Análisis proteómico de las vías de señalización mediadas por la subunidad Gα Pga1 de una proteína G heterotrimérica de Penicillium chrysogenum

The heterotrimeric Gα protein Pga1-mediated signaling pathway regulates the entire developmental program in Penicillium chrysogenum, from spore germination to the formation of conidia. In addition it participates in the regulation of penicillin biosynthesis. We aimed to advance the understanding of this key signaling pathway using a proteomics approach, a powerful tool to identify effectors participating in signal transduction pathways. P. chrysogenum mutants with different levels of activity of the Pga1-mediated signaling pathway were used to perform comparative proteomic analyses by 2DDIGE and LC–MS/MS. Thirty proteins were identified which showed differences in abundance dependent on Pga1 activity level. By modifying the intracellular levels of cAMP we could establish cAMP-dependent and cAMP-independent pathways in Pga1-mediated signaling. Pga1 was shown to regulate abundance of enzymes in primary metabolic pathways involved in ATP, NADPH and cysteine biosynthesis, compounds that are needed for high levels of penicillin production. An in vivo phosphorylated protein containing a pleckstrin homology domain was identified; this protein is a candidate for signal transduction activity. Proteins with possible roles in purine metabolism, protein folding, stress response and morphogenesis were also identified whose abundance was regulated by Pga1 signaling. For phosphoproteomic analyses, two different methodologies were used: 2D-DIGE with phosphoprotein-enriched extracts followed by identification of individual puntos by LC-MS/MS, and a "Label-Free" phosphoproteomics approach with phosphopeptide-enriched extracts (using TiO2 affinity chromatography) submitted to LC-MS/MS. The 2D-DIGE analysis showed changes in abundance of phosphorylated proteins when comparing the strains, but little information on differential phosphorylation. 383 phosphorylated proteins were identified from 8343 total spectra, the Label-Free phosphoproteomic analysis showed changes in the phosphorylation patterns of proteins between strains, which allowed to identify new effectors of the Pga1 signaling pathway. Fifteen probable transcription factors and chromatin remodeling proteins were identified, one of these proteins was 8 Pc16g10400 (protein with a Bromo Adjacent Homology domain and a PHD zinc finger domain), with two phosphorylation sites at S-738 and S-742, of which the S742 site is present only in the Δpga1 strain.La vía de señalización mediada por la subunidad Gα Pga1 de una proteína G heterotrimérica regula el desarrollo en Penicillium chrysogenum, desde la germinación de esporas hasta la formación de conidios. Además participa en la regulación de la biosíntesis de penicilina. Nuestro objetivo fue avanzar en la comprensión de esta vía clave de señalización utilizando un enfoque proteómico, que resulta en una poderosa herramienta para identificar nuevos efectores que participan en las vías de transducción de señales. Para realizar los análisis proteómicos comparativos mediante 2D-DIGE y LCMS/MS se utilizaron mutantes de P. chrysogenum con diferentes niveles de actividad de la vía de señalización mediada por Gα Pga1. Se identificaron treinta proteínas que mostraron cambios significativos en la abundancia de una forma dependiente de la actividad de Gα Pga1. Mediante la modificación de los niveles intracelulares de AMPc logramos establecer vías AMPc-dependiente y AMPcindependiente de Pga1. Se demostró que Gα Pga1 regula la abundancia de enzimas de las vías metabólicas primarias implicadas en la biosíntesis de ATP, NADPH y cisteína, compuestos que son necesarios para la biosíntesis de penicilina. Se identificó una proteína fosforilada in vivo que contiene un dominio Pleckstrin (PH); Esta proteína es candidata para formar parte de vías de transducción de señales. También se identificaron proteínas con posibles funciones en el metabolismo de purinas, plegamiento de proteínas, respuesta al estrés y morfogénesis, cuya abundancia fue regulada por la señalización Gα Pga1. Para los análisis fosfoproteómicos, se utilizaron dos metodologías diferentes: 2DDIGE con extractos enriquecidos de fosfoproteínas seguido de la identificación de los spots (puntos) individuales por LC-MS/MS, y un enfoque fosfoproteómico mediante la metodología "Label free" con extractos enriquecidos de fosfopéptidos (usando cromatografía de afinidad con TiO2) y LC-MS/MS. El análisis 2D-DIGE mostró cambios en la abundancia de proteínas fosforiladas, sin embrago se obtuvo poca información sobre la fosforilación diferencial. En el análisis de "Label free" se identificaron 383 proteínas fosforiladas a partir de 8343 espectros totales, además se logró identificar cambios en los patrones de fosforilación de proteínas entre las 6 condiciones, lo que permitió identificar nuevos efectores de la vía de señalización mediada por Gα Pga1. Se identificaron probables factores de transcripción y proteínas fosforiladas de remodelación de la cromatina, una de estas proteínas fue Pc16g10400 (proteína con un dominio Bromo y tres dominios PHD), con dos sitios de fosforilación en S-738 y S-742, sin embargo la fosforilación en S-742 está presente sólo en la cepa Δpga1

