Estudio del sistema somatostatinérgico cerebral de la rata con encefalomielitis autoinmune experimental

Puebla Jiménez, Lilian, codir.La esclerosis múltiple (EM) es una enfermedad discapacitante que afecta a la vaina de mielina del sistema nervioso. Dicha enfermedad tiene un origen autoinmune y cursa con procesos neuroinflamatorios. Su modelo experimental, la encefalomielitis autoinmune experimental (EAE), imita varias de sus características, como son la sobreproducción de citoquinas proinflamatorias o la muerte neuronal. Tanto los pacientes con EM como los animales con EAE presentan alteraciones motoras y cognitivas. Dado que la somatostatina (SRIF) tiene implicación en la regulación de ambos tipos de procesos, nos planteamos averiguar si la EAE producía algún tipo de alteración en el sistema receptor-efector de la SRIF. Utilizando ratas Lewis hembra, se indujo EAE aguda mediante la inyección de una emulsión de proteína básica de mielina (PBM) en las almohadillas de cada una de las patas posteriores del animal. El análisis de la respuesta humoral del sistema inmune en las ratas inducidas muestra un aumento de los niveles de anticuerpos frente a la PBM, de interferón [gamma] (IFN[gamma]) y de factor de necrosis tumoral [alfa] (TNF[alfa]). En el sistema somatostatinérgico del hipocampo y el estriado de ratas enfermas se encontró que la EAE aguda produce un descenso de la densidad de receptores de SRIF sin afectar a su constante de disociación (Kd) aparente. Dicho descenso se debe, al menos en parte, a una disminución de los niveles proteicos del receptor de SRIF subtipo 2 (sst2). Este descenso está provocado por la disminución en la tasa de transcripción de su ARNm. Dicha disminución podría estar provocada por el aumento de los niveles de TNF[alfa] observados en el estudio. Además, la reducción de los niveles de sst2 conduce a una disminución de la capacidad de la SRIF para inhibir la actividad adenilato ciclasa (AC).Puesto que las experiencias del doctor Nieper realizadas en los años 60 con la sal de calcio, magnesio y potasio de fosfato de etanolamina (PEA) en enfermos de EM sugieren que dicha molécula podría tener algún efecto terapéutico, nos propusimos comprobar si el pretratamiento con PEA, administrado dos días antes de la inducción del modelo y continuado hasta el día del sacrificio es capaz de prevenir las alteraciones del sistema somatostatinérgico provocadas por la EAE aguda. Nuestros resultados demuestran que el PEA es capaz de prevenir tanto las alteraciones inmunológicas como las del sistema somatostatinérgico observadas en este estudio. Este hecho sugiere la realización de experimentos adicionales que confirmen el potencial terapéutico del PEA en el tratamiento de la EM

The CBS domain protein MJ0729 of Methanocaldococcus jannaschii binds DNA

AbstractThe cystathionine beta-synthase (CBS) domains function as regulatory motifs in several proteins. Elucidating how CBS domains exactly work is relevant because several genetic human diseases have been associated with mutations in those motifs. Here, we show, for the first time, that a CBS domain binds calf-thymus DNA and E-boxes recognized by transcription factors. We have carried out the DNA-binding characterization of the CBS domain protein MJ0729 from Methanocaldococcus jannaschii by biochemical and spectroscopic techniques. Binding induces conformational changes in the protein, and involves the sole tryptophan residue. The apparent dissociation constant for the E-boxes is ∼10μM. These results suggest that CBS domains might interact with DNA

The Conformational Stability and Biophysical Properties of the Eukaryotic Thioredoxins of Pisum Sativum Are Not Family-Conserved

Thioredoxins (TRXs) are ubiquitous proteins involved in redox processes. About forty genes encode TRX or TRX-related proteins in plants, grouped in different families according to their subcellular localization. For instance, the h-type TRXs are located in cytoplasm or mitochondria, whereas f-type TRXs have a plastidial origin, although both types of proteins have an eukaryotic origin as opposed to other TRXs. Herein, we study the conformational and the biophysical features of TRXh1, TRXh2 and TRXf from Pisum sativum. The modelled structures of the three proteins show the well-known TRX fold. While sharing similar pH-denaturations features, the chemical and thermal stabilities are different, being PsTRXh1 (Pisum sativum thioredoxin h1) the most stable isoform; moreover, the three proteins follow a three-state denaturation model, during the chemical-denaturations. These differences in the thermal- and chemical-denaturations result from changes, in a broad sense, of the several ASAs (accessible surface areas) of the proteins. Thus, although a strong relationship can be found between the primary amino acid sequence and the structure among TRXs, that between the residue sequence and the conformational stability and biophysical properties is not. We discuss how these differences in the biophysical properties of TRXs determine their unique functions in pea, and we show how residues involved in the biophysical features described (pH-titrations, dimerizations and chemical-denaturations) belong to regions involved in interaction with other proteins. Our results suggest that the sequence demands of protein-protein function are relatively rigid, with different protein-binding pockets (some in common) for each of the three proteins, but the demands of structure and conformational stability per se (as long as there is a maintained core), are less so

