imbalance between them and their endogenous inhibitors is associated with various diseases such as cancer and osteoporosis. Synthetic inactivators could be useful in the treatment of these enzyme-mediated pathological conditions. Therefore, there are ongoing attempts to develop low-molecular weight inactivators for therapeutically relevant cysteine and serine proteases. In the course of this thesis, compounds synthesized in prof. Gütschow's group were investigated as potential inhibitors of selected human proteases. They belong to imidazole compounds derived from N-protected cyclohexylalanine, 2-phenyl-7,8-dihydroimidazo[1,2- a]pyrazin-6(5H)-one derivatives, ,-unsaturated peptidomimetic compounds, carbamates, an N,N-dibenzylcrotonamide derivatives and peptoides. Aims This diploma thesis has been focused on the evaluation of new potential inhibitors against...

Gingipaine sind Cysteinproteinasen des wohl in Ätiologie und Pathogenese der chronischen Erwachsenenparodontitis bedeutsamsten bakteriellen Erregers und zugleich die wichtigsten Virulenzfaktoren der Spezies Porphyromonas gingivalis. Die für diese extrazellulären Produkte kodierenden Gene sind rgpA, rgpB und kgp. Deren Produkte sind entsprechend HRgpA, RgpB und Kgp. HRgpA und RgpB verursachen eine Steigerung der Gefäßpermeabilität durch die Aktivierung des Kallikrein/Kinin-Systems und aktivieren die Blutgerinnung, welche potentiell mit der Synthese der Sulkusflüssigkeit und dem Fortschreiten der Entzündung bis hin zum Verlust des alveolären Knochens assoziiert sind. Offenbar wird dieses durch die Aktivierung von Matrixmetalloproteinasen begünstigt. Kgp ist von den dreien die potenteste fibrinogen-und fibrinabbauende Proteinase und bei der Blutungsneigung der erkrankten Stellen involviert. HRgpA aktiviert besonders Blutgerinnungsfaktoren. Gingipaine stören das Komplementsystem und manipulieren den Zytokinhaushalt der Entzündungskaskaden. Die Gingipaine unterstützen die Kolonisierung von P. gingivalis durch die Bindung zu anderen Bakterien des subgingivalen Biofilms und der Bindung zu epithelialen Zellen. Sie vermögen an Laminin, Fibrinogen, Fibronektin, Hämoglobin und an manchen Typen von Kollagen zu binden. Alle können den Rezeptor CD14 auf Makrophagen abbauen und so die Leukozytenaktivierung hemmen. Sie regulieren die Infektionsintensität, den Bakterienhaushalt, die Aminosäurenaufnahme aus Wirtsproteinen und die Fimbrienreifung. Die Genetik, die Chemie und die virulenzverursachenden Eigenschaften der Gingipaine stehen seit Mitte der 90iger Jahre des letzten Jahrhunderts im Blickpunkt des wissenschaftlichen Interesses. Aufgrund ihrer Schlüsselrolle bei der Pathogenese der Parodontitis und der mikrobiellen Infektion sind die Gingipaine Ziele für die mögliche Entwicklung von Hemmstoffen respektive für Immunisierungsstrategien gegen die chronische Parodontitis.:Erklärungen I Genehmigungsvermerk II Inhaltsverzeichnis III 1. Einleitung und Fragestellung 4 1.1 Parodontitis 4 1.1.1 Epidemiologie 4 1.1.2 Klassifikation 4 1.1.3 Ätiologie und Pathogenese 5 1.1.4 Mikrobiologie 7 1.1.5 Biofilm 8 1.2 Porphyromonas gingivalis 10 1.3 Verwandtschaftsverhältnisse von P. gingivalis 12 1.4 Mit P. gingivalis assoziierte Proteinasen 15 1.5 Fragestellung 16 2. Material und Methoden 17 3. Ergebnisse und Diskussion 18 3.1 Einführung – Gingipaine als Enzyme von P. gingivalis 18 3.1.1 Definition der Gingipaine 18 3.1.2 RgpA- und RgpB-Gen-Gingipaine 19 3.1.3 Kgp-Gen-Gingipain 20 3.2 Bedeutung der Gingipaine für die Pathogenität von P. gingivalis 21 3.2.1 Gingipaine und fimbrienvermittelte Adhäsion, intrazelluläre Invasion und parazellulärer Pfad von P. gingivalis 21 