Métodos espectroscópicos para el análisis de glicoproteínas

El estudio de glicoproteínas demanda el conocimiento de una variedad de aspectos estructurales que, en su conjunto, contribuyen a la descripción de su relación estructura–función. A partir de la búsqueda de propiedades estructurales de la cadena polipeptídica y sus glicanos conjugados, es posible inferir, por ejemplo, el impacto de la pérdida de grupos glicosídicos en la estabilidad de una glicoproteína, el cambio en su funcionalidad debido a mutaciones o incluso el efecto funcional de la unión de un ligando. Debido a esto, los estudios estructurales de glicoproteínas son un punto de partida habitual en el descubrimiento y desarrollo de fármacos contra virus, bacterias y otros agentes patógenos, así como en el diseño de nuevas proteínas terapéuticas para el tratamiento del cáncer y enfermedades autoinmunes. Los estudios estructurales toman una especial relevancia en la demostración de biosimilitud y el control de calidad de glicoproteínas terapéuticas producidas por biotecnología. En la demostración de biosimilitud es necesario realizar un ejercicio de comparabilidad exhaustivo que permita asegurar la similitud en la estructura de una glicoproteína biosimilar respecto de la glicoproteína de referencia. Este ejercicio de comparabilidad también debe realizarse luego de implementarse todo tipo de cambios en los procesos de manufactura de la glicoproteína. Es por esto que las herramientas que brindan información sobre la estructura de proteínas son fundamentales en el control de calidad de glicoproteínas terapéuticas, permiten detectar posibles alteraciones en la estabilidad y agregación del producto farmacéutico, ya sea por adición de excipientes o durante el tiempo de duración del producto...Fil: Masin, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Bertoncini, Carlos Walter. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Empresa Dominzguezlab S.r.l.; Argentin

Charakterisierung der Wechselwirkungen zwischen dem P2X₂ Rezeptor und dem Fe65 Adapterprotein im Rattengehirn

