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Plant proteins that are secreted without a classical signal peptide leader sequence are termed leaderless secretory proteins (LSPs) and are implicated in both plant development and (a)biotic stress responses. In plant proteomics experimental workflows, identification of LSPs is hindered by the possibility of contamination from other subcellar compartments upon purification of the secretome. Applying machine learning algorithms to predict LSPs in plants is also challenging due to the rarity of experimentally validated examples for training purposes. This work attempts to address this issue by establishing criteria for identifying potential plant LSPs based on experimental observations and training random forest classifiers on the putative datasets. The resultant plant protein database LSPDB and bioinformatic prediction tools LSPpred and SPLpred are available at lsppred.lspdb.org. The LSPpred and SPLpred modules are internally validated on the training dataset, with false positives controlled at 5%, and are also able to classify the limited number of established plant LSPs (SPLpred (3/4, LSPpred 4/4). Until such time as a larger set of bona fide (independently experimentally validated) LSPs is established using imaging technologies (light/fluorescence/electron microscopy) to confirm sub-cellular location, these tools represent a bridging method for predicting and identifying plant putative LSPs for subsequent experimental validation.

SARS-CoV-2 is a type of coronavirus responsible for the international outbreak of respiratory illness termed COVID-19 that forced the World Health Organization to declare a pandemic infectious disease situation of international concern at the beginning of 2020. The need for a swift response against COVID-19 prompted to consider different sources to identify bioactive compounds that can be used as therapeutic agents, including available drugs and natural products. Accordingly, this work reports the results of a virtual screening process aimed at identifying antiviral natural product inhibitors of the SARS-CoV-2 Mpro viral protease. For this purpose, ca. 2000 compounds of the Selleck database of Natural Compounds were the subject of an ensemble docking process targeting the Mpro protease. Molecules that showed binding to most of the protein conformations were retained for a further step that involved the computation of the binding free energy of the ligand-Mpro complex along a molecular dynamics trajectory. The compounds that showed a smooth binding free energy behavior were selected for in vitro testing. From the resulting set of compounds, five compounds exhibited an antiviral profile, and they are disclosed in the present work.

The COVID-19 pandemic outbreak prompts an urgent need for efficient therapeutics, and repurposing of known drugs has been extensively used in an attempt to get to anti-SARS-CoV-2 agents in the shortest possible time. The glycoside rutin shows manifold pharmacological activities and, despite its use being limited by its poor solubility in water, it is the active principle of many pharmaceutical preparations. We herein report our in silico and experimental investigations of rutin as a SARS-CoV-2 Mpro inhibitor and of its water solubility improvement obtained by mixing it with l-arginine. Tests of the rutin/l-arginine mixture in a cellular model of SARS-CoV-2 infection highlighted that the mixture still suffers from unfavorable pharmacokinetic properties, but nonetheless, the results of this study suggest that rutin might be a good starting point for hit optimization.

[ES] La presente invención se refiere a un material híbrido que comprende una matriz proteica y nanopartículas (NPs) de una especie de Cu, su proceso para su preparación, así como sus usos. El material de la invención presenta una actividad de inhibición de proteínas de SARS-CoV-2, lo que le confiere capacidad para su uso como agente biocida, así como agente de recubrimiento y desinfectante de materiales seleccionados entre metales, papel, textil y mascarillas quirúrgicas homologadas., [EN] The present invention refers to a hybrid material that includes a protein matrix and nanoparticles (NPS) of a kind of CU, its process for its preparation, as well as its uses. The invention material presents a SARS-COV-2 protein inhibition activity, which gives it the capacity to use as a biocidal agent, as well as coating agent and disinfectant of selected materials between metals, paper, textile and surgical masks approved.

Iron (Fe) and manganese (Mn) are two essential elements for plants that compete for the same uptake transporters and show conflicting interactions at the regulatory level. In order to understand the differential response to both metal deficiencies in plants, two proteomic techniques (two-dimensional gel electrophoresis and label-free shotgun) were used to study the proteome profiles of roots from tomato plants grown under Fe or Mn deficiency. A total of 119 proteins changing in relative abundance were confidently quantified and identified, including 35 and 91 in the cases of Fe deficiency and Mn deficiency, respectively, with 7 of them changing in both deficiencies. The identified proteins were categorized according to function, and GO-enrichment analysis was performed. Data showed that both deficiencies provoked a common and intense cell wall remodelling. However, the response observed for Fe and Mn deficiencies differed greatly in relation to oxidative stress, coumarin production, protein, nitrogen, and energy metabolism.

