Mechanistic insights into the metabolization of S-Sulfocysteine by CHO cells using a multi-omics approach

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Mechanistic insights into the biological activity of S-Sulfocysteine in CHO cells using a multi-omics approach

S-Sulfocysteine (SSC), a bioavailable L-cysteine derivative (Cys), is known to be taken up and metabolized in Chinese hamster ovary (CHO) cells used to produce novel therapeutic biological entities. To gain a deeper mechanistic insight into the SSC biological activity and metabolization, a multi-omics study was performed on industrially relevant CHO-K1 GS cells throughout a fed-batch process, including metabolomic and proteomic profiling combined with multivariate data and pathway analyses. Multi-layered data and enzymatical assays revealed an intracellular SSC/glutathione mixed disulfide formation and glutaredoxin-mediated reduction, releasing Cys and sulfur species. Increased Cys availability was directed towards glutathione and taurine synthesis, while other Cys catabolic pathways were likewise affected, indicating that cells strive to maintain Cys homeostasis and cellular functions

Complement alternative pathway regulation by factor H : proposal of a C3b-H complex by using oxidative footprinting and crosslinking coupled with mass spectrometry

La formation du complexe C3b-H est essentielle au bon fonctionnement de la voie alterne du système du complément puisqu’elle permet d’inhiber la formation du complexe d’attaque membranaire à la surface de nos cellules, préservant l’organisme de toute destruction auto-immune. L’interaction de H avec C3b permet notamment de favoriser l’inactivation protéolytique de C3b par le facteur I, bloquant de ce fait l’activation de la voie alterne. Malgré l’importance du complexe C3b-H, l’interaction entre ces deux protéines à l’échelle moléculaire reste méconnue. En effet, à l’heure actuelle, aucune structure tridimensionnelle de ce complexe n’a pu être obtenue, que ce soit par cristallographie ou par résonance magnétique nucléaire, en raison de sa taille (335 kg.mol-1) et de la flexibilité de H. Cependant, la connaissance de l’organisation de ce complexe est nécessaire à la compréhension de la régulation de la voie alterne et pourrait de plus, se révéler utile quant au développement de thérapies vis-à-vis des maladies auto-immunes tel que le syndrome hémolytique et urémique atypique. Au cours de ces travaux, nous nous sommes donc intéressés à l’étude du complexe C3b-H à l’échelle de l’édifice entier par marquage oxydatif et pontage chimique couplés à la spectrométrie de masse. Ces approches expérimentales nous ont ainsi permis de proposer, à l’échelle de la molécule entière, un modèle tridimensionnel de ce complexe de haut poids moléculaire.The C3b-H complex plays an essential role in regulation of the complement alternative pathway (AP) because it blocks the attack membrane complex (MAC) formation on our self-surface, thus preserving host from autoimmune disease. The C3b-H interaction leads to the proteolytic inactivation of C3b induced by factor I, thus blocking the MAC formation and consequently AP activation. Despite this importance of C3b-H complex for the innate immunity, the mechanism of interaction between these two molecules at the molecular level remains to be established. Indeed, up to date, no three-dimensional structure of this complex is available, either by X-ray crystallography or nuclear magnetic resonance, due to its large size (335 kg.mol-1) and flexibility of H. Yet, these data are required for a better understanding of AP regulation and, might moreover, be useful for the implementation of therapy against autoimmune disorders. In this study, we investigate the C3b-H complex by using oxidative footprinting and cross-linking coupled with mass spectrometry. Our experimental approaches have been successfully applied, allowing us to propose a three-dimensional model of this high molecular complex

Régulation de la voie alterne du complément par le facteur H (élucidation de son assemblage avec C3b par marquage oxydatif et pontage chimique couplés à la spectrométrie de masse)