Die Erregungsüberstragung an chemischen Synapsen des Nervensystems beginnt mit der Freisetzung von Neurotransmittermolekülen aus präsynaptischen Nervenendigungen. Diese Transmittermoleküle aktivieren spezifische Rezeptoren in der postsynaptischen Membran. ATP is ein Neurotransmitter, der zur synaptischen Übertragung im zentralen und peripheren Nervensystem verwendet wird. Dabei bindet ATP an ionotrope Rezeptoren in der postsynaptischen Membran. Diese Rezeptoren nennt man P2X Rezeptoren. In Säugetieren existieren sieben verschiedene P2X Rezeptoruntereinheiten (P2X1 P2X7). Von diesen sind hauptsächlich die P2X2, P2X4 und P2X6 Untereinheiten im Gehirn exprimiert. Mittles goldmarkierter Antikörper konnten die Lokalisation dieser Rezeptoren im äussere Abschnitt der postsynaptischen Spezialisierung elektronenmikroskopisch dargestellt werden. Diese präzise Lokalisierung der P2X Rezeptoren legte die Vermutung nahe, dass P2X Rezeptoren, ähnlich anderern synaptischen Rezeptoren, mit intrazellulären regulatorischen Proteinen interagieren. Bislang jedoch wurden keine entsprechenden Proteine identifiziert.In der vorliegenden Arbeit wurde daher mittels der Hefe-Zwei-Hybrid Methode eine Rattenhirn cDNA Bibliothek mit dem Carboxyterminus der P2X2 Untereinheit nach potentiellen Interaktionspartnern durchsucht. So gelang es das Protein Fe65 als wichtigen neuronalen Interaktionspartner von P2X2 Rezeptoren zu identifizieren. Die Interaktion von Fe65 und P2X2 Untereinheiten wurde zunächst mit Hefe-Zwei-Hybrid Methoden und GST-pulldown Experimenten näher charakterisiert. Dabei zeigte sich, dass Fe65 spezifisch mit P2X2 nicht aber mit anderen P2X Untereinheiten (z.B. P2X4 und P2X7) oder mit einer natürlich vorkommenden Spleissvariant von P2X2 (P2X2(b)) interagiert. Neben Fe65 konnte auch für das verwandte Protein Fe65-like1 nachgewiesen werden, dass es an den P2X2 Carboxyterminus bindet. Durch Generierung von Deletionen und Punktmutationen in den Sequenzen beider Interaktionspartner konnte weiter gezeigt werden, dass die Bindung dieser Proteine zwischen der WW Domäne von Fe65 und einer prolinreichen Sequenz (PPPP) im Carboxyterminus der P2X2 Untereinheit stattfindet.Zwei Antikörper gegen Fe65 wurden generiert, charakterisiert und zur elektronenmikroskopischen Darstellung der subzellulären Lokalisierung von Fe65 eingesetzt. Fe65 wurde dabei in der prä- und postsynaptischen Membran der Synapsen von Schaffer Kollateralen und hippocampalen Pyramidenzellen nachgewiesen. Doppelte Immunogoldmarkierung zeigte ferner, dass P2X2 Untereinheiten und Fe65 im äusseren Abschnitt der postsynaptischen Spezialisierung kolokalisiert sind. Darüberhinaus gelang es beide Proteine gemeinsam aus Membranextrakten von Hirngewebe zu immunopräzipitieren. In der Zusammenschau lassen diese Ergebnisse den Schluss zu das Fe65 und P2X2 Untereinheit in vivo in postsynaptischen Membranen des Zentralnervensystems interagieren.Um zu untersuchen, ob die Bindung von Fe65 die Rezeptoreigenschaften von P2X2 reguliert, wurden elektrophysiologische Untersuchungen an HEK Zellen und Xenopus laevis Oozyten, die diese Proteine einzeln oder zusammen exprimierten, durchgeführt. Während P2X2 Rezeptoren mit Fe65 ähnliche pharmakoligsche Eigenschaften zeigten und Amplitude und Kinetik der Ströme vergleichbar den durch P2X2 Rezeptoren ohne Fe65 vermittelten waren, zeigten sich drastische Unterschiede bei der Permeabilität von P2X2 Rezeptoren mit und ohne Fe65. So wurde die zeit- und aktivierungsabhängige Änderung der Ionenselektivität von P2X2 Rezeptoren durch Assoziation mit Fe65 nahezu komplett unterdrückt. Dies legt die Schlussfolgerung nahe, dass die Bindung von Fe65 die Motilität des P2X2 Carboxyterminus einschränkt und damit die Permeabilität dieser Rezeptoren für grössere organische Kationen reduziert. Dies stellt einen neuartigen Regulationsmechanismus von ionotropen Rezeptoren und damit der ATP-vermittelten synaptischen Erregungsübertragung im Nervensystem dar

Evidence for functional P2X4/P2X7 heteromeric receptors

The cytolytic ionotropic ATP receptor P2X7 has several important roles in immune cell regulation, such as cytokine release, apoptosis, and microbial killing. Although P2X7 receptors are frequently coexpressed with another subtype of P2X receptor, P2X4, they are believed not to form heteromeric assemblies but to function only as homomers. Both receptors play a role in neuropathic pain; therefore, understanding how they coordinate the cellular response to ATP is important for the development of effective pain therapies. Here, we provide biochemical and electrophysiological evidence for an association between P2X4 and P2X7 that increases the diversity of receptor currents mediated via these two subtypes. The heterologously expressed receptors were coimmunoprecipitated from human embryonic kidney (HEK) 293 cells, and the endogenous P2X4 and P2X7 receptors were similarly coimmunoprecipitated from bone marrow-derived macrophages. In HEK293 cells, the fraction of P2X4 receptors biotinylated at the plasma membrane increased 2-fold in the presence of P2X7 although there was no change in overall expression. Coexpression of a dominant-negative P2X4 mutant (C353W) with P2X7, inhibited P2X7 receptor mediated currents by greater than 2-fold, whereas a nonfunctional but non-dominant-negative mutant (S341W) did not. Coexpression of P2X4S341W with P2X7 produced a current that was potentiated by ivermectin and inhibited by 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5-triphosphate (TNP-ATP), whereas expression of P2X7 alone produced a current that was insensitive to both of these compounds at the concentrations used. These results demonstrate a structural and functional interaction between P2X4 and P2X7, which suggests that they associate to form heteromeric receptors