SARS-CoV-2 is a type of coronavirus responsible for the international outbreak of respiratory illness termed COVID-19 that forced the World Health Organization to declare a pandemic infectious disease situation of international concern at the beginning of 2020. The need for a swift response against COVID-19 prompted to consider different sources to identify bioactive compounds that can be used as therapeutic agents, including available drugs and natural products. Accordingly, this work reports the results of a virtual screening process aimed at identifying antiviral natural product inhibitors of the SARS-CoV-2 Mpro viral protease. For this purpose, ca. 2000 compounds of the Selleck database of Natural Compounds were the subject of an ensemble docking process targeting the Mpro protease. Molecules that showed binding to most of the protein conformations were retained for a further step that involved the computation of the binding free energy of the ligand-Mpro complex along a molecular dynamics trajectory. The compounds that showed a smooth binding free energy behavior were selected for in vitro testing. From the resulting set of compounds, five compounds exhibited an antiviral profile, and they are disclosed in the present work., Peer Reviewed, Postprint (author's final draft)

The ability to custom-modify cell surface glycans holds great promise for treatment of a variety of diseases. We propose a glycomimetic ofl-fucose that markedly inhibits the creation of sLeXby FTVI and FTVII, but has no effect on creation of LeXby FTIX. Our findings thus indicate that selective suppression of sLex display can be achieved, and STD-NMR studies surprisingly reveal that the mimetic does not compete with GDP-fucose at the enzymatic binding site.

The ability to custom-modify cell surface glycans holds great promise for treatment of a variety of diseases. We propose a glycomimetic ofl-fucose that markedly inhibits the creation of sLeby FTVI and FTVII, but has no effect on creation of Leby FTIX. Our findings thus indicate that selective suppression of sLex display can be achieved, and STD-NMR studies surprisingly reveal that the mimetic does not compete with GDP-fucose at the enzymatic binding site.

El objetivo general de esta Tesis Doctoral es incrementar el conocimiento existente acerca de los efectos que causan las deficiencias de Fe y Mn y la toxicidad por Mn en los proteomas vegetales implicados en la absorción y el transporte de estos nutrientes. Se pretende dibujar, con un enfoque proteómico, un mapa general de las alteraciones metabólicas producidas por ambos estreses nutricionales en las plantas. El estudio de estos procesos permitirá incrementar el conocimiento existente sobre la homeostasis de estos metales y así asentar las bases para mejorar la calidad y productividad de los cultivos, elaborar posibles estrategias de fitoremediación y desarrollar fertilizantes eficientes y respetuosos con el medio ambiente., Se plantean los siguientes objetivos específicos: 1. Describir la categorización funcional de los perfiles proteicos de los tres fluidos principales en plantas, fluido apoplástico y las savias de xilema y floema, considerando los estudios proteómicos recientemente publicados. 2. Caracterizar, usando técnicas de 2-DE, los perfiles proteicos del fluido apoplástico de remolacha (Beta vulgaris), tanto en plantas control como afectadas por deficiencia de Fe y, en las mismas plantas, tras el suministro de Fe a las raíces. 3. Caracterizar, mediante técnicas de “shotgun proteomics”, los cambios inducidos por las deficiencias de Fe y Mn en el perfil proteico de la savia de xilema de plantas de tomate (Solanum lycopersicum). 4. Caracterizar, mediante técnicas de 2-DE y “shotgun proteomics”, los efectos de la toxicidad de Mn sobre los perfiles proteicos de la raíz y de la savia de xilema de plantas de tomate (Solanum lycopersicum).

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.

Metal toxicity is a common problem in crop species worldwide. Some metals are naturally toxic, whereas others such as manganese (Mn) are essential micro-nutrients for plant growth but can become toxic when in excess. Changes in the composition of the xylem sap, which is the main pathway for ion transport within the plant, is therefore vital to understanding the plant’s response(s) to metal toxicity. In this study we have assessed the effects of exposure of tomato roots to excess Mn on the protein profile of the xylem sap, using a shotgun proteomics approach. Plants were grown in nutrient solution using 4.6 and 300 µM MnCl2 as control and excess Mn treatments, respectively. This approach yielded 668 proteins reliably identified and quantified. Excess Mn caused statistically significant (at p ≤ 0.05) and biologically relevant changes in relative abundance (≥2-fold increases or ≥50% decreases) in 322 proteins, with 82% of them predicted to be secretory using three different prediction tools, with more decreasing than increasing (181 and 82, respectively), suggesting that this metal stress causes an overall deactivation of metabolic pathways. Processes most affected by excess Mn were in the oxido-reductase, polysaccharide and protein metabolism classes. Excess Mn induced changes in hydrolases and peroxidases involved in cell wall degradation and lignin formation, respectively, consistent with the existence of alterations in the cell wall. Protein turnover was also affected, as indicated by the decrease in proteolytic enzymes and protein synthesis-related proteins. Excess Mn modified the redox environment of the xylem sap, with changes in the abundance of oxido-reductase and defense protein classes indicating a stress scenario. Finally, results indicate that excess Mn decreased the amounts of proteins associated with several signaling pathways, including fasciclin-like arabinogalactan-proteins and lipids, as well as proteases, which may be involved in the release of sign

