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14 pags., 4 figs., Background: Amyloids are ordered, insoluble protein aggregates, characterized by a cross-β sheet quaternary structure in which molecules in a β-strand conformation are stacked along the filament axis via intermolecular interactions. While amyloids are typically associated with pathological conditions, functional amyloids have also been identified and are present in a wide variety of organisms ranging from bacteria to humans. The cytoplasmic polyadenylation element-binding (CPEB) prion-like protein is an mRNA-binding translation regulator, whose neuronal isoforms undergo activity-dependent aggregation, a process that has emerged as a plausible biochemical substrate for memory maintenance. CPEB aggregation is driven by prion-like domains (PLD) that are divergent in sequence across species, and it remains unknown whether such divergent PLDs follow a similar aggregating assembly pathway. Here, we describe the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD and compare them to those of the Drosophila ortholog, Orb2 PLD. Results: Using in vitro single-molecule and bulk biophysical methods, we find transient oligomers and mature amyloid-like filaments that suggest similarities in the late stages of the assembly pathway for both ApCPEB and Orb2 PLDs. However, while prior to aggregation the Orb2 PLD monomer remains mainly as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising α-helical structures that evolve to coiled-coil species, indicating structural differences at the beginning of their amyloid assembly pathways. Conclusion: Our results indicate that divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms., The work was funded by two joint grants from the Ministry of Economy and Competitiveness to MCV (SAF2013-49179-C2-1-R and SAF2016-76678-C2-1-R) and DVL (SAF2013-49179-C2-2-R and SAF2016-76678-C2-2-R) and grants of the Ministry of Economy and Science (BFU2015-70072-R) and the CIBER de Enfermedades Respiratorias (CIBERES; ISCIII) to MM.

11 pags., 6 figs., Low transfection efficiency is a major challenge to overcome in non-viral approaches to reach clinical practice. Our aim was to explore new strategies to achieve more efficient non-viral gene therapies for clinical applications and in particular, for retinal diseases. Cationic niosomes and three GFP-encoding genetic materials consisting on minicircle (2.3 kb), its parental plasmid (3.5 kb) and a larger plasmid (5.5 kb) were combined to form nioplexes. Once fully physicochemically characterized, in vitro experiments in ARPE-19 retina epithelial cells showed that transfection efficiency of minicircle nioplexes doubled that of plasmids ones, maintaining good cell viability in all cases. Transfections in retinal primary cells and injections of nioplexes in rat retinas confirmed the higher capacity of cationic niosomes vectoring minicircle to deliver the genetic material into retina cells. Therefore, nioplexes based on cationic niosomes vectoring minicircle DNA represent a potential tool for the treatment of inherited retinal diseases., This work was supported by the Basque Country Government (Department of Education, University and Research, pre-doctoral grant PRE_2016_2_0302 and Consolidated Groups, IT907-16), the Ikerbasque Foundation for Science from the Basque Country, the Spanish Grant MAT 2015-69976-C3-1, SAF2013-42347-R, and by the Research Chair“Bidons Egara”

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammation and impaired airway immunity, providing an opportunistic platform for nontypeable Haemophilus influenzae (NTHi) infection. In this context, therapies targeting not only overactive inflammation without significant adverse effects, but also infection are of interest. Increasing evidence suggests that polyphenols, plant secondary metabolites with anti-inflammatory and antimicrobial properties, may be protective. Here, a Cistus salviifolius plant extract containing quercetin, myricetin, and punicalagin was shown to reduce NTHi viability. Analysis of these polyphenols revealed that quercetin has a bactericidal effect on NTHi, does not display synergies, and that bacteria do not seem to develop resistance. Moreover, quercetin lowered NTHi airway epithelial invasion through a mechanism likely involving inhibition of Akt phosphorylation, and reduced the expression of bacterially-induced proinflammatory markers il-8, cxcl-1, il-6, pde4b, and tnf&alpha, We further tested quercetin&rsquo, s effect on NTHi murine pulmonary infection, showing a moderate reduction in bacterial counts and significantly reduced expression of proinflammatory genes, compared to untreated mice. Quercetin administration during NTHi infection on a zebrafish septicemia infection model system showed a bacterial clearing effect without signs of host toxicity. In conclusion, this study highlights the therapeutic potential of the xenohormetic molecule quercetin against NTHi infection.

The study of leaf litter as a resource for shredders has emerged as a key topic in trophic links in ecology. However, thus far, most studies have emphasized the leaf quality as one of the main determinants of shredder behaviour and growth without simultaneously considering the leaf quantity availability. Nevertheless, the combined effects of leaf quantity and quality on shredder behaviour and growth is particularly crucial to further understand how ecosystem functioning may respond to the increasing flow intermittency due to climate change. In this study, we explore how changes in the leaf litter quality and quantity influence the feeding preferences and growth of an invertebrate shredder (Potamophylax latipennis). To do so, we used black poplar leaves conditioned in two streams with different flow regimens as a food resource. Afterwards, using a microcosm approach, we offered leaf discs that varied in terms of leaf quantity and quality to P. latipennis. Our results showed that flow intermittency had a negative effect on the quality of the food resource, and a lower quality had a negative effect on the consumption and growth rates of P. latipennis. Furthermore, we found that P. latipennis fed selectively on higher quality leaves even though the availability (quantity) of this resource was lower. In the context of climate change, with higher aridity/drier conditions/scenarios, our findings suggest that a decrease in the availability (quantity) of high-quality resources could potentially threaten links in global fluvial food webs and thus threaten ecosystem functioning.

