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The global health emergency generated by coronavirus disease 2019 (COVID-19) has prompted the search for preventive and therapeutic treatments for its pathogen, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). There are many potential targets for drug discovery and development to tackle this disease. One of these targets is the main protease, Mpro or 3CLpro, which is highly conserved among coronaviruses. 3CLpro is an essential player in the viral replication cycle, processing the large viral polyproteins and rendering the individual proteins functional. We report a biophysical characterization of the structural stability and the catalytic activity of 3CLpro from SARS-CoV-2, from which a suitable experimental in vitro molecular screening procedure has been designed. By screening of a small chemical library consisting of about 150 compounds, the natural product quercetin was identified as reasonably potent inhibitor of SARS-CoV-2 3CLpro (K i  ~ 7 μM). Quercetin could be shown to interact with 3CLpro using biophysical techniques and bind to the active site in molecular simulations. Quercetin, with well-known pharmacokinetic and ADMET properties, can be considered as a good candidate for further optimization and development, or repositioned for COVID-19 therapeutic treatment., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The aim of this work was to assess the effects of manganese (Mn) toxicity on the proteome of tomato roots using two proteomic approaches, shotgun and two-dimensional electrophoresis. The shotgun approach yielded 367 reliable proteins, whereas the 2-DE approach detected 340 consistent spots. The 2-DE method found 54 proteins changing in relative abundance in the excess Mn treatment, whereas the shotgun detected changes in 118 proteins. Only 7% of the differential proteins were found by both methods, illustrating their complementary nature. Metabolic pathways most affected were protein metabolism, oxido-reductases and signaling. Results support that Mn toxicity alters the protein turnover and impairs energy production in roots, leading to changes in glycolysis, pyruvate metabolism, TCA and oxidative phosphorylation. Excess Mn also induced changes in peroxidases and hydrolases participating in cell wall lignification and suberization and activated plant defense mechanisms, with changes occurring via pathogenesis-related proteins as well as peroxidases. Finally, Mn toxicity elicited regulatory mechanisms and affected the abundance of root nutrient reservoir proteins. The overall analysis of the differential root proteome upon Mn toxicity suggests a general slowdown of metabolic activities, especially energy production, cell wall integrity and protein turnover, which occurs in parallel with increases in stress related proteins., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The aim of this work was to study the effects of Fe and Mn deficiencies on the xylem sap proteome of tomato using a shotgun proteomic approach, with the final goal of elucidating plant response mechanisms to these stresses. This approach yielded 643 proteins reliably identified and quantified with 70% of them predicted as secretory. Iron and Mn deficiencies caused statistically significant and biologically relevant abundance changes in 119 and 118 xylem sap proteins, respectively. In both deficiencies, metabolic pathways most affected were protein metabolism, stress/oxidoreductases and cell wall modifications. First, results suggest that Fe deficiency elicited more stress responses than Mn deficiency, based on the changes in oxidative and proteolytic enzymes. Second, both nutrient deficiencies affect the secondary cell wall metabolism, with changes in Fe deficiency occurring via peroxidase activity, and in Mn deficiency involving peroxidase, Cu-oxidase and fasciclin-like arabinogalactan proteins. Third, the primary cell wall metabolism was affected by both nutrient deficiencies, with changes following opposite directions as judged from the abundances of several glycoside-hydrolases with endo-glycolytic activities and pectin esterases. Fourth, signaling pathways via xylem involving CLE and/or lipids as well as changes in phosphorylation and N-glycosylation also play a role in the responses to these stresses. Biological significance In spite of being essential for the delivery of nutrients to the shoots, our knowledge of xylem responses to nutrient deficiencies is very limited. The present work applies a shotgun proteomic approach to unravel the effects of Fe and Mn deficiencies on the xylem sap proteome. Overall, Fe deficiency seems to elicit more stress in the xylem sap proteome than Mn deficiency, based on the changes measured in proteolytic and oxido-reductase proteins, whereas both nutrients exert modifications in the composition of the primary and secondary cell wall. Cell wall modifications could affect the mechanical and permeability properties of the xylem sap vessels, and therefore ultimately affect solute transport and distribution to the leaves. Results also suggest that signaling cascades involving lipid and peptides might play a role in nutrient stress signaling and pinpoint interesting candidates for future studies. Finally, both nutrient deficiencies seem to affect phosphorylation and glycosylation processes, again following an opposite pattern., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Unlabelled: Iron deficiency is a yield-limiting factor with major implications for crop production, especially in soils with high CaCO3. Because stems are essential for the delivery of nutrients to the shoots, the aim of this work was to study the effects of Fe deficiency on the stem proteome of Medicago truncatula. Two-dimensional electrophoresis separation of stem protein extracts resolved 276 consistent spots in the whole experiment. Iron deficiency in absence or presence of CaCO3 caused significant changes in relative abundance in 10 and 31 spots, respectively, and 80% of them were identified by mass spectrometry. Overall results indicate that Fe deficiency by itself has a mild effect on the stem proteome, whereas Fe deficiency in the presence of CaCO3 has a stronger impact and causes changes in a larger number of proteins, including increases in stress and protein metabolism related proteins not observed in the absence of CaCO3. Both treatments resulted in increases in cell wall related proteins, which were more intense in the presence of CaCO3. The increases induced by Fe-deficiency in the lignin per protein ratio and changes in the lignin monomer composition, assessed by pyrolysis-gas chromatography-mass spectrometry and microscopy, respectively, further support the existence of cell wall alterations., Biological Significance: In spite of being essential for the delivery of nutrients to the shoots, our knowledge of stem responses to nutrient deficiencies is very limited. The present work applies 2-DE techniques to unravel the response of this understudied tissue to Fe deficiency. Proteomics data, complemented with mineral, lignin and microscopy analyses, indicate that stems respond to Fe deficiency by increasing stress and defense related proteins, probably in response of mineral and osmotic unbalances, and eliciting significant changes in cell wall composition. The changes observed are likely to ultimately affect solute transport and distribution to the leaves., (Copyright © 2016 Elsevier B.V. All rights reserved.)