RESUMEN
Snake venoms are complex cocktails of non-toxic and toxic molecules that work synergistically for the envenoming outcome. Alongside the immediate consequences, chronic manifestations and long-term sequelae can occur. Recently, extracellular vesicles (EVs) were found in snake venom. EVs mediate cellular communication through long distances, delivering proteins and nucleic acids that modulate the recipient cell's function. However, the biological roles of snake venom EVs, including possible cross-organism communication, are still unknown. This knowledge may expand the understanding of envenoming mechanisms. In the present study, we isolated and characterized the EVs from Bothrops jararaca venom (Bj-EVs), giving insights into their biological roles. Fresh venom was submitted to differential centrifugation, resulting in two EV populations with typical morphology and size range. Several conserved EV markers and a subset of venom related EV markers, represented mainly by processing enzymes, were identified by proteomic analysis. The most abundant protein family observed in Bj-EVs was 5'-nucleotidase, known to be immunosuppressive and a low abundant and ubiquitous toxin in snake venoms. Additionally, we demonstrated that mammalian cells efficiently internalize Bj-EVs. The commercial antibothropic antivenom partially recognizes Bj-EVs and inhibits cellular EV uptake. Based on the proteomic results and the in vitro interaction assays using macrophages and muscle cells, we propose that Bj-EVs may be involved not only in venom production and processing but also in host immune modulation and long-term effects of envenoming.
Asunto(s)
Bothrops , Venenos de Crotálidos , Vesículas Extracelulares , Animales , Venenos de Crotálidos/química , Proteómica , Proteínas , Venenos de Serpiente , MamíferosRESUMEN
Tritrichomonas foetus is a protist that causes bovine trichomoniasis and presents a well-developed Golgi. There are very few studies concerning the Golgi in trichomonads. In this work, monoclonal antibodies were raised against Golgi of T. foetus and used as a tool on morphologic and biochemical studies of this organelle. Among the antibodies produced, one was named mAb anti-Golgi 20.3, which recognized specifically the Golgi complex by fluorescence and electron microscopy. By immunoblotting this antibody recognized two proteins with 60 and 66 kDa that were identified as putative beta-tubulin and adenosine triphosphatase, respectively. The mAb 20.3 also recognized the Golgi complex of the Trichomonas vaginalis, a human parasite. In addition, the nucleotide coding sequences of these proteins were identified and included in the T. foetus database, and the 3D structure of the proteins was predicted. In conclusion, this study indicated: (1) adenosine triphosphatase is present in the Golgi, (2) ATPase is conserved between T. foetus and T. vaginalis, (3) there is new information concerning the nucleic acid sequences and protein structures of adenosine triphosphatase and beta-tubulin from T. foetus and (4) the mAb anti-Golgi 20.3 is a good Golgi marker and can be used in future studies.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Aparato de Golgi/ultraestructura , Infecciones Protozoarias en Animales/parasitología , Tritrichomonas foetus/ultraestructura , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Bovinos , Femenino , Aparato de Golgi/química , Aparato de Golgi/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión/veterinaria , Microscopía Fluorescente/veterinaria , Modelos Moleculares , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Alineación de Secuencia/veterinaria , Análisis de Secuencia de ADN/veterinaria , Trichomonas vaginalis/enzimología , Trichomonas vaginalis/inmunología , Tritrichomonas foetus/enzimología , Tritrichomonas foetus/genética , Tritrichomonas foetus/inmunologíaRESUMEN
Deletion of SIT4 phosphatase decreased the pyruvate decarboxylase activity, which is essential for directing the glucose flux to ethanol production. Concomitantly, a reduction in the fermentative capacity was observed. As pyruvate decarboxylase expression was not altered, its post-translational phosphorylation was studied. Immunoblot analyses using anti-phosphoserine antibodies against the affinity-purified Pdc1p showed that Pdc1p is a phosphoenzyme. Dephosphorylation of Pdc1p by alkaline phosphatase inhibited activity by 50%. Moreover, phosphorylation of Pdc1p was dependent on the growth phase, being hyperphosphorylated in the logarithmic phase, which showed to be dependent on the presence of SIT4. A comparison of the kinetic parameters of pyruvate decarboxylase in total protein extracts from WT yeast and the Δsit4 mutant revealed that the apparent K(m) values of the cofactor thiamin pyrophosphate (TPP) were 81 and 205 µM, respectively, with V(max) values of 0.294 and 0.173 µmol mg⻹ min⻹, respectively. Treatment of the purified enzyme with alkaline phosphatase increased the K(m) for TPP from 20 to 84 µM and for pyruvate from 2.3 to 4.6 mM, while the V(max) changed from 0.806 to 0.673 µmol mg⻹ min⻹. These results suggest that the Pdc1p phosphorylation dependent on SIT4 occurs at residues that change the apparent affinity for TPP and pyruvate.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Proteína Fosfatasa 2/metabolismo , Piruvato Descarboxilasa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Coenzimas/metabolismo , Eliminación de Gen , Cinética , Fosforilación , Unión Proteica , Ácido Pirúvico/metabolismo , Saccharomyces cerevisiae/genética , Tiamina Pirofosfato/metabolismoRESUMEN