15 pags, 8 figs, Murine adenovirus 2 (MAdV-2) infects cells of the mouse gastrointestinal tract. Like human adenoviruses, it is a member of the genus Mastadenovirus, family Adenoviridae. The MAdV-2 genome has a single fibre gene that expresses a 787 residue-long protein. Through analogy to other adenovirus fibre proteins, it is expected that the carboxy-terminal virus-distal head domain of the fibre is responsible for binding to the host cell, although the natural receptor is unknown. The putative head domain has little sequence identity to adenovirus fibres of known structure. In this report, we present high-resolution crystal structures of the carboxy-terminal part of the MAdV-2 fibre. The structures reveal a domain with the typical adenovirus fibre head topology and a domain containing two triple ß-spiral repeats of the shaft domain. Through glycan microarray profiling, saturation transfer difference nuclear magnetic resonance spectroscopy, isothermal titration calorimetry and site-directed mutagenesis, we show that the fibre specifically binds to the monosaccharide N-acetylglucosamine (GlcNAc). The crystal structure of the complex reveals that GlcNAc binds between the AB and CD loops at the top of each of the three monomers of the MAdV-2 fibre head. However, infection competition assays show that soluble GlcNAc monosaccharide and natural GlcNAc-containing polymers do not inhibit infection by MAdV-2. Furthermore, site-directed mutation of the GlcNAc-binding residues does not prevent the inhibition of infection by soluble fibre protein. On the other hand, we show that the MAdV-2 fibre protein binds GlcNAc-containing mucin glycans, which suggests that the MAdV-2 fibre protein may play a role in viral mucin penetration in the mouse gut., This research was sponsored by grant BFU2014-53425-P (to M. J. v. R.), coordinated grants CTQ2015-64597-P-C02-01 and CTQ2015-64597-P-C02-02 (to J. J. B. and F. J. C., respectively), grant BFU2015-70052-R (to M. M.) and the Spanish Adenovirus Network (AdenoNet, BIO2015-68990-REDT), all from the Spanish Agencia Estatal de Investigación. Financial support to M. M. from the CIBER of Respiratory Diseases (CIBERES) from the Spanish Institute of Health Carlos III is also acknowledged. These grants are co-financed by the European Regional Development Fund of the European Union. A. K. S. and T. H. N. were recipients of pre-doctoral fellowships from La Caixa and CSIC-VAST, respectively. The expression vectors were designed and created in Hungary, and this was financed by the Hungarian Scientific Research Fund (OTKA K100163). M. K. thanks Enterprise Ireland for a Commercialisation Fund grant (CF/2015/0089), A. K. acknowledges the National University of Ireland for a Cancer Care West Hardiman PhD scholarship and L. J. acknowledges the EU FP7 programme in support of the GlycoHIT consortium (grant no. 260600). This work was supported by R01 AI104920 (to J. G. S.) from the National Institute for Allergy and Infectious Diseases (www.niaid.nih.gov). S. S. W. was also supported by the Helen Riaboff Whiteley Endowment to the University of Washington and by Public Health Service, National Research Service Awards T32 AI083203 from the National Institute for Allergy and Infectious Diseases and T32 GM007270 from the National Institute of General Medical Sciences.

Streptococcus suis is a Gram-positive bacterium that infects humans and various animals, causing human mortality rates ranging from 5 to 20%, as well as important losses for the swine industry. In addition, there is no effective vaccine for S. suis and isolates with increasing antibiotic multiresistance are emerging worldwide. Facing this situation, wild type or engineered bacteriophage lysins constitute a promising alternative to conventional antibiotics. In this study, we have constructed a new chimeric lysin, Csl2, by fusing the catalytic domain of Cpl-7 lysozyme to the CW-7 repeats of LySMP lysin from an S. suis phage. Csl2 efficiently kills different S. suis strains and shows noticeable activity against a few streptococci of the mitis group. Specifically, 15 μg/ml Csl2 killed 4.3 logs of S. suis serotype 2 S735 strain in 60 min, in a buffer containing 150 mM NaCl and 10 mM CaCl, at pH 6.0. We have set up a protocol to form a good biofilm with the non-encapsulated S. suis mutant strain BD101, and the use of 30 μg/ml Csl2 was enough for dispersing such biofilms and reducing 1-2 logs the number of planktonic bacteria. In vitro results have been validated in an adult zebrafish model of infection.