Producción y purificación de β-N-acetilhexosaminidasa en cultivo sumergido de Lecanicillium lecanii inmovilizado en espuma de poliuretano

El objetivo del presente trabajo fue producir y purificar la enzima Nacetilhexosaminidasa (Nhasa) a partir de un cultivo sumergido del hongo Lecanicillium lecanii inmovilizado en espuma de poliuretano, para lo cual se utilizaron las condiciones reportadas por Carrasco y col. (2009) y se probaron dos protocolos de purificación. Inicialmente, se llevó a cabo la cinética de producción Nhasa, para lo que se utilizaron, como inóculo, biopartículas (hongo inmovilizado) en un biorreactor y como control se utilizaron esporas libres. Se encontró que la actividad volumétrica al utilizar biopartículas fue cuatro veces mayor (48.82±6.14 U/L), con respecto al control (11.2±0.26 U/L). De igual modo la mayor actividad específica se obtuvo al utilizar como inóculo las biopartículas obteniéndose 0.25±0.07 U/mg de proteína, mientras que, al inocular el reactor con esporas, solo fue de 0.028±0.0007 U/mg de proteína. En cuanto a la actividad Nhasa/g de sustratos sólidos iniciales (SSI), la mayor actividad se obtuvo en el cultivo con las biopartículas (2.73±0.34 U/gSSI) a diferencia del cultivo inoculado con esporas (0.62±0.014 U/gSSI). Posteriormente, se diseño una estrategia de purificación de una Nhasa a partir del extracto crudo enzimático obtenido del cultivo sumergido inoculado con biopartículas. Para tal fin, se precipitaron las proteínas con 40% de saturación con sulfato de amonio, obteniendo una actividad específica de 4.22±0.6 U/mg de proteína, el sobrenadante se llevo hasta 80% de saturación, obteniéndose en este segundo precipitado una actividad específica de 2±0.18 U/mg de proteína. Los precipitados de 40 y 80% fueron inyectados en una columna de exclusión molecular (Sephacryl™ S-100 High Resolution), separándose fracciones con actividades específicas de 7.92 U/mg y de 0.44 U/mg con factores de purificación de 2.55 y 0.14 para los precipitados al 40% y 80% de El objetivo del presente trabajo fue producir y purificar la enzima Nacetilhexosaminidasa (Nhasa) a partir de un cultivo sumergido del hongo Lecanicillium lecanii inmovilizado en espuma de poliuretano, para lo cual se utilizaron las condiciones reportadas por Carrasco y col. (2009) y se probaron dos protocolos de purificación. Inicialmente, se llevó a cabo la cinética de producción Nhasa, para lo que se utilizaron, como inóculo, biopartículas (hongo inmovilizado) en un biorreactor y como control se utilizaron esporas libres. Se encontró que la actividad volumétrica al utilizar biopartículas fue cuatro veces mayor (48.82±6.14 U/L), con respecto al control (11.2±0.26 U/L). De igual modo la mayor actividad específica se obtuvo al utilizar como inóculo las biopartículas obteniéndose 0.25±0.07 U/mg de proteína, mientras que, al inocular el reactor con esporas, solo fue de 0.028±0.0007 U/mg de proteína. En cuanto a la actividad Nhasa/g de sustratos sólidos iniciales (SSI), la mayor actividad se obtuvo en el cultivo con las biopartículas (2.73±0.34 U/gSSI) a diferencia del cultivo inoculado con esporas (0.62±0.014 U/gSSI). Posteriormente, se diseño una estrategia de purificación de una Nhasa a partir del extracto crudo enzimático obtenido del cultivo sumergido inoculado con biopartículas. Para tal fin, se precipitaron las proteínas con 40% de saturación con sulfato de amonio, obteniendo una actividad específica de 4.22±0.6 U/mg de proteína, el sobrenadante se llevo hasta 80% de saturación, obteniéndose en este segundo precipitado una actividad específica de 2±0.18 U/mg de proteína. Los precipitados de 40 y 80% fueron inyectados en una columna de exclusión molecular (Sephacryl™ S-100 High Resolution), separándose fracciones con actividades específicas de 7.92 U/mg y de 0.44 U/mg con factores de purificación de 2.55 y 0.14 para los precipitados al 40% y 80% de 6The aim of this study was the production in submerged culture of the enzyme Nacetylhexosaminidase (Nhase) of the entomopathogenic fungus Lecanicillium lecanii immobilized in polyurethane foam and its purification. In a previous work, the conditions of immobilization and production were established (Carrasco et al., 2009), tested two purification protocols Initially kinetics of Nhase activity were carried out in a bioreactor using bioparticles and spores (free cells) as inocula, the former yields a maximum volumetric activity of 48.82± 6.14 U/L, whereas the latter was 11.2 ± 0.26 U/L i.e. more than