RESUMEN

In recent decades, the role played by extracellular vesicles in physiological and pathological processes has attracted attention. Extracellular vesicles are released by different types of cells and carry molecules that could become biomarkers for the diagnosis of diseases. Extracellular vesicles are also moldable tools for the controlled release of bioactive substances in clinical and therapeutic applications. However, one of the significant challenges when studying these exciting and versatile vesicles is the purification process, which presents significant difficulties in terms of lack of purity, yield, and reproducibility, reflected in unreliable data. Therefore, our objective in the present study was to compare the proteomic profile of serum-derived EVs purified using ExoQuick™ (Systems Biosciences), Total Isolation Kit (Life Technologies), Ultracentrifugation, and Ultrafiltration. Each technique utilized for purification has shown different concentrations and populations of purified particles. The results showed marked differences in distribution, size, and protein content, demonstrating the need to develop reproducible and reliable protocols to isolate extracellular vesicles for their clinical application.

RESUMEN

Suppressor of gamma response 1 (SOG1) is a member of the NAC domain family transcription factors of the DNA damage response (DDR) signaling in the plant's genome. SOG1 is directly involved in transcriptional response to DNA damage, cell cycle checkpoints and ATR or ATM-mediated activation of the DNA damage responses and repair functioning in programmed cell death and regulation of end reduplication. Different mutations in the SOG1 protein lead to severe diseases and, ultimately, cell death. Single nucleotide polymorphisms (SNPs) are an important type of genetic alteration that cause different diseases or programmed cell death. The current study applied different computational approaches to Arabidopsis thaliana L. SOG1 protein to identify the potential deleterious nsSNPs and monitor their impact on the structure, function and protein stability. Various bioinformatics tools were applied to analyze the retrieved 34 nsSNPs and interestingly extracted four deleterious nsSNPs, that is, ensvath13968004 (Q166L), tmp18998388 (P159L), ensvath01103049 (K199N) and tmp18998295 (Y190F). For example, homology modeling, conservation and conformational analysis of the mutant's models were considered to scrutinize the deviations of these variants from the native SOG1 structure. All atoms molecular dynamic simulation confirmed the significance of these mutations on the protein stability, residual and structural conformation, compactness, surface conformation, dominant motion, Gibbs free energy distribution and dynamic effects. Similarly, protein-protein interaction revealed that SOG1 operates as a hub-linking cluster of various proteins, and any changes in the SOG1 might result in the disassociation of several signal transduction cascades.Communicated by Ramaswamy H. Sarma.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Simulación de Dinámica Molecular , Factores de Transcripción/genética , Daño del ADN , Mutación , Polimorfismo de Nucleótido Simple , Proteínas de Arabidopsis/genética

RESUMEN

Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness - which is limited to the parasite's adult form - and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. Fourteen of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.

Asunto(s)

Descubrimiento de Drogas/métodos , Purina-Nucleósido Fosforilasa/química , Purina-Nucleósido Fosforilasa/metabolismo , Piridinas/metabolismo , Pirimidinas/metabolismo , Schistosoma mansoni/enzimología , Tiazoles/metabolismo , Animales , Dominio Catalítico , Cristalización , Cristalografía por Rayos X/métodos , Dimetilsulfóxido/farmacología , Evaluación Preclínica de Medicamentos/métodos , Modelos Moleculares , Conformación Proteica en Hélice alfa , Purina-Nucleósido Fosforilasa/genética , Esquistosomiasis mansoni/tratamiento farmacológico , Esquistosomiasis mansoni/parasitología

RESUMEN

RNA interference (RNAi)-mediated gene silencing can be used to control specific insect pest populations. Unfortunately, the variable efficiency in the knockdown levels of target genes has narrowed the applicability of this technology to a few species. Here, we examine the current state of knowledge regarding the miRNA (micro RNA) and siRNA (small interfering RNA) pathways in insects and investigate the structural variability at key protein domains of the RNAi machinery. Our goal was to correlate domain variability with mechanisms affecting the gene silencing efficiency. To this end, the protein domains of 168 insect species, encompassing the orders Coleoptera, Diptera, Hemiptera, Hymenoptera, and Lepidoptera, were analysed using our pipeline, which takes advantage of meticulous structure-based sequence alignments. We used phylogenetic inference and the evolutionary rate coefficient (K) to outline the variability across domain regions and surfaces. Our results show that four domains, namely dsrm, Helicase, PAZ and Ribonuclease III, are the main contributors of protein variability in the RNAi machinery across different insect orders. We discuss the potential roles of these domains in regulating RNAi-mediated gene silencing and the role of loop regions in fine-tuning RNAi efficiency. Additionally, we identified several order-specific singularities which indicate that lepidopterans have evolved differently from other insect orders, possibly due to constant coevolution with plants and viruses. In conclusion, our results highlight several variability hotspots that deserve further investigation in order to improve the application of RNAi technology in the control of insect pests.