Stability and binding of the phosphorylated species of the N-terminal domain of enzyme I and the histidine phosphocarrier protein from the Streptomyces coelicolor phosphoenolpyruvate:sugar phosphotransferase system

The phosphotransferase system (PTS) is involved in the use of carbon sources in bacteria. It is formed by two general proteins: enzyme I (EI) and the histidine phosphocarrier (HPr), and various sugar-specific permeases. EI is formed by two domains, with the N-terminal domain (EIN) being responsible for the binding to HPr. In low-G + C Gram-positive bacteria, HPr becomes phosphorylated not only by phosphoenolpyruvate (PEP) at the active-site histidine, but also by ATP at a serine. In this work, we have characterized: (i) the stability and binding affinities between the active-site-histidine phosphorylated species of HPr and the EIN from Streptomyces coelicolor; and (ii) the stability and binding affinities of the species involving the phosphorylation at the regulatory serine of HPrsc. Our results show that the phosphorylated active-site species of both proteins are less stable than the unphosphorylated counterparts. Conversely, the Hpr-S47D, which mimics phosphorylation at the regulatory serine, is more stable than wild-type HPrsc due to helical N-capping effects, as suggested by the modeled structure of the protein. Binding among the phosphorylated and unphosphorylated species is always entropically driven, but the affinity and the enthalpy vary widely

Insights into the mechanism of activation of the phosphorylation-independent response regulator NblR. Role of residues Cys69 and Cys96

Cyanobacteria respond to environmental stress conditions by adjusting their photosynthesis machinery. In Synechococcus sp. PCC 7942, phycobilisome degradation and other acclimation responses after nutrient or high light stress require activation by the phosphorylation-independent response regulator NblR. Structural modelling of its receiver domain suggested a role for Cys69 and Cys96 on activation of NblR. Here, we investigate this hypothesis by engineering Cys to Ala substitutions. In vivo and in vitro analyses indicated that mutations Cys69Ala and/or Cys96Ala have a minor impact on NblR function, structure, size, or oligomerization state of the protein, and that Cys69 and Cys96 do not seem to form disulphide bridges. Our results argue against the predicted involvement of Cys69 and Cys96 on NblR activation by redox sensing.This work was supported by the Spanish Ministerio de Ciencia e Innovación (grants BFU2009-07371 to A.C., BIO2009-10872 and BIO2010-15424 to A.M. and SAF2008-05742-C02-01 and CSD2008-00005 to J.L.N.) and the Generalitat Valenciana (grants ACOMP2006/083 and ACOMP2011/211 to A.C., ACOMP2010/114 and ACOMP2011/113 to J.L.N.). M.L. López-Redondo was a fellow of the Fundación Mutua Madrileña Automovilística

Non-canonical residues of the marginally stable monomeric ubiquitin conjugase from goldfish are involved in binding to the C terminus of Ring 1B

E2 ubiquitin conjugases are ∼ 20 kDa enzymes involved in ubiquitination processes in eukaryotes. The E2s are responsible for the transference of ubiquitin (Ub) to E3 enzymes, which finally transfer Ub to diverse target proteins, labelling them for degradation, localization and regulation. Although their functions are relatively well-characterized, their conformational stabilities are poorly known. In this work, we have used, as a model for our biophysical and binding studies, the E2-C from Carassius auratus (goldfish), a homologue of the human ubiquitin conjugase UbcH10. E2-C ca was a monomeric protein with an elongated shape; moreover, the protein was only marginally stable within a narrow pH range (from 6.0 to 8.0). We also explored the binding of E2-C ca towards non-canonical E3 ligases. Binding of E2-C ca to the C terminus of murine Ring 1B (C-Ring1B), which does not contain the RING finger of the whole Ring1B, occurred with an affinity of ∼ 400 nM, as shown by fluorescence and ITC. Furthermore, binding of E2-C ca to C-Ring1B did not occur at its canonical E2-loops, since residues M43 and F53, far away from those loops, were involved in binding. Thus, the C-Ring1B-interacting region of E2-C ca comprises the first β-strand and nearby residues.We deeply thank Prof. T. Tokumoto the kind gift of the E2-Cca clone. We thank the three referees for discussions, ideas and helpful suggestions, which have improved this manuscript. We thank May García, María del Carmen Fuster and Javier Casanova for technical assistance. This work was supported by the Spanish Ministerio de Ciencia e Innovación (MCINN) (CTQ2011-24393, CSD2008-00005 to JLN), and an intramural BIFI grant. The authors declare they do not have any competing interest.Peer Reviewe