This is an open access article distributed under the terms of the Creative Commons Attribution License., Calmodulin (CaM) phosphorylated at different serine/threonine and tyrosine residues is known to exert differential regulatory effects on a variety of CaM-binding enzymes as compared to non-phosphorylated CaM. In this report we describe the preparation and characterization of a series of phospho-(Y)-mimetic CaM mutants in which either one or the two tyrosine residues present in CaM (Y99 and Y138) were substituted to aspartic acid or glutamic acid. It was expected that the negative charge of the respective carboxyl group of these amino acids mimics the negative charge of phosphate and reproduce the effects that distinct phospho-(Y)-CaM species may have on target proteins. We describe some physicochemical properties of these CaM mutants as compared to wild type CaM, after their expression in Escherichia coli and purification to homogeneity, including: i) changes in their electrophoretic mobility in the absence and presence of Ca2+; ii) ultraviolet (UV) light absorption spectra, far- and near-UV circular dichroism data; iii) thermal stability in the absence and presence of Ca2+; and iv) Tb3+-emitted fluorescence upon tyrosine excitation. We also describe some biochemical properties of these CaM mutants, such as their differential phosphorylation by the tyrosine kinase c-Src, and their action as compared to wild type CaM, on the activity of two CaM-dependent enzymes: cyclic nucleotide phosphodiesterase 1 (PDE1) and endothelial nitric oxide synthase (eNOS) assayed in vitro., This work was funded by grants to AV from the Secretaría de Estado de Investigación, Desarrollo e Innovación (SAF2011-23494) and the Consejería de Educación de la Comunidad de Madrid (S2011/BMD-2349), and grant to AV and DS from the European Commission (contract PITN-GA-2011-289033 DYNANO). Additional funding to DS and MM was provided by the Centro de Investigación Biomédica en Red de Enfermedades Respiratorias. SRS was funded by a Marie Curie contract from the European Commission and VS and GB were supported by fellowship and grants (03-00-6057-2005 and PG-03-8728-2013) from the Consejo de Desarrollo Científico y Humanístico de la Universidad Central de Venezuela.

Spermadhesins are a family of seminal plasma proteins composed of a single CUB domain, which appear to be involved in various aspects of the fertilization process in pigs. PSP-I and PSP-II, the most abundant porcine spermadhesins, occur in seminal plasma as noncovalent heterodimers devoid of heparin-binding capability. Of note is the stability of this dimer, which is significantly affected by physiologically relevant conditions such as Zn2+ ions. Here, we show that PSP-I and PSP-II when separated appear to conserve the overall fold of the CUB domain observed in the crystal structure of the PSP-I/PSP-II heterodimer, as concluded from gel filtration, analytical ultracentrifugation, differential scanning calorimetry, and circular dichroism analyses. However, Zn2+ concentrations in the range of those found in boar seminal plasma induce the unfolding and self-association of PSP-I, apparently as a consequence of the exposure of hydrophobic core residues, whereas they have no effect on PSP-II. Remarkably, Zn2+-denatured and self-associated (but not structured monomeric) PSP-I is retained on a heparin column, resembling the behavior of free PSP-I and homologous spermadhesins of the heparin-binding fraction of boar seminal plasma, which also exhibit different aggregation states. Thus, the modulation of the structural organization and heparin-binding ability of PSP-I by Zn2+ might be a physiological phenomenon in seminal plasma.

Spermadhesins are a family of 12-16 kDa proteins with a single CUB domain. PSP-I and PSP-II, the most abundant boar spermadhesins, are present in seminal plasma as a noncovalent heterodimer. Dimerization markedly affects the binding ability of the subunits. Notably, heparin and mannose 6-phosphate binding abilities of PSP-II are abolished, indicating that the corresponding binding sites may be located at (or near) the dimer interface. Pursuing the hypothesis that cryptic binding sites in PSP-I/PSP-II may be exposed in specific physiological environments, we examined the influence of Zn2+ and acidic pH on the heterodimer stability. According to near-UV CD spectra, the core native fold is preserved in the presence of physiological concentrations of Zn2+, a cation unusually abundant in boar seminal plasma. However, the thermostability of the heterodimer decreases significantly, as observed by CD and differential scanning calorimetry. The effect is Zn2+-specific and is reversed by EDTA. Destabilization is also observed at acidic pH. Gel filtration analysis using radioiodinated PSP-I/PSP-II reveals that dissociation of the heterodimer at low (nanomolar) protein concentrations is promoted by both Zn2+ and acidic pH. Although the integrity of the heterodimer in seminal plasma seems to be guaranteed by its high concentration, dissociation may be facilitated in the female genital tract because of dilution of the protein in the intraluminal fluids of the cervix and the uterus, and the acidic fluid of the uterotubal junction. Such a mechanism may be relevant in the regulation of uterine immune reactions.

We have analyzed the in vitro disassembly of the capsid of the minute virus of mice, and the stability of capsid chimeras carrying heterologous epitope insertions. Upon heating in a physiological buffer, empty capsids formed by 60 copies of protein VP2 underwent first a reversible conformational change with a small enthalpy change detected by fluorescence. This change was associated with, but not limited to, externalization of the VP2 N terminus. Irreversible capsid dissociation as detected by changes in fluorescence, hemagglutination activity, and electrophoretic mobility occurred at much higher temperatures. Differential scanning calorimetry in the same conditions indicated that the dissociation/denaturation transition involved a high enthalpy change and proceeded through one or more intermediates. In contrast, in the presence of 1.5 M guanidinium chloride, heat-induced disassembly fitted a two-state irreversible process. Both thermally and chemically induced dissociation/denaturation yielded a form that had lost a part of the tertiary structure, but still retained the native secondary structure. Data from chemical dissociation indicates this form may correspond to a molten globule-like monomeric state of the capsid protein. All five antigenic peptide insertions attempted in exposed loops, despite being perhaps among the least disruptive, led to defects in folding/assembly of the capsid and, in most cases, to reduced capsid stability against thermal dissociation. The results with one of the simplest viral capsids reveal a complex pathway for disassembly, and a reduction in capsid assembly and stability upon insertion of peptides, even within the most exposed capsid loops.