3.2.2 Effekte auf die Integrität des Bindegewebes 24 3.2.3 Aktivierung des Kallikrein-Kinin-Systems 25 3.2.4 Aktivierende Einflüsse auf das Gerinnungssystem 27 3.2.5 Hemmende Einflüsse auf das Fibrinogen-Fibrin-System 28 3.2.6 Beeinträchtigung der Immunabwehr des Wirts 28 3.3 Gingipaine im biochemischen Regulationsmechanismus von P. gingivalis 35 3.3.1 Die Rolle des proteolytischen Systems von P. gingivalis beim Nährstofferwerb 35 3.3.2 Gingipainreifung und die Steuerung des intrazellulären Haushalts von P. gingivalis 39 3.3.3 Regulation der Proteinaseexpression von P. gingivalis 40 3.3.4 Gingipaine als bakterielle Hämagglutinine, Adhäsine und hämoglobinbindende Proteine 42 3.3.5 Proteinasegenmutationen bei P. gingivalis und ihre enzymatische Aktivität 42 3.4 Gingipaine und systemische Effekte 44 3.5 Synergismen mit anderen Spezies und quorum sensing 45 3.6 Hemmstoffe der Gingipaine 46 3.7 Immunisierung gegen Gingipaine 48 4. Zusammenfassung / Summary 52 5. Literaturverzeichnis 53 Anhang 87 Abbildungsverzeichnis 87 Tabellenverzeichnis 87 Danksagung 88 Lebenslauf 89 / Gingipains are cysteine proteases of the probably most important bacterial pathogen of the adult periodontitis in the field of etiology as well as pathology. At the time, they are the most important virulence factors of the species Porphyromonas gingivalis. The encoding genes of this extracellular products are rgpA, rgpB and kgp. Their products are corresponding HRgpA, RgpB and Kgp. HRgpA and RgpB induce vascular permeability enhancement through activation of the kallikrein/kinin system and activate the blood coagulation, processes potentially associated with gingival crevicular fluid synthesis and progression of inflammation which ultimately can lead to alveolar bone loss. Obviously, this will be favoured by matrix metalloproteinases. Kgp is the most potent fibrinogen/fibrin degrading enzyme of the three gingipains involved in the bleeding tendency at the diseased sites. HRgpA especially activates coagulation factors. Gingipains disturb the complement system and manipulate the cytokine network of the inflammation cascades. Gingipains support colonizing of P. gingivalis due to their connection to other bacteria of the subgingival biofilm and to epithelial cells. They are able to bind to laminin, fibrinogen, fibronectin, hemoglobin and some types of collagen. All of them are able to degrade macrophage CD14 receptor, thus preventing activation of the leukocytes. They regulate the intensity of infection and the bacterial housekeeping, including amino acid uptake from host proteins and fimbriae maturation. Genetics, chemistry and the virulence inducing properties of gingipains are the focus of scientific attention since the middle of the nineties of the last century. Due to their key role in pathogenesis of periodontitis and microbial infection the gingipains are targets for the possible development of inhibitory substances, respectively for immunization strategies against chronic periodontitis.:Erklärungen I Genehmigungsvermerk II Inhaltsverzeichnis III 1. Einleitung und Fragestellung 4 1.1 Parodontitis 4 1.1.1 Epidemiologie 4 1.1.2 Klassifikation 4 1.1.3 Ätiologie und Pathogenese 5 1.1.4 Mikrobiologie 7 1.1.5 Biofilm 8 1.2 Porphyromonas gingivalis 10 1.3 Verwandtschaftsverhältnisse von P. gingivalis 12 1.4 Mit P. gingivalis assoziierte Proteinasen 15 1.5 Fragestellung 16 2. Material und Methoden 17 3. Ergebnisse und Diskussion 18 3.1 Einführung – Gingipaine als Enzyme von P. gingivalis 18 3.1.1 Definition der Gingipaine 18 3.1.2 RgpA- und RgpB-Gen-Gingipaine 19 3.1.3 Kgp-Gen-Gingipain 20 3.2 Bedeutung der Gingipaine für die Pathogenität