El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / SARS-CoV-2 main protease is a common target for inhibition assays due to its high conservation among coronaviruses. Since flavonoids show antiviral activity, several in silico works have proposed them as potential SARS-CoV-2 main protease inhibitors. Nonetheless, there is reason to doubt certain results given the lack of consideration for flavonoid promiscuity or main protease plasticity, usage of short library sizes, absence of control molecules and/or the limitation of the methodology to a single target site. Here, we report a virtual screening study where dorsilurin E, euchrenone a11, sanggenol O and CHEMBL2171598 are proposed to inhibit main protease through different pathways. Remarkably, novel structural mechanisms were observed after sanggenol O and CHEMBL2171598 bound to experimentally proven allosteric sites. The former drastically affected the active site, while the latter triggered a hinge movement which has been previously reported for an inactive SARS-CoV main protease mutant. The use of a curated database of 4.8 k flavonoids, combining two well-known docking software (AutoDock Vina and AutoDock4.2), molecular dynamics and MMPBSA, guaranteed an adequate analysis and robust interpretation. These criteria can be considered for future screening campaigns against SARS-CoV-2 main protease. / Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica / Revisión por pares

Le colza (Brassica napus L.) est une plante de grande culture particulièrement exigeante en azote (N) et caractérisée par une faible Efficience d’Usage de l’Azote (EUA), principalement due à une mauvaise Efficience de Remobilisation de l’Azote (ERA) au cours de la sénescence foliaire. L’optimisation de l’ERA est donc un enjeu majeur pour améliorer le bilan agro-environnemental de cette culture. La dégradation des protéines étant l’un des processus clés de la remobilisation de l’N associée à la sénescence, l’objectif de ce travail est d’identifier et caractériser les principaux acteurs de la protéolyse lors de la sénescence naturelle ou induite par une limitation en N. Chez la plante modèle Arabidopsis thaliana cultivée en condition de forte disponibilité en N (HN), nous avons montré que SAG12 est une protéase majeure lors de la sénescence foliaire afin d’assurer la remobilisation de l’N essentielle à l’élaboration du rendement et au remplissage en N des graines. En effet, nos travaux montrent que le rôle de SAG12 est central et qu’en absence de son activité, d’autres protéases appartenant au CPs, mais aussi à d’autres classes (protéases à aspartate ; APs), sont sollicitées pour soutenir la remobilisation de l’N foliaire. Dans ce contexte, AED1 (une APs CND41-like) est particulièrement intéressante puisqu’elle semble collaborer étroitement avec SAG12. Par ailleurs, cette étude met en évidence pour la première fois une localisation racinaire de SAG12. Dans cet organe, le rôle de SAG12 est crucial pour remobiliser l’N des racines pour maintenir le rendement et la teneur en N des graines lorsque les plantes sont soumises à une limitation en N. Un autre volet de ce travail a consisté à étudier d’autres moyens de réguler l’activité protéolytique en se focalisant notamment sur des systèmes anti-protéolytiques. Cette étude suggère qu’une Water Soluble Chlorophyll binding Protein (WSCP), la protéine WSCP1, porte effectivement une double fonction de protection des chlorophylles et d’inhibiteur de protéases à sérine, ce qui en fait un potentiel candidat pour prolonger la durée de vie des feuilles et ainsi réduire l’asynchronisme entre la période de vidage des feuilles et la période de remplissage en N des graines.L’ensemble de ces travaux permet de proposer de nouveaux candidats pertinents pour la sélection de variétés de colza présentant une remobilisation efficiente de l’N dans un contexte de limitation des intrants azotés. / Rapeseed (Brassica napus L.) is a field crop plant that is particularly requiring nitrogen (N) and characterized by a low Nitrogen Use Efficiency (NUE), mainly due to a poor Nitrogen Remobilization Efficiency (NRE) during foliar senescence. The optimization of NRE is therefore a major challenge to improve the agro-environmental balance of this crop. Since protein degradation is one of the key processes in the remobilization of N associated with senescence, the objective of this work is to identify and characterize the main actors of proteolysis during natural or induced by N limitation senescence. In the plant model Arabidopsis thaliana grown under high N conditions (HN), we have shown that SAG12 is a major protease during leaf senescence to ensure the remobilization of N essential for yield and seed N filling. Indeed, our work shows that the role of SAG12 is pivotal and that in the absence of its activity, other proteases belonging to the CPs but also to other classes (aspartate proteases; APs) are requested to support the remobilization of foliar N. In this context, AED1 (a CND41-like APs) is particularly interesting since it seems to collaborate closely with SAG12. In addition, this study shows for the first time a root localization of SAG12. In this organ, the role of SAG12 is crucial, in particular to remobilize N from the roots to sustain yield and N content of the seeds when plants face an N limitation. Another aspect of this work was to study other ways of regulating proteolytic activity, focusing in particular on anti-proteolytic systems. This study suggests that a Water Soluble Chlorophyll binding Protein (WSCP), the WSCP1 protein, has effectively a dual function of chlorophyll protection and serine proteases inhibition, which make it a potential candidate to extend leaf lifespan and thus, reduce asynchronism between leaf emptying time and the N seed filling time.Altogether, these results allow to suggest new relevant candidates for the selection of rapeseed varieties with an efficient N remobilization in a context of nitrogen input limitation.