fourfold the activity with bioparticules. The highest specific activity was also found when the bioparticles was used as inoculum (0.25±0.07 U/mg protein). Indeed, the highest Nhase/g of initial solid substrates (SSI) activity was determined in the culture using bioparticles (2.73 ± 0.34 U/gSSI), compared with the culture inoculated with spores (0.62 ± 0.014 U/gSSI). Purification of Nhase from crude enzymatic extract of submerged culture with bioparticles was carried out. Proteins were salted out at 40% saturation with ammonium sulfate obtaining a specific activity of 4.22 ± 0.6 U/mg, the supernatant was brought to 80% saturation with ammonium sulfate (2±0.18 U/mg). Each of these fractions were injected into a size exclusion chromatography column (Sephacryl™ S-100 High Resolution), obtaining specific activities of 7.92 U/mg and 0.44 U/mg with purification factor of 2.55 and 0.14 for the fraction precipitated at 40 and 80% saturation, respectively. Subsequently, the fractions with activity were subjected to ion exchange chromatography (Macro-Prep High Q support), nevertheless there was not a good separation and above all the enzymatic activity was absent. Based on the above, the purification protocol was modified, Nhase activities were determined as 88.2±10.78 Nhase U/mg protein, 16.58±2.26 U/mg The aim of this study was the production in submerged culture of the enzyme Nacetylhexosaminidase (Nhase) of the entomopathogenic fungus Lecanicillium lecanii immobilized in polyurethane foam and its purification. In a previous work, the conditions of immobilization and production were established (Carrasco et al., 2009), tested two purification protocols Initially kinetics of Nhase activity were carried out in a bioreactor using bioparticles and spores (free cells) as inocula, the former yields a maximum volumetric activity of 48.82± 6.14 U/L, whereas the latter was 11.2 ± 0.26 U/L i.e. more than fourfold the activity with bioparticules. The highest specific activity was also found when the bioparticles was used as inoculum (0.25±0.07 U/mg protein). Indeed, the highest Nhase/g of initial solid substrates (SSI) activity was determined in the culture using bioparticles (2.73 ± 0.34 U/gSSI), compared with the culture inoculated with spores (0.62 ± 0.014 U/gSSI). Purification of Nhase from crude enzymatic extract of submerged culture with bioparticles was carried out. Proteins were salted out at 40% saturation with ammonium sulfate obtaining a specific activity of 4.22 ± 0.6 U/mg, the supernatant was brought to 80% saturation with ammonium sulfate (2±0.18 U/mg). Each of these fractions were injected into a size exclusion chromatography column (Sephacryl™ S-100 High Resolution), obtaining specific activities of 7.92 U/mg and 0.44 U/mg with purification factor of 2.55 and 0.14 for the fraction precipitated at 40 and 80% saturation, respectively. Subsequently, the fractions with activity were subjected to ion exchange chromatography (Macro-Prep High Q support), nevertheless there was not a good separation and above all the enzymatic activity was absent. Based on the above, the purification protocol was modified, Nhase activities were determined as 88.2±10.78 Nhase U/mg protein, 16.58±2.26 U/mg nd 1.69±0.3 U/mg for precipitates of 40, 60 and 80% of saturation with ammonium sulfate, respectively. The accumulated enzyme activity in the precipitates with ammonium sulfate among 60 and 80% did not show significant differences, thus for the further work it was employed 60% saturation. Precipitated fraction was dissolved in Tris-HCl 50mM, NaCl 0.15M buffer pH 7.8 and ultrafiltrate (molecular weight cut off 10 KDa). The retentate was mixed up with 4% (v/v) Triton X-100 and 10% (v/v) acetonitrile and injected into size exclusion chromatography column (Sephacryl™ S-100 High Resolution). The fractions with activity were 10 (0.88 U/mg U/mg protein) and 11 (1.98 U/mg U/mg), with a purification factor of 72.12 and 162.16, respectively. Fractions 10 and 11 were injected in an ion exchange column (DEAE-Sepharose fast-flow). Fractions with Nhase activity were subjected to SDS-PAGE, showing only two bands between 50-75 KDa for fractions 20-30, fractions 31-33 observed a single band for the fraction 10 and also a single band in fractions 30-36 for the fraction 11. The purification of Nhase from a crude enzymatic extract of the immobilized Lecanicilliun lecanii was achieved after only three steps of purification. The purified Nhase is most active at pH 40°C and 6.