Asunto(s)

Silenciador del Gen , Proteínas de Insectos/metabolismo , Insectos/clasificación , Insectos/genética , MicroARNs/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Animales , Proteínas de Insectos/genética , Insectos/metabolismo , Filogenia , Dominios Proteicos

RESUMEN

BACKGROUND: Drought is one of the most harmful abiotic stresses for plants, leading to reduced productivity of several economically important crops and, consequently, considerable losses in the agricultural sector. When plants are exposed to stressful conditions, such as drought and high salinity, they modulate the expression of genes that lead to developmental, biochemical, and physiological changes, which help to overcome the deleterious effects of adverse circumstances. Thus, the search for new specific gene promoter sequences has proved to be a powerful biotechnological strategy to control the expression of key genes involved in water deprivation or multiple stress responses. RESULTS: This study aimed to identify and characterize the GmRD26 promoter (pGmRD26), which is involved in the regulation of plant responses to drought stress. The expression profile of the GmRD26 gene was investigated by qRT-PCR under normal and stress conditions in Williams 82, BR16 and Embrapa48 soybean-cultivars. Our data confirm that GmRD26 is induced under water deficit with different induction folds between analyzed cultivars, which display different genetic background and physiological behaviour under drought. The characterization of the GmRD26 promoter was performed under simulated stress conditions with abscisic acid (ABA), polyethylene glycol (PEG) and drought (air dry) on A. thaliana plants containing the complete construct of pGmRD26::GUS (2.054 bp) and two promoter modules, pGmRD26A::GUS (909 pb) and pGmRD26B::GUS (435 bp), controlling the expression of the ß-glucuronidase (uidA) gene. Analysis of GUS activity has demonstrated that pGmRD26 and pGmRD26A induce strong reporter gene expression, as the pAtRD29 positive control promoter under ABA and PEG treatment. CONCLUSIONS: The full-length promoter pGmRD26 and the pGmRD26A module provides an improved uidA transcription capacity when compared with the other promoter module, especially in response to polyethylene glycol and drought treatments. These data indicate that pGmRD26A may become a promising biotechnological asset with potential use in the development of modified drought-tolerant plants or other plants designed for stress responses.

Asunto(s)

Ácido Abscísico/farmacología , Glycine max/genética , Biotecnología/métodos , Sequías , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/genética , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Glycine max/efectos de los fármacos , Estrés Fisiológico/genética , Estrés Fisiológico/fisiología

RESUMEN

Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, SmPNP2. Thermofluorescence screening of different ligands suggested cytidine and cytosine are potential ligands. The binding of cytosine and cytidine were confirmed by isothermal titration calorimetry, with a KD of 27 µM for cytosine, and a KM of 76.3 µM for cytidine. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystal structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capacity of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site thus permitting an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site enhances our understanding of the correlation between structure and nucleotide selectivity, thus suggesting new ways to analyse PNP activity.

Asunto(s)

Nucleósidos/metabolismo , Purina-Nucleósido Fosforilasa/química , Purina-Nucleósido Fosforilasa/metabolismo , Schistosoma mansoni/enzimología , Schistosoma mansoni/genética , Adenosina/metabolismo , Animales , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Citidina/metabolismo , Citosina/metabolismo , Proteínas del Helminto/química , Proteínas del Helminto/metabolismo , Inosina/metabolismo , Cinética , Modelos Moleculares , Mutación , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Purina-Nucleósido Fosforilasa/genética , Schistosoma mansoni/química , Especificidad por Sustrato

RESUMEN

The exploration of emerging host organisms for the economic and efficient production of protein microbicides against HIV is urgently needed in resource-poor areas worldwide. In this study, the production of the novel HIV entry inhibitor candidate, griffithsin (GRFT), was investigated using Nicotiana benthamiana as the expression platform based on a non-viral vector. To increase the yield of recombinant GRFT, the RNA silencing defense mechanism of N. benthamiana was abolished by using three gene silencing suppressors. A transient expression system was used by transferring the GRFT gene, which encodes 122 amino acids, under the control of the enhanced CaMV 35S promoter. The presence of correctly assembled GRFT in transgenic leaves was confirmed using immunoglobulin-specific sandwich ELISA. The data demonstrated that the use of three gene silencing suppressors allowed the highest accumulation of GRFT, with a yield of 400 µg g-1 fresh weight, and this amount was reduced to 287 µg g-1 after purification, representing a recovery of 71.75%. The analysis also showed that the ability of GRFT expressed in N. benthamiana to bind to glycoprotein 120 is close to that of the GRFT protein purified from E. coli. Whole-cell assays using purified GRFT showed that our purified GRFT was potently active against HIV. This study provides the first high-level production of the HIV-1 entry inhibitor griffithsin with a non-viral expression system and illustrates the robustness of the co-agroinfiltration expression system improved through the use of three gene silencing suppressors.