Structural and functional interaction between P2X(4), P2X(7) and pannexin-1

Activation of P2X7 receptors triggers an increase in membrane permeability to large molecules and, in immune cells, the processing and release of inflammatory cytokines. The hemi-channel Pannexin-1 (Px1) was recently shown to interact with P2X7 and to be involved in both processes [1]. There is also evidence that the P2X4 receptor interacts with P2X7 [2]. In this study, we set out to further investigate the trafficking and functional interaction between P2X7, Px1 and P2X4. Proteins were overexpressed in HEK293 cells, and we used siRNA and carbenoxolone (CBX) to inhibit Px1. In HEK293 cells transiently overexpressing P2X7, incubation with CBX produced a dose-dependent inhibition of ethidium uptake evoked by ATP or BzATP. Surprisingly, in HEK293 cells expressing recombinant Px1 as well as P2X7, there was a reduction in the rate of ethidium uptake compared to cells transfected with P2X7 alone, and also incubation with CBX enhanced rather than inhibited BzATP-evoked ethidium uptake. The expression levels of P2X7 receptors in wild-type and Px1 stable cells were very similar, yet the fraction of P2X7 at the cell surface was slightly increased in Px1-cells as determined by biotinylation and selective cross-linking of surface receptors. The role of the cytoskeleton in large pore formation and in P2X7 coupling to Px1 was also examined. As shown previously, disruption of actin filaments with latrunculin had no effect on ethidium uptake; in contrast, depolymerisation of microtubules with colchicine resulted in a significant increase in both the rate and maximal dye uptake in response to ATP. The effects of co-expressing P2X4 on the interaction between P2X7 and Px1 are being investigated

Poly-(pentaerythritol adipate)-co-(poly ethylene glycol) Elastomers for the Sustained Release of Paclitaxel

Over the past 10 years poly-(polyol alkanoate)s elastomers have intensively been investigated due to their extraordinary potential for soft tissue engineering applications. In this work, a family of novel hyperbranched elastomers based on pentaerythritol and adipic acid, modified with PEG of 200 and 400 Da, is synthesized. The polymers are obtained by a simple 2 steps thermal process that avoids the use of toxic catalysts. Noteworthy, elastomers properties can be finely tuned by adjusting the molar ratio of monomers and curing conditions. The elastomers, which are fully characterized by standard methods, show excellent results in the in vitro controlled release of Paclitaxel. Cell culture assays indicate that all materials are able to inhibit lung cancer cell proliferation and that the release of Paclitaxel reduce cancer cell viability, indicating that the elastomers prepared hereby are good candidates for their use as eluting systems in long term cancer treatment.Fil: Navarro, Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Minari, Roque Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Ceaglio, Natalia Analia. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; ArgentinaFil: Masin, Marianela. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Laboratorio de Cultivos Celulares; ArgentinaFil: Vaillard, Santiago Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentin

A pore residue of the KCNQ3 potassium M-channel subunit controls surface expression

KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) are the principal subunits underlying the potassium M-current, which exerts a strong control on neuronal excitability. KCNQ3 subunits coassemble with KCNQ2 to form functional heteromeric channels that are specifically transported to the axonal initial segment and nodes of Ranvier. In contrast, there is no evidence for functional homomeric KCNQ3 channels in neurons, and it appears that these are inefficiently trafficked to the plasma membrane. Among eukaryotic potassium channels, the KCNQ3 subunit is unusual because it has an alanine in place of a threonine at the pore inner vestibule, three residues upstream of the GYG signature sequence of the selectivity filter. This residue is critical for the potentiation of the current after heteromerization, but the mechanism is unknown. We report that the presence of this uncommon residue at position 315 has a strong impact on the stability of the homotetramers and on channel trafficking. Wild-type KCNQ3 expressed alone is retained within the endoplasmic reticulum, and this mechanism is overcome by the substitution of threonine for Ala315. KCNQ3 subunits require assembly with KCNQ2 to exit this compartment, whereas KCNQ3-A315T is no longer dependent on KCNQ2 to form channels that are efficiently trafficked to the plasma membrane. The presence of this alanine, therefore, plays an important role in regulating the subunit composition of functional M-channels expressed at the surface of neurons