Chemotherapy. All rights reserved. Objectives: Streptococcus pneumoniae is becoming increasingly antibiotic resistant worldwide and new antimicrobials are urgently needed. Our aim was new chimeric phage endolysins, or lysins, with improved bactericidal activity by swapping the structural components of two pneumococcal phage lysozymes: Cpl-1 (the best lysin tested to date) and Cpl-7S. Methods: The bactericidal effects of four new chimeric lysins were checked against several bacteria. The purified enzymes were added at different concentrations to resuspended bacteria and viable cells were measured after 1 h. Killing capacity of the most active lysin, Cpl-711, was tested in a mouse bacteraemia model, following mouse survival after injecting different amounts (25-500 mg) of enzyme. The capacity of Cpl-711 to reduce pneumococcal biofilm formation was also studied. Results: The chimera Cpl-711 substantially improved the killing activity of the parental phage lysozymes, Cpl-1 and Cpl-7S, against pneumococcal bacteria, including multiresistant strains. Specifically, 5 mg/mL Cpl-711 killed ≥7.5 log of pneumococcal R6 strain. Cpl-711 also reduced pneumococcal biofilm formation and killed 4 log of the bacterial population at 1 mg/mL. Mice challenged intraperitoneally with D39_IU pneumococcal strain were protected by treatment with a single intraperitoneal injection of Cpl-711 1 h later, resulting in about 50% greater protection than with Cpl-1. Conclusions: Domain swapping among phage lysins allows the construction of new chimeric enzymes with high bactericidal activity and a different substrate range. Cpl-711, the most powerful endolysin against pneumococci, offers a promising therapeutic perspective for the treatment of multiresistant pneumococcal infections.

J. Jesús Casas et al., Terrestrial plant litter is important in sustaining stream food webs in forested headwaters. Leaf litter quality often decreases when native species are replaced by introduced species, and a lower quality of leaf litter inputs may alter litter decomposition at sites afforested with non-native species. However, since detritivore composition and resource use plasticity may depend on the prevalent litter inputs, the extent of the alteration in decomposition can vary between streams. We tested 2 hypotheses using 2 native and 3 introduced species of tree differing in quality in 4 Iberian regions with contrasting vegetational traits: 1) decomposition rates of all plant species would be higher in regions where streams normally receive litter inputs of lower rather than higher quality; 2) a higher resource-use plasticity of detritivores in regions vegetated with plants of lower litter quality will cause a greater evenness in decomposition rates among plant species compared to regions where streams normally receive higher-quality plant litter inputs. Results showed a highly consistent interspecific ranking of decomposition rates across regions driven by litter quality, and a significant regional effect. Hypothesis 1 was supported: decomposition rates of the five litter types were generally higher in streams from regions vegetated with species producing leaf litter of low quality, possibly due to the profusion of caddisfly shredders in their communities. Hypothesis 2 was not supported: the relative differences in decomposition rates among leaf litter species remained essentially unaltered across regions. Our results suggest that, even in regions where detritivores can be comparatively efficient using resources of low quality, caution is needed particularly when afforestation programs introduce plant species of lower litter quality than the native species. © 2013 Elsevier B.V., This research was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) projects IMPARIOS (CGL2007-66664-C04) and TEMPERIOS (CGL2010-22129-C04).JJC and MM contributed to this manuscript during tenure of the grants RIBARID (CGL2012-39635) and CARBONET (CGL2011-30474-C02-02) (MINECO), respectively.

Actinoporins are water-soluble proteins with the ability to form pores upon insertion into biological membranes. They constitute a family of proteins with high degree of sequence identities but different hemolytic activities, suggesting that minor conformational arrangements result in major functional changes. A good example of this situation is the sea anemone Stichodactyla helianthus which produces two very similar actinoporins, sticholysins I (StnI) and II (StnII), but of very different hemolytic efficiency. Within this idea, given that the high resolution three-dimensional structure of StnII is already known, we have now solved that one corresponding to StnI in order to analyze the influence of particular residues on the conformation and activity of these proteins. In addition, random mutagenesis has been also used to produce five less hemolytic variants of StnI. All these mutations map to functionally relevant regions because they are probably involved in conformational changes associated with pore formation, which take place after membrane binding, and involve long-distance rearrangements of the polypeptide chain of actinoporins., This work was supported by the Spanish Ministerio de Ciencia e Innovación through projects CTQ2008-00080/BQU, CTQ2011-22514, and BFU2009-10185, and one FPU fellowship granted to S.G.-L.