La formation du complexe C3b-H est essentielle au bon fonctionnement de la voie alterne du système du complément puisqu elle permet d inhiber la formation du complexe d attaque membranaire à la surface de nos cellules, préservant l organisme de toute destruction auto-immune. L interaction de H avec C3b permet notamment de favoriser l inactivation protéolytique de C3b par le facteur I, bloquant de ce fait l activation de la voie alterne. Malgré l importance du complexe C3b-H, l interaction entre ces deux protéines à l échelle moléculaire reste méconnue. En effet, à l heure actuelle, aucune structure tridimensionnelle de ce complexe n a pu être obtenue, que ce soit par cristallographie ou par résonance magnétique nucléaire, en raison de sa taille (335 kg.mol-1) et de la flexibilité de H. Cependant, la connaissance de l organisation de ce complexe est nécessaire à la compréhension de la régulation de la voie alterne et pourrait de plus, se révéler utile quant au développement de thérapies vis-à-vis des maladies auto-immunes tel que le syndrome hémolytique et urémique atypique. Au cours de ces travaux, nous nous sommes donc intéressés à l étude du complexe C3b-H à l échelle de l édifice entier par marquage oxydatif et pontage chimique couplés à la spectrométrie de masse. Ces approches expérimentales nous ont ainsi permis de proposer, à l échelle de la molécule entière, un modèle tridimensionnel de ce complexe de haut poids moléculaire.The C3b-H complex plays an essential role in regulation of the complement alternative pathway (AP) because it blocks the attack membrane complex (MAC) formation on our self-surface, thus preserving host from autoimmune disease. The C3b-H interaction leads to the proteolytic inactivation of C3b induced by factor I, thus blocking the MAC formation and consequently AP activation. Despite this importance of C3b-H complex for the innate immunity, the mechanism of interaction between these two molecules at the molecular level remains to be established. Indeed, up to date, no three-dimensional structure of this complex is available, either by X-ray crystallography or nuclear magnetic resonance, due to its large size (335 kg.mol-1) and flexibility of H. Yet, these data are required for a better understanding of AP regulation and, might moreover, be useful for the implementation of therapy against autoimmune disorders. In this study, we investigate the C3b-H complex by using oxidative footprinting and cross-linking coupled with mass spectrometry. Our experimental approaches have been successfully applied, allowing us to propose a three-dimensional model of this high molecular complex.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

Apport de la spectrométrie de masse MALDI-TOF à l'étude de la dystrophie musculaire des ceintures

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Mechanistic insights into the biological activity of S-Sulfocysteine in CHO cells using a multi-omics approach

S-Sulfocysteine (SSC), a bioavailable L-cysteine derivative (Cys), is known to be taken up and metabolized in Chinese hamster ovary (CHO) cells used to produce novel therapeutic biological entities. To gain a deeper mechanistic insight into the SSC biological activity and metabolization, a multi-omics study was performed on industrially relevant CHO-K1 GS cells throughout a fed-batch process, including metabolomic and proteomic profiling combined with multivariate data and pathway analyses. Multi-layered data and enzymatical assays revealed an intracellular SSC/glutathione mixed disulfide formation and glutaredoxin-mediated reduction, releasing Cys and sulfur species. Increased Cys availability was directed towards glutathione and taurine synthesis, while other Cys catabolic pathways were likewise affected, indicating that cells strive to maintain Cys homeostasis and cellular functions

How does broiler range use impact forage intake, outdoor excretion and gaseous emissions?

International audienceFree range enables broiler chickens to express natural behaviours, eat grass, worms or insects outdoors, but also induces excretion outside the poultry house. To understand how the outdoor run is impacted by range use, we studied three strains of intermediate to slow-growing chickens: JA757 (734 animals, average daily gain (ADG): 36 g/day, outdoor density (OD): 0.26-0.29 chicken/m², rearing duration (RD): 86 days), S757N (735 animals, ADG: 26 g/day, OD: 0.26-0.29 chicken/m² and RD: 100 days) and a dual-purpose (DP) crossbreed (771 animals, ADG: 16 g/day, OD: 0.27- 0.31 chicken/m² and RD: 121 days). Birds had access to a grassy outdoor run with mature trees from 36 days of age. We collected data on the time spent outdoor by broilers using Radio Frequency IDentification (RFID) chips, on the composition and amount of manure and on grass growth to evaluate the impact of range use on grass consumption and outdoor nitrogen (N) / phosphorus (P) excretions. RFID showed that S757N chickens spent about twice as much time outdoor per day than the JA757 and DP ones. Similarly, the S757N chickens also consumed about twice as much grass per day than JA757 and the DP ones. Consistently, S757N chicken outdoor excretions of N and P per day of outdoor access were about twice as much as that of the DP chickens while only about 30% greater than that of JA757. Therefore, we confirmed the relationship between range use and foraging, but we showed strain-dependant outdoor excretion in relation to growth rate and feed intake. A finer understanding of how genetics impact foraging behaviour can help maximize the use of the outdoor run without compromising resource intake and excretion on outdoor areas. The project PPILOW has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N°816172