Polypeptide GalNAc-transferase T3 (GalNAc-T3) regulates fibroblast growth factor 23 (FGF23) by O-glycosylating Thr178 in a furin proprotein processing motif RHT178R↓S. FGF23 regulates phosphate homeostasis and deficiency in GALNT3 or FGF23 results in hyperphosphatemia and familial tumoral calcinosis. We explored the molecular mechanism for GalNAc-T3 glycosylation of FGF23 using engineered cell models and biophysical studies including kinetics, molecular dynamics and X-ray crystallography of GalNAc-T3 complexed to glycopeptide substrates. GalNAc-T3 uses a lectin domain mediated mechanism to glycosylate Thr178 requiring previous glycosylation at Thr171. Notably, Thr178 is a poor substrate site with limiting glycosylation due to substrate clashes leading to destabilization of the catalytic domain flexible loop. We suggest GalNAc-T3 specificity for FGF23 and its ability to control circulating levels of intact FGF23 is achieved by FGF23 being a poor substrate. GalNAc-T3’s structure further reveals the molecular bases for reported disease-causing mutations. Our findings provide an insight into how GalNAc-T isoenzymes achieve isoenzyme-specific nonredundant functions.

Background: Current efforts in the identification of new biomarkers are directed towards an accurate differentiation between benign and premalignant cysts. Thermal Liquid Biopsy (TLB) has been previously applied to inflammatory and tumor diseases and could offer an interesting point of view in this type of pathology. Methods: In this work, twenty patients (12 males and 8 females, average ages 62) diagnosed with a pancreatic cyst benign (10) and premalignant (10) cyst lesions were recruited, and biological samples were obtained during the endoscopic ultrasonography procedure. Results: Proteomic content of cyst liquid samples was studied and several common proteins in the different groups were identified. TLB cyst liquid profiles reflected protein content. Also, TLB serum score was able to discriminate between healthy and cysts patients (71% sensitivity and 98% specificity) and between benign and premalignant cysts (75% sensitivity and 67% specificity). Conclusions: TLB analysis of plasmatic serum sample, a quick, simple and non-invasive technique that can be easily implemented, reports valuable information on the observed pancreatic lesion. These preliminary results set the basis for a larger study to refine TLB serum score and move closer to the clinical application of TLB providing useful information to the gastroenterologist during patient diagnosis.

This article contains consolidated proteomic data obtained from xylem sap collected from tomato plants grown in Fe- and Mn-sufficient control, as well as Fe-deficient and Mn-deficient conditions. Data presented here cover proteins identified and quantified by shotgun proteomics and Progenesis LC-MS analyses: proteins identified with at least two peptides and showing changes statistically significant (ANOVA; p ≤ 0.05) and above a biologically relevant selected threshold (fold ≥ 2) between treatments are listed. The comparison between Fe-deficient, Mn-deficient and control xylem sap samples using a multivariate statistical data analysis (Principal Component Analysis, PCA) is also included. Data included in this article are discussed in depth in the research article entitled “Effects of Fe and Mn deficiencies on the protein profiles of tomato (Solanum lycopersicum) xylem sap as revealed by shotgun analyses” [1]. This dataset is made available to support the cited study as well to extend analyses at a later stage.

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

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.

The phloem sap, xylem sap and apoplastic fluid play key roles in long and short distance transport of signals and nutrients, and act as a barrier against local and systemic pathogen infection. Among other components, these plant fluids contain proteins which are likely to be important players in their functionalities. However, detailed information about their proteomes is only starting to arise due to the difficulties inherent to the collection methods. This review compiles the proteomic information available to date in these three plant fluids, and compares the proteomes obtained in different plant species in order to shed light into conserved functions in each plant fluid. Inter-species comparisons indicate that all these fluids contain the protein machinery for selfmaintenance and defense, including proteins related to cell wall metabolism, pathogen defense, proteolysis, and redox response. These analyses also revealed that proteins may play more relevant roles in signaling in the phloem sap and apoplastic fluid than in the xylem sap. A comparison of the proteomes of the three fluids indicates that although functional categories are somewhat similar, proteins involved are likely to be fluid-specific, except for a small group of proteins present in the three fluids, which may have a universal role, especially in cell wall maintenance and defense.