von P. gingivalis 21 3.2.1 Gingipaine und fimbrienvermittelte Adhäsion, intrazelluläre Invasion und parazellulärer Pfad von P. gingivalis 21 3.2.2 Effekte auf die Integrität des Bindegewebes 24 3.2.3 Aktivierung des Kallikrein-Kinin-Systems 25 3.2.4 Aktivierende Einflüsse auf das Gerinnungssystem 27 3.2.5 Hemmende Einflüsse auf das Fibrinogen-Fibrin-System 28 3.2.6 Beeinträchtigung der Immunabwehr des Wirts 28 3.3 Gingipaine im biochemischen Regulationsmechanismus von P. gingivalis 35 3.3.1 Die Rolle des proteolytischen Systems von P. gingivalis beim Nährstofferwerb 35 3.3.2 Gingipainreifung und die Steuerung des intrazellulären Haushalts von P. gingivalis 39 3.3.3 Regulation der Proteinaseexpression von P. gingivalis 40 3.3.4 Gingipaine als bakterielle Hämagglutinine, Adhäsine und hämoglobinbindende Proteine 42 3.3.5 Proteinasegenmutationen bei P. gingivalis und ihre enzymatische Aktivität 42 3.4 Gingipaine und systemische Effekte 44 3.5 Synergismen mit anderen Spezies und quorum sensing 45 3.6 Hemmstoffe der Gingipaine 46 3.7 Immunisierung gegen Gingipaine 48 4. Zusammenfassung / Summary 52 5. Literaturverzeichnis 53 Anhang 87 Abbildungsverzeichnis 87 Tabellenverzeichnis 87 Danksagung 88 Lebenslauf 89

La vie a émergé sur Terre il y a probablement 3,8 milliards d'années, sur une planète largement recouverte d'eau. Ce travail porte sur la synthèse prébiotique de peptides, en particulier de peptides riches en thiol. Nous avons étudié les réactions des aminonitriles (les premiers produits de la réaction de Strecker) avec la cystéine et l'homocystéine. Elles conduisent à la formation de cycles à 5 ou 6 chaînons qui sont ensuite hydrolysés pour donner les dipeptides correspondants (aa-Cys ou aa-Hcy). Les dipeptides contenant un thiol obtenus sont capables de favoriser la formation de chaînes peptidiques plus longues via des liaisons thioesters et de favoriser la formation de certains hétérocycles. L'homocystéine nitrile se cyclise dans l'eau pour former l'homocystéine thiolactone, qui présente une double réactivité, la thiolactone est ouverte par des amines puis on observe une condensation de l'aminothiol ainsi formé avec les nitriles. Le nitrile de cystéine et le thioester de S-éthyle de la cystéine conduisent à la formation de polycystéine, tandis que les molécules de type Cys-aa-CN donnent des polypeptides linéaires et cycliques. Nos résultats soutiennent l’hypothèse que des peptides contenant des thiols auraient joué un rôle important dans les premiers stades du développement de la vie. / Life emerged on Earth probably about 3.8 billion years ago, on a planet that was largely covered by water. This work focuses on the prebiotic synthesis of peptides, especially thiol-rich ones. We studied the reactions of aminonitriles (the first products of the Strecker reaction) with cysteine and homocysteine. These reactions lead to the formation of 5- or 6-membered rings which are then hydrolysed to give the corresponding dipeptides (aa-Cys or aa-Hcy). The obtained thiol-containing dipeptides are able to promote the formation of longer peptide chains via thioesters bonds, and to promote the formation of some heterocycles. Homocysteine nitrile cyclizes in water to form homocysteine thiolactone, which shows a double reactivity, thiolactone opening by amines followed by aminothiol condensation reaction with nitriles. Cysteine nitrile and the S-ethyl thioester of cysteine lead to the formation of polycysteine, while Cys-aa-CN molecules gives linear and cyclic polypeptides. Our results support the hypothesis that thiol-containing peptides would have been important molecules in the early stages of life development.