Phage endolysins are murein hydrolases that break the bacterial cell wall to provoke lysis and release of phage progeny.Recently, these enzymes have also been recognized as powerful and specific antibacterial agents when added exogenously. In the pneumococcal system, most cell wall associated murein hydrolases reported so far depend on choline for activity, and Cpl-7 lysozyme constitutes a remarkable exception. Here, we report the improvement of the killing activity of the Cpl-7 endolysin by inversion of the sign of the charge of the cell wall-binding module (from-14.93 to-3.0 at neutral pH). The engineered variant, Cpl-7S, has 15 amino acid substitutions and an improved lytic activity against Streptococcus pneumoniae (including multiresistant strains), Streptococcus pyogenes, and other pathogens. Moreover, we have demonstrated that a single 25-μg dose of Cpl-7S significantly increased the survival rate of zebrafish embryos infected with S. pneumoniae or S. pyogenes, confirming the killing effect of Cpl-7S in vivo. Interestingly, Cpl-7S, in combination with 0.01% carvacrol (an essential oil), was also found to efficiently kill Gram-negative bacteria such as Escherichia coli and Pseudomonas putida, an effect not described previously. Our findings provide a strategy to improve the lytic activity of phage endolysins based on facilitating their pass through the negatively charged bacterial envelope, and thereby their interaction with the cell wall target, by modulating the net charge of the cell wall-binding modules. Copyright © 2013, American Society for Microbiology. All Rights Reserved., This research was funded by grants from the Ministerio de Ciencia e Innovación (MICINN) to P. García (SAF2009-10824) and E. García (SAF2012-39444-C02-01) and from the MICINN (BFU2009-10052 and BF42012-36825) and the Consejería de Educación de la Comunidad de Madrid (S2010/BMD/2457) to M. Menéndez. Additional funding was provided by the CIBER de Enfermedades Respiratorias (CIBERES), an initiative of the Instituto de Salud Carlos III (ISCIII). Roberto Díez-Martínez was the recipient of a fellowship from the MICINN (FPI program).We declare a competing financial interest. We are coinventors on a Spanish patent application (no. P201330777) covering the results contained in this article. Any potential income generated by exploitation of the patent rights and received by our employers, the CSIC, and CIBERES shall be shared with us according to Spanish law

37 p-.8 fig.-3 tab., The comparatively small number of members of the family of adhesion/growth-regulatory galectins in chicken predestines this system as an attractive model to study divergence of these lectins after gene duplications. Expression profiling of the three homodimeric (proto-type) chicken galectins (CG-1A, CG-1B and CG-2) has raised evidence for distinct functionalities, explaining the interest in a detailed crystallographic analysis of CG-2. As revealed here, marked differences are found in the ligand-binding site and in the contact pattern within the homodimer interface, underlying a characteristic orientation of the two subunits. Notably, a distinctive trimer of dimers that is unique in all galectin crystal structures reported to date forms the core unit of the crystallographic assembly. Combination with spectroscopic and thermodynamic measurements, and comparisons with CG-1A and CG-1B, identifies differential changes in circular-dichroism spectra in the presence of lactose, reflecting the far-reaching impact of the ligand on hydrodynamic behavior, and inter-galectin differences in both the entropy and the enthalpy of binding. This structural information is a salient step to complete the analysis of the full set of galectins from this model organism., This work has been kindly supported by grants BFU2009-10052, BFU2011-24615, BFU2012-36825 and CSD2009-00088 from the Spanish Ministry of Science and Innovation, the CIBER of Respiratory Diseases (CIBERES), an initiative from ISCIII, the Regional Government of Madrid (S2010/BMD-2353), and funding from the European Union’s Seventh Framework Program FP7/2007-2013 under REA grant agreements no. 260600 (“GlycoHIT”) and 317297 (“GLYCOPHARM”).

The CUB domain is a widespread 110-amino-acid module found in functionally diverse, often developmentally regulated proteins, for which an antiparallel beta-barrel topology similar to that in immunoglobulin V domains has been predicted. Spermadhesins have been proposed as a subgroup of this protein family built up by a single CUB domain architecture. To test the proposed structural model, we have analyzed the structural organization of two members of the spermadhesin protein family, porcine seminal plasma proteins I/II (PSP-I/PSP-II) heterodimer and bovine acidic seminal fluid protein (aSFP) homodimer, using differential scanning calorimetry, far-ultraviolet circular dichroism and Fourier-transform infrared spectroscopy. Thermal unfolding of PSP-I/PSP-II and aSFP were irreversible and followed a one-step process with transition temperatures (Tm) of 60.5 degrees C and 78.6 degrees C, respectively. The calorimetric enthalpy changes (delta Hcat) of thermal denaturation were 439 kJ/mol for PSP-I/PSP-II and 660 kJ/mol for aSFP dimer. Analysis of the calorimetric curves of PSP-I/PSP-II showed that the entire dimer constituted the cooperative unfolding unit. Fourier-transform infrared spectroscopy and deconvolution of circular dichroic spectra using a convex constraint analysis indicated that beta-structure and turns are the major structural element of both PSP-I/PSP-II (53% of beta-sheet, 21% of turns) and aSFP (44% of beta-sheet, 36% of turns), and that the porcine and the bovine proteins contain little, if any, alpha-helical structure. Taken together, our results indicate that the porcine and the bovine spermadhesin molecules are probably all-beta-structure proteins, and would support a beta-barrel topology like that predicted for the CUB domain. Other beta-structure folds, such as the Greek-key pattern characteristic of many carbohydrate-binding protein domains cannot be eliminated. Finally, the same combination of biophysical techniques was used to characterize the residual secondary structure of thermally denatured forms of PSP-I/PSP-II and aSFP, and to emphasize the aggregation tendency of these forms.

Amyloidogenic neurodegenerative diseases are incurable conditions with high social impact that are typically caused by specific, largely disordered proteins. However, the underlying molecular mechanism remains elusive to established techniques. A favored hypothesis postulates that a critical conformational change in the monomer (an ideal therapeutic target) in these >neurotoxic proteins> triggers the pathogenic cascade. We use force spectroscopy and a novel methodology for unequivocal single-molecule identification to demonstrate a rich conformational polymorphism in the monomer of four representative neurotoxic proteins. This polymorphism strongly correlates with amyloidogenesis and neurotoxicity: it is absent in a fibrillization-incompetent mutant, favored by familial-disease mutations and diminished by a surprisingly promiscuous inhibitor of the critical monomeric β-conformational change, neurotoxicity, and neurodegeneration. Hence, we postulate that specific mechanostable conformers are the cause of these diseases, representing important new early-diagnostic and therapeutic targets. The demonstrated ability to inhibit the conformational heterogeneity of these proteins by a single pharmacological agent reveals common features in the monomer and suggests a common pathway to diagnose, prevent, halt, or reverse multiple neurodegenerative diseases. © 2012 Hervás et al.