Effects of single and continuous administration of amyloid ß-peptide (25-35) on adenylyl cyclase activity and the somatostatinergic system in the rat frontal and parietal cortex

It is unknown whether the amyloid β-peptide (Aβ), a principal component found in extracellular neuritic plaques in the brain of patients with Alzheimer’s disease (AD), is capable of altering adenylyl cyclase (AC) activity and the somatostatin (SRIF) receptor-effector system in the cerebral cortex of the patients. Therefore, the objective of this study was to investigate the effect of the β fragment, β (25–35), on AC activity and the somatostatinergic system in the rat frontoparietal cortex. A single dose of β (25–35) (10μg) injected intracerebroventricularly significantly decreased the density of SRIF receptors (27.4%) and increased their affinity (32.2%) in the frontoparietal cortex. The inhibitory effect of SRIF on basal and forskolin (FK)-stimulated AC activity was significantly lower in the β (25–35)-treated rats when compared with controls. β (25–35) did not modify Giα1, Giα2 nor Giα3 levels in membranes from the frontoparietal cortex. Continuous infusion of the peptide induced a decrease in the SRIF receptor density in this brain area to a similar extent as that observed 14 days after the single administration of the peptide. Likewise, this treatment decreased the SRIF receptor density in the frontal cortex (15.3%) and parietal cortex (27.2%). This effect was accompanied by a decrease in the SRIF-mediated inhibition of FK-stimulated AC activity (from 41.6% to 25.6%) in the frontal cortex as well by a decrease in basal AC activity (from 36.9% to 31.6%) and FK-stimulated AC activity (from 35.6% to 27.1%) in the parietal cortex. Continuous infusion of Aβ (25–35) had no effect on Giα1, Giα2 or Giα3 levels in membranes from frontal and parietal cortex. However, this treatment caused a decrease in SRIF-like immunoreactivity content in the parietal (38.9%) and frontal (20.4%) cortex. These results suggest that Aβ might be involved in the alterations of somatostatinergic system reported in AD

Gly-Pro-Glu protects β-amyloid-induced somatostatin depletion in the rat cortex

The effect of Gly-Pro-Glu (GPE) on the somatostatinergic system of the temporal cortex in amyloid β-peptide (Aβ) treated rats was investigated. Intracerebroventricular Aβ25-35 administration for 14 days (300 pmol/day) to ovariectomized rats produced a marked reduction in somatostatin (SRIF) content, SRIF receptor density and reduced the inhibitory effect of SRIF on adenylyl cyclase activity. I.p. injection of three doses (300 μg) of GPE on days 0, 6 and 12 resulted in a partial recovery of the parameters affected by Aβ25-35 administration. These results indicate that GPE may have an in vivo effect protecting the temporal cortical somatostatinergic system from Aβ insult

Study of Comminution Kinetics in an Electrofragmentation Lab-Scale Device

A significant challenge in mineral raw materials comminution is the improvement of process energy efficiency. Conventional comminution techniques, although globally used, are far from being considered power-efficient. The use of high-voltage electric pulses in comminution is a concept that is worthy of study; despite its lack of industrial-scale validation after several decades of lab-scale research, it seems promising as a pretreatment leading to energy savings. In this article, the Cumulative Kinetic Model methodology is adapted to model the comminution effect in an electrofragmentation device, and study a dunite rock ore. The results show that product particle size distribution (PSD) can be predicted with reasonable accuracy using the proposed model

The isolated N terminus of Ring1B is a well-folded, monomeric fragment with native-like structure

The Polycomb group (PcG) proteins assemble into Polycomb repressive complexes (PRCs), PRC1 and PRC2, which act as general transcriptional repressors. PRC1 comprises a variety of biochemical entities endowed with histone H2A monoubiquitylation activity conferred by really interesting new gene (RING) finger E3 ubiquitin ligases Ring1A and Ring1B. All PRC1 complexes contain Ring1 proteins which are essential for Polycomb epigenetic regulation. We have been able to express the isolated N-terminal region of Ring1B, N-Ring1B, comprising the first 221 residues of the 334-residue-long Ring1B. This fragment contains the 41-residue-long RING finger motif, and flanking sequences that form an interacting platform for PcG and non-PcG proteins. We found that the N-Ring1B is a well-folded, monomeric fragment, with native-like structure which unfolds irreversibly. The protein is capable of binding to an ubiquitin-conjugase protein (with an 85% of sequence similarity to the Ring1B physiological partner) with moderate affinity. © The Author 2013.Peer Reviewe