Spiders produce a diverse number of silk proteins that are well-known for their extraordinary mechanical and biological properties. Dragline silk has been the most prominent focus of research because of its exceptional high tensile strength and extensibility. In our research, we have focused on the characterization of small molecular weight proteins found within dragline and tubuliform silks. Within the black widow spider, Lactrodectus hesperus, these proteins have been named Cysteine-Rich Protein (CRP) and determined to be a family of five individual proteins. The small protein identified within the tubuliform silks has been named Egg Case Protein 3 (ECP-3). In this study, recombinant expression of ECP-3 in the pET-19b-SUMO vector was to facilitate purification and development of an immunological reagent. Using western blot analysis, we have demonstrated that ECP-3 is efficiently expressed in bacteria. We also investigated CRP1 protein and its ability to bind MaSp1 components using pull down assays to determine potential interactions. No substantial biochemical evidence was produced to demonstrate protein-protein interactions between the two. Additionally, we show that using RT-PCR analysis from mRNAs collected from the major ampullate gland that transcript levels for CRP-family members from non-silked and a silked spider are different. CRP2 and CRP4 mRNA levels were shown to increase upon silking. Overall, the major findings of this thesis involved characterizing the ECP-3 protein found within tubuliform silks as well as determining the expression patterns for CRP-family members.

Spider silk displays a unique balance of high tensile strength and extensibility, making it one of the toughest materials on the planet. Dragline silk, also known as the lifeline of the spider, represents one of the best studied fiber types and many labs are attempting to produce synthetic dragline silk fibers for commercial applications. In these studies, we develop a minifibroin for expression studies in bacteria. Using recombinant DNA methodology and protein expression studies, we develop a natural minifibroin that contains the highly conserved N- and C-terminal domains, along with several internal block repeats of MaSp1. We also characterize a family of small cysteine-rich proteins (CRPs) and demonstrate that these factors are present within the spinning dope of the major ampullate gland using MS analysis. Biochemical studies and characterization of one of the family members, CRP1, demonstrate that this factor can self-polymerize into higher molecular weight complexes under oxidizing conditions, but can be converted into a monomeric species under reducing conditions. Self-polymerization of CRP1 is also shown to be independent of pH and salt concentration, two important chemical cues that help fibroin aggregation. Overall, our data demonstrate that the polymerization state of CRP1 is dependent upon redox state, suggesting that the redox environment during fiber extrusion may help regulate the oligomerization of CRP molecules during dragline silk production.

A previous study identified Enterococcus faecalis as one of two bacteria known to have the selD gene and other selenium related genes without having the genes necessary to make selenocysteine or selenouridine. EF2570, a gene in the cluster, was later shown to be upregulated during biofilm formation and also responsible for a selenite- and molybdate-dependent increase in biofilm formation in vitro. The protein encoded was identified as a selenium dependent molybdenum hydroxylase (SDMH), enzymes that contain a labile selenium atom required for activity. While the process of inserting selenocysteine into a protein is well known, the process by which a SDMH acquires a labile selenium atom has not yet been described. To begin unraveling this pathway, the nifS-like EF2568 from the gene cluster will be characterized. Some NifS-like proteins have been shown to have selenocysteine lyase activity, providing a source of selenium for selenophosphate synthetase, the selD gene product. Study of EF2568 has shown that it specifically reacts with L-selenocysteine to form selenide and alanine with L-cysteine inhibiting the reaction. Guided by homology to the well-characterized human and E. coli NifS-like proteins, mutants of the active site and substrate discerning residues were also characterized for activity with L-selenocysteine and L-cysteine. While mutation of the residue at position 112 thought to be responsible for substrate specificity did not affect reactivity of the enzyme with L-cysteine, it did affect reactivity with L-selenocysteine. Studying the characteristics of this novel group II selenocysteine lyase will provide a foundation for studying the remaining pathway.