Microorganisms use specialized systems to export virulence factors into host cells. Secretion of effector proteins into the extracellular environment has been described in Trypanosoma cruzi; however, a comprehensive proteomic analysis of the secretome and the secretion mechanisms involved remain elusive. Here, we present evidence that T. cruzi releases proteins associated with vesicles that are formed by at least two different mechanisms. Transmission electron microscopy showed larger vesicles budding from the plasma membrane of noninfective epimastigotes and infective metacyclic trypomastigotes, as well as smaller vesicles within the flagellar pocket of both forms. Parasite conditioned culture supernatant was fractionated and characterized by morphological, immunochemical, and proteomic analyses. Three fractions were obtained by differential ultracentrifugation: the first enriched in larger vesicles resembling ectosomes, the second enriched in smaller vesicles resembling exosomes, and a third fraction enriched in soluble proteins not associated with extracellular vesicles. Label-free quantitative proteomic analysis revealed a rich collection of proteins involved in metabolism, signaling, nucleic acid binding, and parasite survival and virulence. These findings support the notion that T. cruzi uses different secretion pathways to excrete/secrete proteins. Moreover, our results suggest that metacyclic forms may use extracellular vesicles to deliver cargo into host cells.
Asunto(s)
Membrana Celular/metabolismo , Flagelos/metabolismo , Estadios del Ciclo de Vida/fisiología , Proteínas Protozoarias/aislamiento & purificación , Trypanosoma cruzi/fisiología , Factores de Virulencia/aislamiento & purificación , Animales , Transporte Biológico , Células Cultivadas , Cromatografía Liquida , Medios de Cultivo Condicionados/química , Ratones , Microscopía Electrónica de Transmisión , Proteómica , Proteínas Protozoarias/metabolismo , Vesículas Secretoras , Espectrometría de Masas en Tándem , UltracentrifugaciónRESUMEN
Virus diseases are significant threats to modern agriculture and their control remains a challenge to the management of cultivation. The main virus resistance strategies are based on either natural resistance or engineered virus-resistant plants. Recent progress in understanding the molecular mechanisms underlying the roles of resistance genes has promoted the development of new anti-virus strategies. Engineered plants, in particular plants expressing RNA-silencing nucleotides, are becoming increasingly important and are likely to provide more effective strategies in future. A general discussion on the biotechnology of plant responses to virus infection is followed by recent advances in engineered plant resistance.
As viroses são problemas importantes para a agricultura moderna e o seu controle representa um desafio para o manejo de áreas cultivadas. As principais estratégias de resistência a vírus se baseiam em mecanismos naturais ou em engenharia genética. Recentemente, a maior compreensão dos mecanismos moleculares envolvidos na função de genes de resistência da planta facilitou o desenvolvimento de novas estratégias antivirais. Plantas modificadas geneticamente, em particular aquelas expressando a via de silenciamento de RNA, são alvo de interesse crescente e representam a possibilidade de estratégias futuras mais efetivas. Neste trabalho são discutidos diferentes aspectos relacionados à resistência a viroses em plantas. Adicionalmente, a perspectiva de aplicação biotecnológica das diferentes vias de resistência é apresentada.
RESUMEN
INTRODUCTION: A variety of sample preparation protocols for plant proteomic analysis using two-dimensional gel electrophoresis (2-DE) have been reported. However, they usually have to be adapted and further optimised for the analysis of plant species not previously studied. OBJECTIVE: This work aimed to evaluate different sample preparation protocols for analysing Carica papaya L. leaf proteins through 2-DE. METHODOLOGY: Four sample preparation methods were tested: (1) phenol extraction and methanol-ammonium acetate precipitation; (2) no precipitation fractionation; and the traditional trichloroacetic acid-acetone precipitation either (3) with or (4) without protein fractionation. The samples were analysed for their compatibility with SDS-PAGE (1-DE) and 2-DE. Fifteen selected protein spots were trypsinised and analysed by matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS), followed by a protein search using the NCBInr database to accurately identify all proteins. RESULTS: Methods number 3 and 4 resulted in large quantities of protein with good 1-DE separation and were chosen for 2-DE analysis. However, only the TCA method without fractionation (no. 4) proved to be useful. Spot number and resolution advances were achieved, which included having an additional solubilisation step in the conventional TCA method. Moreover, most of the theoretical and experimental protein molecular weight and pI data had similar values, suggesting good focusing and, most importantly, limited protein degradation. CONCLUSION: The described sample preparation method allows the proteomic analysis of papaya leaves by 2-DE and mass spectrometry (MALDI-TOF-MS/MS). The methods presented can be a starting point for the optimisation of sample preparation protocols for other plant species.