Gemini surfactants have two polar head groups and two hydrocarbon tails. Compared with conventional surfactants, geminis have much lower (οM vs. mM) critical micelle concentrations and possess slower (ms vs οs) monomer micelle kinetics. The structure of the gemini surfactants studied is [HOCH 2CH 2-, CH 3-, CH 3(CH 2) 15-N +-(CH 2) s-N +-(CH 2) 15CH 3,-CH 3,-CH 2CH 2OH]·2Br - where s=4, 5, or 6. Our objective is to reveal the effect of these cationic gemini surfactants on the structure and stability of two model proteins: Ribonuclease A (RNase A) and Hen Egg White Lysozyme (HEWL). 2D 1H NMR and Circular Dichroism (CD) spectroscopies show that the conformation of RNase A and HEWL is unaffected at low to neutral pH where these proteins are positively charged, although hydrogen exchange shows that RNase A's conformational stability is slightly lowered. At alkaline pH, where these proteins lose their net positive charge, fluorescence and CD spectroscopies and ITC experiments show that they do interact with gemini surfactants, and multiple protein•gemini complexes are observed. Based on the results, we conclude that these cationic gemini surfactants neither interact strongly with nor severely destabilize these well folded proteins in physiological conditions, and we advance that they can serve as useful membrane mimetics for studying the interactions between membrane components and positively charged proteins. © 2011 Elsevier Inc.

11 páginas, 8 figuras -- PAGS nrs. 5445-5455, Thioglycosides offer the advantage over O-glycosides to be resistant to hydrolysis. Based on initial evidence of this recognition ability for glycosyldisulfides by screening dynamic combinatorial libraries, we have now systematically studied dithiodigalactoside on a plant toxin (Viscum album agglutinin) and five human lectins (adhesion/growth-regulatory galectins with medical relevance e.g. in tumor progression and spread). Inhibition assays with surface-presented neoglycoprotein and in solution monitored by saturation transfer difference NMR spectroscopy, flanked by epitope mapping, as well as isothermal titration calorimetry revealed binding properties to VAA (K(a): 1560 +/- 20 M (1)). They were reflected by the structural model and the affinity on the level of toxin-exposed cells. In comparison, galectins were considerably less reactive, with intrafamily grading down to very minor reactivity for tandem-repeat-type galectins, as quantitated by radioassays for both domains of galectin-4. Model building indicated contact formation to be restricted to only one galactose moiety, in contrast to thiodigalactoside. The tested glycosyldisulfide exhibits selectivity between the plant toxin and the tested human lectins, and also between these proteins. Therefore, glycosyldisulfides have potential as chemical platform for inhibitor design., This work has been generously supported by grants CTQ2009-08536, SAF2008-00945, BFU2006-10288 and BFU2009-10052 from the Spanish Ministry, an EC Marie Curie Research Training Network grant (contract no. MRTN-CT-2005-019561), the EC research program GlycoHIT (contract ID 260600), the Verein zur Forderung des biologisch-technologischen Fortschritts in der Medizin e. V. and the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII). We thank Drs B. Friday and S. Namirha for inspiring discussions as well as M. V. Lopez-Moyano for excellent technical assistance.

MDL 27048 [trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2- methyl-2-propen-1-one] fluoresces when bound to tubulin but not in solution. This effect has been investigated and found to be mimicked by viscous solvents. Therefore, MDL 27048 appears to be a fluorescent compound whose intramolecular rotational relaxation varies as a function of microenvironment viscosity. The binding parameters of MDL 27048 to tubulin have been firmly established by fluorescence of the ligand, quenching of the protein fluorescence, and gel equilibrium chromatography. The apparent binding equilibrium constant was (2.75 +/- 0.45) x 10(6)M-1, and the binding site number was 0.81 +/- 0.12 (10 mM sodium phosphate-0.1 mM GTP, pH 7.0, at 25 degrees C). The binding is exothermic. The binding of MDL 27048 overlaps the colchicine and podophyllotoxin binding sites. Binding of MDL 27048 to the colchicine site was also measured by competition with MTC [2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one] , a well-characterized reversibly binding probe of the colchicine site [Andreu et al. (1984) Biochemistry 23, 1742-1752; Bane et al., (1984) J. Biol. Chem. 259, 7391-7398]. In contrast with close analogues of colchicine, MDL 27048 and podophyllotoxin neither affected the far-ultraviolet circular dichroism spectrum of tubulin, within experimental error, nor induced tubulin GTPase activity. Like podophyllotoxin, an excess of MDL 27048 over tubulin induced no abnormal cooperative polymerization of tubulin, which is characteristic of colchicine binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Human tandem-repeat-type galectin-9 is a potent adhesion/growth-regulatory effector via lectin capacity of its N- and C-terminal domains. This bioactivity prompted further crystallographic study of the N-domain, combined with analysis in solution. Binding of lactose markedly increased the N-domain's resistance to thermal denaturation. Crystallography revealed its intimate contact profile, besides detecting an extension of the beta-sandwich fold by an antiparallel beta-strand F0 aligned to the C-terminal F1 strand. Ligand accommodation in its low-energy conformation leads to a movement of Arg87's side chain. As consequence, the ligand's glucose moiety and Arg87 become hydrogen bonded. The resulting predictions for spatial parameters in solution were verified by determining (a) the pattern of magnetization transfer from the protein to protons of lactose and Forssman disaccharide by NMR spectroscopy and (b) the ellipticity changes at wavelengths characteristic for Trp/Tyr residues in near-UV CD spectroscopy. Whereas solid-phase assays confirmed a previously noted tendency for homo- and heterotypic aggregation, gel filtration and ultracentrifugation disclosed monomeric status in solution, in line with crystallographic data. Using cell mutants with defects in glycosylation, this lectin domain was shown to preferentially bind N-glycans without alpha2,3-sialylation. Since proximal promoter sequences were delineated to diverge markedly among galectin genes and resulting differences in expression profiles were exemplarily documented immunohistochemically, the intrafamily diversification appears to have assigned this protein to a characteristic expression and activity profile among galectins. Our data thus take the crystallographic information to the level of the lectin in solution and in tissues by a strategic combination of spectroscopic and cell/histochemical assays.