Predicting protein associations with long noncoding RNAs

Only a small fraction of the human transcriptome (∼1%) encodes proteins, but a large portion of transcripts is long noncoding RNAs (lncRNAs) and is an unexplored component of mammalian genomes. Here we introduce a method to perform large-scale predictions of protein-RNA associations. Our algorithm, ‘fast predictions of RNA and protein interactions and domains at the Center for Genomic Regulation, Barcelona, Catalonia’ (catRAPID), evaluates the interaction propensities of polypeptide and nucleotide chains using their physicochemical properties. The algorithm is freely available at http://big.crg.cat/ gene_function_and_evolution/services/catrapid

Artichoke extracts with potential application in chemoprevention and inflammatory processes

The aim of this work is to study three cultivars of artichoke (Cynara cardunculus var. scolymus): Gauchito, Guri and Oro Verde in terms of their in vitro chemoprevention and anti-inflammatory properties. These cultivars show good productive performance. The phenolic composition of their fresh leaves and edible bracts was analyzed by high performance liquid chromatography and high resolution mass spectrometry (HPLC-HRMS), showing mainly caffeoylquinic acids and flavonoids. Caffeoylquinic acids were quantified and the highest content was found in Gauchito cultivar. In this cultivar, the content of dicaffeoylquinic acids in fresh bracts was six times higher than that in fresh leaves (10064.5 ± 378.3 mg/kg versus 1451.0 ± 209.3 mg/kg respectively). Luteolin flavonoids were detected in leaves. The extracts from fresh bracts and leaves were assessed in their in vitro bioactivity against human neuroblastoma cells (SH-SY5Y). Inhibition of SH-SY5Y cells proliferation by Gauchito and Guri leaf extracts (8 µg/mL) was higher than 50 %. The leaf extracts of the same cultivars showed an inhibitory effect on human interferon IFN-I, decreasing its activity 50% at 40 µg/mL. Interestingly, the bract extracts did not show in vitro bioactivity at these concentrations, nor did the pure compounds chlorogenic acid, cynarin, apigenin and luteolin (at 2 µg/mL). These results suggest that Gauchito and Guri leaf extracts have potential for human neuroblastoma chemoprevention and treatment of inflammatory processes

A Functional P2X7 Splice Variant with an Alternative Transmembrane Domain 1 Escapes Gene Inactivation in P2X7 Knock-out Mice*

The ATP-activated P2X7 receptor channel is involved in immune function and inflammatory pain and represents an important drug target. Here we describe a new P2X7 splice variant (P2X7(k)), containing an alternative intracellular N terminus and first transmembrane domain encoded by a novel exon 1 in the rodent P2rx7 gene. Whole cell patch clamp recordings of the rat isoform expressed in HEK293 cells revealed an 8-fold higher sensitivity to the agonist Bz-ATP and much slower deactivation kinetics when compared with the P2X7(a) receptor. Permeability measurements in Xenopus oocytes show a high permeability for N-methyl-d-glucamine immediately upon activation, suggesting that the P2X7(k) channel is constitutively dilated upon opening. The rates of agonist-induced dye uptake and membrane blebbing in HEK cells were also increased. PCR analyses and biochemical analysis by SDS-PAGE and BN-PAGE indicate that the P2X7(k) variant escapes gene deletion in one of the available P2X7−/− mice strains and is strongly expressed in the spleen. Taken together, we describe a novel P2X7 isoform with distinct functional properties that contributes to the diversity of P2X7 receptor signaling. Its presence in one of the P2X7−/− strains has important implications for our understanding of the role of this receptor in health and disease