Vaspin is an adipokine which improves glucose metabolism and insulin sensitivity in obesity. Kallikrein 7 (KLK7) is the first known protease target inhibited by vaspin and a potential target for the treatment of metabolic disorders. Here, we tested the hypothesis that inhibition of KLK7 in adipose tissue may beneficially affect glucose metabolism and adipose tissue function. Therefore, we have inactivated the Klk7 gene in adipose tissue using conditional gene-targeting strategies in mice. Klk7-deficient mice (ATKlk7 −/−) exhibited less weight gain, predominant expansion of subcutaneous adipose tissue and improved whole body insulin sensitivity under a high fat diet (HFD). ATKlk7 −/− mice displayed higher energy expenditure and food intake, most likely due to altered adipokine secretion including lower circulating leptin. Pro-inflammatory cytokine expression was significantly reduced in combination with an increased percentage of alternatively activated (anti-inflammatory) M2 macrophages in epigonadal adipose tissue of ATKlk7 −/−. Taken together, by attenuating adipose tissue inflammation, altering adipokine secretion and epigonadal adipose tissue expansion, Klk7 deficiency in adipose tissue partially ameliorates the adverse effects of HFD-induced obesity. In summary, we provide first evidence for a previously unrecognized role of KLK7 in adipose tissue with effects on whole body energy expenditure and insulin sensitivity.

This work deals with the preparation of combinatorial libraries of peptides serving as platforms for proteolytic phenotyping. The primary objective was to prepare a solid phase fluorogenic peptide library and screen proteases by fluorescence. Further, the possibility of preparing solid phase DNA-encoded libraries was studied. Due to the non-reactivity of the specific proteases with the solid phase peptides, the solid phase was completely abandoned and DNA-encoded peptide library was prepared in the solution. Using this model of DNA-encoded dipeptide with terminal biotin, the new principle of testing proteolytic activities of proteases was verified. A combinatorial library of DNA-encoded hexapeptides was also prepared. Despite the low yield of the library, the possibility of DNA encoding, the amplifiability of the prepared molecules and the possibility of biotin-based separation were verified. The integrity of the hexapeptide sequence and the protease testing is the subject of further study.

Prolyl endopeptidase SmPEP from the blood fluke Schistosoma mansoni is investigated here for the first time. This enzyme is potentially interesting as a drug target for the treatment of schistosomiasis. SmPEP was detected in the extract of adult worms by enzyme activity and immunoreactivity. Enzymatically active SmPEP was produced in the E. coli expression system and was chromatographically purified. The pH optimum of recombinant SmPEP was about 8. Substrate specificity analysis revealed that SmPEP cleaved peptide substrates by endopeptidase activity, however, macromolecular substrates were not fragmented. The residue preferences in the positions P3 to P1' were determined using synthetic fluorogenic peptide substrates. SmPEP was found to be highly sensitive to the inhibition by Z-Ala-Pro-CMK and Z-Arg-Pro-CHO. Primary screening of crystallization conditions for recombinant SmPEP was performed. " (In Czech)"

Digestive proteolysis in termites has not been studied yet. In this diploma thesis, proteolytic enzymes of the digestive tract of two significant pest species Reticulitermes santonensis and Coptotermes formosanus (Rhinotermitidae) were analyzed. Proteases were identified and quantified in gut compartments using a panel of specific substrates and inhibitors. Major proteases were localized in the midgut and were classified as endogenous serine proteases of trypsin type. Minor cysteine proteases were detected in the paunch and were most likely produced by symbionts. The trypsin protease from R. santonensis was chromatographically isolated and its N-terminal sequence was identified. The physiological importance of the digestive trypsin proteases was demonstrated using selective inhibitors tested in vivo with C. formosanus. Based on the analysis of proteases from additional 12 termite species, a general scheme of digestive proteolysis in the order Isoptera was proposed. (In Czech)