Amyloids are ordered protein aggregates that are typically associated with neurodegenerative diseases and cognitive impairment. By contrast, the amyloid-like state of the neuronal RNA binding protein Orb2 in Drosophila was recently implicated in memory consolidation, but it remains unclear what features of this functional amyloid-like protein give rise to such diametrically opposed behaviour. Here, using an array of biophysical, cell biological and behavioural assays we have characterized the structural features of Orb2 from the monomer to the amyloid state. Surprisingly, we find that Orb2 shares many structural traits with pathological amyloids, including the intermediate toxic oligomeric species, which can be sequestered in vivo in hetero-oligomers by pathological amyloids. However, unlike pathological amyloids, Orb2 rapidly forms amyloids and its toxic intermediates are extremely transient, indicating that kinetic parameters differentiate this functional amyloid from pathological amyloids. We also observed that a well-known anti-amyloidogenic peptide interferes with long-term memory in Drosophila. These results provide structural insights into how the amyloid-like state of the Orb2 protein can stabilize memory and be nontoxic. They also provide insight into how amyloid-based diseases may affect memory processes., This research was supported by funds from SAF2013-49179-C2-1-R JPND_CD_FP-688- 059 (AC14/00037 ISCIII) to MCV, SIMR to KS, SAF2013-49179-C2-2-R JPND_CD_FP-688-059 (AC14/00037 ISCIII) to DVL, BFU2012-36825, S2011/BMD-2457 (Comunidad de Madrid)

6 páginas, 3 figuras., Thermodynamic evidence for the selective Ca2+-mediated self-aggregation via carbohydrate−carbohydrate interactions of gold glyconanoparticles functionalized with the disaccharides lactose (lacto-Au) and maltose (malto-Au), or the biologically relevant trisaccharide Lewis X (LeX−Au), was obtained by isothermal titration calorimetry. The aggregation process was also directly visualized by atomic force microscopy. It was shown in the case of the trisaccharide Lewis X that the Ca2+-mediated aggregation is a slow process that takes place with a decrease in enthalpy of 160 ± 30 kcal mol-1, while the heat evolved in the case of lactose and maltose glyconanoparticles was very low and thermal equilibrium was quickly achieved. Measurements in the presence of Mg2+ and Na+ cations confirm the selectivity for Ca2+ of LeX−Au glyconanoparticles. The relevance of this result to cell−cell adhesion process mediated by carbohydrate−carbohydrate interactions is discussed., This research was supported by the Spanish Ministerio de Ciencia y Tecnolgia (BQU2002-03734) and the EU (HPRN-CT-2002-00190). P.E. thanks the EU and the Spanish Ministerio de Educacion y Ciencia (MEC) for financial support. J.M.F. thanks the MEC for financial support.

Septo-optic dysplasia (SOD) is loosely defined as any combination of optic nerve hypoplasia, pituitary gland hypoplasia, and midline abnormalities of the brain. Recent studies have shown that this rare disease has its origin in key mutations in Hesx-1, a protein that plays a critical role in normal development of the forebrain, eyes, and other anterior structures during embryogenesis. R53C mutation in the Hesx-1 homeodomain has recently been identified in some patients with SOD. However, no detailed description of the effect of this mutation on the protein structure, stability, and function has been reported so far. The impact of R53C substitution on Hesx-1 homeodomain structure, stability, and DNA-binding properties was analyzed by using a combination of NMR spectroscopy, molecular modeling and circular dichroism experiments. Although R53C mutation has very slight effects on protein structure, it has a profound impact on homeodomain stability. We show that intramolecular disulfide formation can be easily accomplished in the mutated homeodomain, which suggests that such bond formation could take place in vivo. This modification has a key effect on the homeodomain structure and stability, mainly through its effect on helix I/helix III packing. Finally, CD titrations allowed us to establish the energy cost of the R53C substitution with respect to Hesx-1-homeodomain-DNA complex stability. Detailed structural models are provided for the wild-type human Hesx-1 homeodomain and the R53C mutant, which is associated with SOD in humans. The reported effect of R53C mutation on protein stability and DNA-binding properties, together with the significant structural perturbations induced by disulfide formation in the mutated polypeptide might explain the loss of activity of the mutant in vivo.