Cathepsins have been proposed as biomarkers of chemical exposure in the zebrafish embryo model but it is unclear whether they can also be used to detect sublethal stress. The present study evaluates three cathepsin types as candidate biomarkers in zebrafish embryos. In addition to other functions, cathepsins are also involved in yolk lysosomal processes for the internal nutrition of embryos of oviparous animals until external feeding starts. The baseline enzyme activity of cathepsin types H, C and L during the embryonic development of zebrafish in the first 96 h post fertilisation was studied. Secondly, the effect of leupeptin, a known cathepsin inhibitor, and four embryotoxic xenobiotic compounds with different modes of action (phenanthrene—baseline toxicity; rotenone—an inhibitor of electron transport chain in mitochondria; DNOC (Dinitro-ortho-cresol)—an inhibitor of ATP synthesis; and tebuconazole—a sterol biosynthesis inhibitor) on in vivo cathepsin H, C and L total activities have been tested. The positive control leupeptin showed effects on cathepsin L at a 20-fold lower concentration compared to the respective LC50 (0.4 mM) of the zebrafish embryo assay (FET). The observed effects on the enzyme activity of the four other xenobiotics were not or just slightly more sensitive (factor of 1.5 to 3), but the differences did not reach statistical significance. Results of this study indicate that the analysed cathepsins are not susceptible to toxins other than the known peptide-like inhibitors. However, specific cathepsin inhibitors might be identified using the zebrafish embryo.

Le développement d'une réponse antivirale contre les virus, incluant le virus de l'immunodéficience humaine (VIH), le virus de la grippe, le virus respiratoire syncytial (VRS) ou le SARS-CoV-2, repose sur l'activation d'adaptateurs intracellulaires qui conduisent à la production d'interférons et de cytokines proinflammatoires. Une activation correctement équilibrée de ces voies permet à la cellule de monter un état antiviral, essentiel pour restreindre la réplication et la propagation du virus. Les acides nucléiques viraux à base d'ADN, présents à l’intérieur de la cellule, peuvent être reconnus par la GMP-AMP Synthase cyclique (cGAS), qui transduit ensuite le signal via l'adaptateur Stimulateur des gènes d'interféron (STING). D'autre part, les acides nucléiques viraux à ARN sont reconnus par les récepteurs de type RIG-I (RLR), qui interagissent ensuite avec l'adaptateur de signalisation antivirale mitochondrial (MAVS). STING et MAVS sont des protéines pivots de signalisation localisées dans des compartiments membranaires, régulées par plusieurs modifications post-traductionnelles (PTM) et, lors de l'activation, elles subissent des événements de polymérisation allant jusqu'à la formation d'agrégats fonctionnels. Des données soutiennent un rôle des espèces réactives de l'oxygène (ROS) dans la régulation des voies dépendantes de STING et MAVS, mais les mécanismes restent mal définis. Les ROS sont connus pour modifier la structure et l'activité des protéines de signalisation via des PTMs oxydatives réversibles sur des cystéines (Cys ox-PTM). Les Cys ox-PTM réversibles consistent en une variété de modifications, les plus étudiées étant la S-sulfénylation (Cys-SOH), la S-glutathionylation (Cys-SSG) et le disulfure (S-S). Afin d’identifier les Cys ox-PTM qui affectent les protéines de signalisation impliquées dans la réponse antivirale, nous avons effectué une identification à l'échelle du protéome des Cys ox-PTM induites par les ROS en utilisant un marquage bioswitch à base de maléimide couplé à la spectrométrie de masse. Nous avons identifié 2720 sites Cys ox-PTM uniques englobant 1473 protéines avec une abondance, localisation et fonctions distinctes. Parmi ceux-ci, nous avons découvert l'oxydation de STING sur la Cys148 et Cys206. Cette dernière étant inductible par le stress oxydatif ou par le ligand naturel 2'3'-cGAMP et joue un rôle inhibiteur pour empêcher l'hyperactivation de STING par la formation de polymères inactifs contenant des liaisons