RESUMEN

RNA interference (RNAi) approaches have been applied as a biotechnological tool for controlling plant insect pests via selective gene down regulation. However, the inefficiency of RNAi mechanism in insects is associated with several barriers, including dsRNA delivery and uptake by the cell, dsRNA interaction with the cellular membrane receptor and dsRNA exposure to insect gut nucleases during feeding. The cotton boll weevil (Anthonomus grandis) is a coleopteran in which RNAi-mediated gene silencing does not function efficiently through dsRNA feeding, and the factors involved in the mechanism remain unknown. Herein, we identified three nucleases in the cotton boll weevil transcriptome denoted AgraNuc1, AgraNuc2, and AgraNuc3, and the influences of these nucleases on the gene silencing of A. grandis chitin synthase II (AgraChSII) were evaluated through oral dsRNA feeding trials. A phylogenetic analysis showed that all three nucleases share high similarity with the DNA/RNA non-specific endonuclease family of other insects. These nucleases were found to be mainly expressed in the posterior midgut region of the insect. Two days after nuclease RNAi-mediated gene silencing, dsRNA degradation by the gut juice was substantially reduced. Notably, after nucleases gene silencing, the orally delivered dsRNA against the AgraChSII gene resulted in improved gene silencing efficiency when compared to the control (non-silenced nucleases). The data presented here demonstrates that A. grandis midgut nucleases are effectively one of the main barriers to dsRNA delivery and emphasize the need to develop novel RNAi delivery strategies focusing on protecting the dsRNA from gut nucleases and enhancing its oral delivery and uptake to crop insect pests.

Asunto(s)

Endonucleasas/metabolismo , Silenciador del Gen , Control de Insectos/métodos , Gorgojos/genética , Animales , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcriptoma

RESUMEN

A current metagenomics focus is to interpret and transform collected genomic data into biological information. By combining structural, functional and genomic data we have assessed a novel bacterial protein selected from a carbohydrate-related activity screen in a microbial metagenomic library from Capra hircus (domestic goat) gut. This uncharacterized protein was predicted as a bacterial cell wall-modifying enzyme (CWME) and shown to contain four domains: an N-terminal, a cysteine protease, a peptidoglycan-binding and an SH3 bacterial domain. We successfully cloned, expressed and purified this putative cysteine protease (PCP), which presented autoproteolytic activity and inhibition by protease inhibitors. We observed cell wall hydrolytic activity and ampicillin binding capacity, a characteristic of most bacterial CWME. Fluorimetric binding analysis yielded a Kb of 1.8 × 105 M-1 for ampicillin. Small-angle X-ray scattering (SAXS) showed a maximum particle dimension of 95 Å with a real-space Rg of 28.35 Å. The elongated molecular envelope corroborates the dynamic light scattering (DLS) estimated size. Furthermore, homology modeling and SAXS allowed the construction of a model that explains the stability and secondary structural changes observed by circular dichroism (CD). In short, we report a novel cell wall-modifying autoproteolytic PCP with insight into its biochemical, biophysical and structural features.

Asunto(s)

Ampicilina/metabolismo , Bacterias/enzimología , Clonación Molecular/métodos , Proteasas de Cisteína/química , Proteasas de Cisteína/metabolismo , Cabras/microbiología , Animales , Bacterias/química , Bacterias/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pared Celular/enzimología , Pared Celular/genética , Proteasas de Cisteína/genética , Hidrólisis , Metagenoma , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Estabilidad Proteica , Estructura Secundaria de Proteína , Dispersión del Ángulo Pequeño , Difracción de Rayos X

RESUMEN

BACKGROUND: Tospovirus is a plant-infecting genus within the family Bunyaviridae, which also includes four animal-infecting genera: Hantavirus, Nairovirus, Phlebovirus and Orthobunyavirus. Compared to these members, the structures of Tospovirus proteins still are poorly understood. Despite multiple studies have attempted to identify candidate N protein regions involved in RNA binding and protein multimerization for tospovirus using yeast two-hybrid systems (Y2HS) and site-directed mutagenesis, the tospovirus ribonucleocapsids (RNPs) remains largely uncharacterized at the molecular level and the lack of structural information prevents detailed insight into these interactions. RESULTS: Here we used the nucleoprotein structure of LACV (La Crosse virus-Orthobunyavirus) and molecular dynamics simulations to access the structure and dynamics of the nucleoprotein from tospovirus GRSV (Groundnut ringspot virus). The resulting model is a monomer composed by a flexible N-terminal and C-terminal arms and a globular domain with a positively charged groove in which RNA is deeply encompassed. This model allowed identifying the candidate amino acids residues involved in RNA interaction and N-N multimerization. Moreover, most residues predicted to be involved in these interactions are highly conserved among tospoviruses. CONCLUSIONS: Crucially, the interaction model proposed here for GRSV N is further corroborated by the all available mutational studies on TSWV (Tomato spotted wilt virus) N, so far. Our data will help designing further and more accurate mutational and functional studies of tospovirus N proteins. In addition, the proposed model may shed light on the mechanisms of RNP shaping and could allow the identification of essential amino acid residues as potential targets for tospovirus control strategies.

Asunto(s)

Nucleoproteínas/química , Tospovirus/química , Secuencia de Aminoácidos , Secuencia de Bases , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Nucleoproteínas/genética , Alineación de Secuencia , Tospovirus/genética