NeVOmics: An Enrichment Tool for Gene Ontology and Functional Network Analysis and Visualization of Data from OMICs Technologies

The increasing number of OMICs studies demands bioinformatic tools that aid in the analysis of large sets of genes or proteins to understand their roles in the cell and establish functional networks and pathways. In the last decade, over-representation or enrichment tools have played a successful role in the functional analysis of large gene/protein lists, which is evidenced by thousands of publications citing these tools. However, in most cases the results of these analyses are long lists of biological terms associated to proteins that are difficult to digest and interpret. Here we present NeVOmics, Network-based Visualization for Omics, a functional enrichment analysis tool that identifies statistically over-represented biological terms within a given gene/protein set. This tool provides a hypergeometric distribution test to calculate significantly enriched biological terms, and facilitates analysis on cluster distribution and relationship of proteins to processes and pathways. NeVOmics is adapted to use updated information from the two main annotation databases: Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG). NeVOmics compares favorably to other Gene Ontology and enrichment tools regarding coverage in the identification of biological terms. NeVOmics can also build different network-based graphical representations from the enrichment results, which makes it an integrative tool that greatly facilitates interpretation of results obtained by OMICs approaches. NeVOmics is freely accessible at https://github.com/bioinfproject/bioinfo/

Ribosomes: The New Role of Ribosomal Proteins as Natural Antimicrobials

Moonlighting proteins are those capable of performing more than one biochemical or biophysical function within the same polypeptide chain. They have been a recent focus of research due to their potential applications in the health, pharmacological, and nutritional sciences. Among them, some ribosomal proteins involved in assembly and protein translation have also shown other functionalities, including inhibiting infectious bacteria, viruses, parasites, fungi, and tumor cells. Therefore, they may be considered antimicrobial peptides (AMPs). However, information regarding the mechanism of action of ribosomal proteins as AMPs is not yet fully understood. Researchers have suggested that the antimicrobial activity of ribosomal proteins may be associated with an increase in intracellular reactive oxidative species (ROS) in target cells, which, in turn, could affect membrane integrity and cause their inactivation and death. Moreover, the global overuse of antibiotics has resulted in an increase in pathogenic bacteria resistant to common antibiotics. Therefore, AMPs such as ribosomal proteins may have potential applications in the pharmaceutical and food industries in the place of antibiotics. This article provides an overview of the potential roles of ribosomes and AMP ribosomal proteins in conjunction with their potential applications

Probiotic Properties and Proteomic Analysis of <i>Pediococcus pentosaceus</i> 1101