intermoléculaires S-S. En outre, nous avons observé que MAVS était également capable de former des polymères intermoléculaires contenant des S-S en réponse à une infection par des virus à ARN, et que l'ancrage de la protéine à la membrane était essentiel pour la formation de ces polymères. Le couplage du marquage par bioswitch à base de maléimide à l'analyse par immunoblot a confirmé que MAVS était oxydé pendant une infection avec un virus à ARN. Nous avons également constaté que des Cys situées dans des positions clés pour la formation de polymères de MAVS actifs étaient essentielles pour transduire la signalisation en aval et finalement activer le promoteur IFNβ en réponse à l'infection virale. Nos études établissent un mécanisme direct par lequel les ROS contrôlent la réponse immunitaire innée cGAS/STING et RLRs/MAVS-dépendante. Ils offrent un nouveau terrain pour la conception de thérapies ciblant des adaptateurs pertinents pour les infections virales, telles que la vaccination, mais aussi pour les troubles auto-immuns et inflammatoires. / The development of an antiviral response against viruses, including Human Immunodeficiency Virus (HIV), influenza, Respiratory Syncytial Virus (RSV) or SARS-CoV-2, relies on the activation of intracellular adaptors that ultimately lead to the production of interferons and proinflammatory cytokines. Properly balanced activation of these pathways allows the cell to mount an antiviral state, essential to restrict virus replication and spreading. Intracellular DNA viral nucleic acids can be recognized by the cyclic GMP-AMP Synthase (cGAS), which then transduces the signal through the Stimulator of Interferon Genes (STING) adaptor. On the other hand, RNA viral nucleic acids are recognized by the RIG-I-like Receptors (RLRs), which then interact with the Mitochondrial Antiviral Signaling (MAVS) adaptor. STING and MAVS are signaling hub proteins localized in membranous compartments, regulated by several posttranslational modifications (PTMs) and, upon activation, they undergo polymerization events going as far as the formation of functional aggregates. Compelling evidence supports a role of reactive oxygen species (ROS) in the regulation of STING and MAVS-dependent pathways, but the mechanisms remain ill-defined. ROS are known to modify signaling protein structure and activity through reversible oxidative PTM of Cysteines (Cys ox-PTM). Reversible Cys ox-PTMs consist of a variety of modifications, the most widely studied being S-sulfenylation (Cys-SOH), S-glutathionylation (Cys-SSG) and disulfide (S-S). To unveil the Cys ox-PTMs that affect signaling proteins involved in the antiviral response, we performed a proteome wide identification of the Cys ox-PTMs induced by ROS using maleimide-based bioswitch labeling coupled to mass spectrometry. We identified 2720 unique Cys ox-PTM sites encompassing 1473 proteins with distinct abundance, location, and functions. Among these, we uncovered the oxidation of STING at Cys148 and Cys206. The latter was inducible by oxidative stress or by the natural ligand 2’3’-cGAMP and plays an inhibitory role to prevent STING hyperactivation through the formation of inactive polymers containing intermolecular S-S bonds. Further, we observed that MAVS was also able to form intermolecular S-S containing polymers in response to RNA virus infection, and that the anchoring of the protein to the membrane was essential for these polymers to form. Maleimide-based bioswitch labeling couple to immunoblot analysis confirmed that MAVS was oxidized during RNA virus infection. We also found that Cys located in positions key for the formation of active polymers were essential for MAVS to transduce downstream signaling and ultimately activate IFNβ promoter in response to virus infection. Our studies establish a direct mechanism by which ROS control the cGAS/STING and the RLRs/MAVS-dependent innate immune response. They provide new ground for the design of therapies targeting adaptors relevant to viral infections, such as vaccination, but also to autoimmune and inflammatory disorders.