Proteases are important enzymes in the biotechnology due to their specific cleavage of substrates. HRV 3C, sortase A and TEV are some examples of cysteine proteases which become more of use lately in applications as removal of affinity tags (3C/TEV) and labelling of proteins (sortase). Here an investigation was made on the proteases by displaying them on two different prokaryotic hosts; E. coli and S. carnosus and to use these to cleave away affinity proteins (Affibody molecule) from other cells with an incorporated cleavage site. Constructs were cloned and incorporated into expressing strains which were then cultivated and induced. Analysis of surface expression was done by flow cytometer. Cleavage was made by cultivating combinations with cleavable bacteria and bacteria displaying proteases. A functional protease would lead to the presence of Affibody molecules in the supernatant. Flow cytomtery analysis was first made to inevstigate signal difference in Affibody binding by the addition of flurophores. Secondly SDS-PAGE was made on the centrifuged supernatant to investigate the presence of a product. Finally analysis of the bacteria was made by examining the reaction with soluble substrate and comparing activity with soluble enzyme. All of the enzymes were able to be displayed on the surface of bacteria with a clear separation from control. The cleavage analysis showed however varying results yet no clear evidence of product. Best flow cytometer results were seen for 3C but SDS-PAGE/MS did not show any cleaved product. For Sortase SDS-PAGE showed positive result but analysis with MS showed no product. TEV was concluded not to be funcional at all hence the failing to cleave soluble substrate  when condition seemed near optimal and faulty flow cytometer data. Even though the lack of success there is still many further studies that can be done on the proteases in order to prove its absence/presence  of activity.

No description available.

Trichomonas vagina/is, one of the most common sexually transmitted diseases, has been shown to increase patients' susceptibility to HIV infection and cervical cancer; moreover, resistance to metronidazole is increasing, and new drug targets must be identified in order to combat resistant strains. T vagina/is expresses cysteine proteases that have been implicated in vaginal epithelial apoptosis as well as immune system evasion. In the past the various cysteine proteases have been studied as a group, and the following work examines, one specific protease, TvCP2, in detail through Western blot analysis, immunofluorescent staining, and recombinant expression. The experiments 5 presented here suggest that aT l-CP2 over-expressing transfectant line processes CP2 and sequesters it in cellular compartments. Previous data gives strong evidence of the secretion of cysteine protease CP4 and hints at the possibility of CP2 secretion as well; however, our results show no co-localization between CP2 and CP4 in T l-CP2 over expressing transfectants, suggesting separate trafficking and different roles. To better characterize CP2 function, we attempted to express active, recombinant protein. Although Pichia pastoris serves as a reliable expression vehicle, a processing event following translation ofTvCP2 appears to have cleaved the pro-domain and, along with it, the a-secretion signal, trapping active TvCP2 within the cellular pellet. A thioreoxintagged version ofTvCP2 has been expressed in E. coli, and preliminary experiments show it may auto-activate under certain conditions, but further experimentation is required to confirm the presence of active CP2 within the fraction purified from these cells.

For the past 40 years, antiangiogenic approaches have been of major interest in the development of methods to cure and prevent cancer. Angiogenesis, the development of blood vessels from pre-existing vascularization, is essential for cancer growth and spread of metastasis through the delivery of nutrients and oxygen essential to sustain the metabolic activity of these malignant cells. Blocking access to blood will cause cancerous cells to assume a dormant state creating inactive micro-tumors innocuous to the host. Angiostatin, the internal fragment of the fibrinolytic zymogen plasminogen, has shown great potential in reducing cancer size and number of metastatic colonies in animal models. Owing to the success of these preliminary results angiostatin is currently on clinical trials. Plasminogen is known to be transferred from blood to milk during lactation. The objectives of this research were to: 1) investigate the ability of various proteases in cleaving plasminogen, both from human and bovine sources, and consequently release the angiostatin like fragment; 2) determine the anticancer activity of bovine angiostatin; 3) examine ability of the antiangiogenic fragment to survive digestion; 4) purify the fragment of interest through column chromatography. Production of angiostatin was tested through hydrolysis of plasminogen via Bacillus Polymyxa protease (or dispase I), elastase, lactic acid bacteria and Bacilli originated enzymes. Once proteases capable of angiostatin like peptide production were identified, and sequence analysis of the fragments obtained conducted to confirm that bovine angiostatin was indeed produced, ability of angiostatin, both human and bovine, in inhibiting malignant melanoma as well as colon cancer cells was evaluated in vitro. From the results obtained we can confirm that bovine angiostatin inhibitory activity on cancerous cells is similar to that observed for human angiostatin. Analysis of bovine angiostatin survival through in vitro human digestion model was also examined. Results show good possibility of angiostatin surviving digestion, even if confirmation of these results is required through further in vivo studies. Additionally, digestive enzymes such as trypsin and α-chymotrypsin showed ability in cleaving plasminogen directly to release a 25kDa fragment. Knowing that each kringle has some degree of anticancer activity it would be of interest to further study the possibility of angiostatin related fragments to be produced during milk digestion. Finally, affinity chromatography through L-lysine used to purify human angiostatin resulted to be an adequate method for bovine angiostatin purification. Preliminary results obtained from this study open a new area worth investigating to uncover the potential of using bovine angiostatin in the development of novel food products capable of cancer prevention.