Identification of Novel Short Ragweed Pollen Allergens Using Combined Transcriptomic and Immunoproteomic Approaches

<div><p>Background</p><p>Allergy to short ragweed (<i>Ambrosia artemisiifolia</i>) pollen is a serious and expanding health problem in North America and Europe. Whereas only 10 short ragweed pollen allergens are officially recorded, patterns of IgE reactivity observed in ragweed allergic patients suggest that other allergens contribute to allergenicity. The objective of the present study was to identify novel allergens following extensive characterization of the transcriptome and proteome of short ragweed pollen.</p><p>Methods</p><p>Following a Proteomics-Informed-by-Transcriptomics approach, a comprehensive transcriptomic data set was built up from RNA-seq analysis of short ragweed pollen. Mass spectrometry-based proteomic analyses and IgE reactivity profiling after high resolution 2D-gel electrophoresis were then combined to identify novel allergens.</p><p>Results</p><p>Short ragweed pollen transcripts were assembled after deep RNA sequencing and used to inform proteomic analyses, thus leading to the identification of 573 proteins in the short ragweed pollen. Patterns of IgE reactivity of individual sera from 22 allergic patients were assessed using an aqueous short ragweed pollen extract resolved over 2D-gels. Combined with information derived from the annotated pollen proteome, those analyses revealed the presence of multiple unreported IgE reactive proteins, including new Amb a 1 and Amb a 3 isoallergens as well as 7 novel candidate allergens reacting with IgEs from 20–70% of patients. The latter encompass members of the carbonic anhydrase, enolase, galactose oxidase, GDP dissociation inhibitor, pathogenesis related-17, polygalacturonase and UDP-glucose pyrophosphorylase families.</p><p>Conclusions</p><p>We extended the list of allergens identified in short ragweed pollen. These findings have implications for both diagnosis and allergen immunotherapy purposes.</p></div

Structural and functional characterization of the major allergen amb a 11 from short ragweed pollen

Allergy to the short ragweed (Ambrosia artemisiifolia) pollen is a major health problem. The ragweed allergen repertoire has been recently expanded with the identification of Amb a 11, a new major allergen belonging to the cysteine protease family. To better characterize Amb a 11, a recombinant proform of the molecule with a preserved active site was produced in Escherichia coli, refolded, and processed in vitro into a mature enzyme. The enzymatic activity is revealed by maturation following an autocatalytic processing resulting in the cleavage of both N- and C-terminal propeptides. The 2.05-angstrom resolution crystal structure of pro-Amb a 11 shows an overall typical C1A cysteine protease fold with a network of molecular interactions between the N-terminal propeptide and the catalytic triad of the enzyme. The allergenicity of Amb a 11 was confirmed in a murine sensitization model, resulting in airway inflammation, production of serum IgEs, and induction of Th2 immune responses. Of note, inflammatory responses were higher with the mature form, demonstrating that the cysteine protease activity critically contributes to the allergenicity of the molecule. Collectively, our results clearly demonstrate that Amb a 11 is a bona fide cysteine protease exhibiting a strong allergenicity. As such, it should be considered as an important molecule for diagnosis and immunotherapy of ragweed pollen allergy

IgE reactivity of recombinant Der f 36 and Der p 36.

<p>Recombinant non-glycosylated Der f 36 and Der p 36 were produced in <i>P</i>. <i>pastoris</i>. IgE reactivity was assessed by western blot using a pool of sera from HDM-sensitized individuals. Culture supernatant from a mock strain was used as a negative control.</p