Pediococcus pentosaceus 1101 was identified by using 16S rRNA and MALDI-Biotyper. The strain was exposed to conditions that resemble the gastrointestinal tract (GT) to evaluate its probiotic properties. That included the growth kinetics, proteolytic and inhibitory activities within a pH range, survival at low pH and in the presence of bile salts, antagonistic activity, cell-adhesion properties, and antibiotic resistance. The evaluation was followed by a genomic and proteomic analysis that involved the identification of proteins obtained under control and gastrointestinal conditions. The strain showed antagonistic activity against Gram-negative and Gram-positive bacteria, high resistance to acidity (87% logarithmic survival rate, pH 2) and bile salts (99% logarithmic survival rate, 0.5% w/v), and hydrophobic binding, as well as sensitivity to penicillin, amoxicillin, and chloramphenicol. On the other hand, P. pentosaceus 1101 has a genome size of 1.76 Mbp, with 1754 coding sequences, 55 rRNAs, and 33 tRNAs. The proteomic analysis showed that 120 proteins were involved in mechanisms in which the strain senses the effects of acid and bile salts. Moreover, the strain produces at least one lytic enzyme (N-acetylmuramoyl-L-alanine amidase; 32 kDa) that may be related to the antimicrobial activity. Therefore, proteins identified might be a key factor when it comes to the adaptation of P. pentosaceus 1101 into the GT and associated with its technological and probiotic properties

Overview of recent TJ-II stellarator results

The main results obtained in the TJ-II stellarator in the last two years are reported. The most important topics investigated have been modelling and validation of impurity transport, validation of gyrokinetic simulations, turbulence characterisation, effect of magnetic configuration on transport, fuelling with pellet injection, fast particles and liquid metal plasma facing components. As regards impurity transport research, a number of working lines exploring several recently discovered effects have been developed: the effect of tangential drifts on stellarator neoclassical transport, the impurity flux driven by electric fields tangent to magnetic surfaces and attempts of experimental validation with Doppler reflectometry of the variation of the radial electric field on the flux surface. Concerning gyrokinetic simulations, two validation activities have been performed, the comparison with measurements of zonal flow relaxation in pellet-induced fast transients and the comparison with experimental poloidal variation of fluctuations amplitude. The impact of radial electric fields on turbulence spreading in the edge and scrape-off layer has been also experimentally characterized using a 2D Langmuir probe array. Another remarkable piece of work has been the investigation of the radial propagation of small temperature perturbations using transfer entropy. Research on the physics and modelling of plasma core fuelling with pellet and tracer-encapsulated solid-pellet injection has produced also relevant results. Neutral beam injection driven Alfvénic activity and its possible control by electron cyclotron current drive has been examined as well in TJ-II. Finally, recent results on alternative plasma facing components based on liquid metals are also presented.ISSN:0029-5515ISSN:1741-432