10 p.-2 tab.-7 fig., Ceragenin CSA-13, a cationic steroid, is here reported to show a concentration-dependent bactericidal/bacteriolytic activity against pathogenic streptococci, including multidrug-resistant Streptococcus pneumoniae. The autolysis promoted by CSA- 13 in pneumococcal cultures appears to be due to the triggering of the major S. pneumoniae autolysin LytA, an Nacetylmuramoyl-L-alanine amidase. CSA-13 also disintegrated pneumococcal biofilms in a very efficient manner, although at concentrations slightly higher than those required for bactericidal activity on planktonic bacteria. CSA-13 has little hemolytic activity which should allow testing its antibacterial efficacy in animal models., This work was supported by Dirección General de Investigación Científica y Técnica (grant numbers SAF2009-10824, BFU2009-10052, BFU2012-36825,and SAF2012-39444-C02-01) and Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), which is an initiative of the ISCIII.

The MAL proteolipid, an integral membrane protein with selective residence in glycolipid- and cholesterol-enriched membrane (GEM) microdomains, has recently been identified as being an element of the integral protein machinery necessary for apical transport in MDCK cells. With the use of a recombinant baculovirus, we have expressed and purified polyhistidine-tagged MAL to determine whether MAL has special lipid requirements for becoming incorporated into membranes. In contrast with caveolin-1, a component of GEMs that requires cholesterol for its integration into artificial membranes, MAL incorporation took place with dimyristoylphosphatidylcholine as the only lipid component. The presence of cholesterol, sphingomyelin, or galactocerebrosides did not affect the efficiency of this process. These results indicated that MAL is compatible with membranes containing either only phospholipids or also glycolipids and cholesterol and are consistent with the reported requirement of a sorting event for the specific targeting of MAL to GEM microdomains.

11 p.-12 fig.-6 tab., Tubulin liganded with GTP at the N-site in the alpha-subunit and with GDP at the E-site in the beta-subunit (GDP-tubulin) reversibly binds one high affinity Mg2+ cation (Kb = 1.1 x 10(7) M-1), whereas tubulin liganded with GTP at both subunits (GTP-tubulin) binds one more high affinity Mg2+. The two cation binding loci are identified as nucleotide sites N and E, respectively. Mg2+ at the N-site controls the stability and structure of the alphabeta-tubulin dimer. Mg2+ dissociation is followed by the slow release of bound nucleotide and functional inactivation. Mg2+ bound to the N-site significantly increases the thermal stability of the GDP-tubulin dimer (by 10 degrees C and approximately 50 kcal mol-1 of experimental enthalpy change). However, the thermal stability of Mg2+-liganded GDP- and GTP-tubulin is the same. Mg2+ binding to the N-site is linked to the alphabeta-dimer formation. The binding of Mg2+ to the alpha-subunit communicates a marked enhancement of fluorescence to a colchicine analogue bound to the beta-subunit. Colchicine, in turn, thermally stabilizes Mg2+-depleted tubulin. The tubulin properties described would be simply explained if the N-site and the colchicine site are at the alpha-beta dimerization interface. It follows that the E-site would be at the beta-end of the tubulin dimer, consistent with the known functional role of the E nucleotide gamma-phosphate and coordinated cation controlling microtubule stability., This work was supported by Dirección General de Enseñanza Superior Grants PB93-0114 (to M. M.), PB95-0120 (to G. R.), and PB95-0116 (to J. M. A.).

Differential scanning calorimetry has been used to characterize the thermal denaturation of gastric (H+,K+)-ATPase. The excess heat capacity function of (H+,K+)-ATPase in highly oriented gastric vesicles displays two peaks at 53.9 degrees C (Tm1) and 61.8 degrees C (Tm2). Its thermal denaturation is an irreversible process that does not exhibit kinetic control and can be resolved in two independent two-state processes. They can be assigned to two cooperative domains located in the cytoplasmic loops of the alpha-subunit, according to the disappearance of the endothermic signal upon removal of these regions by proteinase K digestion. Analysis of the thermal-induced unfolding of the enzyme trapped in different catalytic cycle intermediates has allowed us to get insight into the E1-E2 conformational change. In the E1 forms both transitions are always observed. As Tm1 is shifted to Tm2 by vanadate and ATP interaction, the unfolding mechanism changes from two independent to two sequential two-state transitions, revealing interdomain interactions. Stabilization of the E2 forms results in the disappearance of the second transition at saturation by K+, Mg2+-ATP, and Mg2+-vanadate as well as in significant changes in Tm2 and DeltaH1. The catalytic domain melts following a process in which intermolecular interactions either in the native or in the unfolded state might be involved. Interestingly, the E2-vanadate-K+ form displays intermediate properties between the E1 and E2 conformational families.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT, E.C.2.4.2.8) from Artemia cysts exhibits maximum activity at 70°C. Its thermal stability has been examined following enzymatic activity as a function of temperature. Cold-induced renaturation experiments of samples heated at increasing temperatures showed that reversibility of thermal inactivation depends on the incubation time and final temperature. Prolonged incubation of the thermoinactivated enzyme at 0°C did not afford any further increase of the catalytic activity at 37°C. The complex substrate PRPP:Mg protects HGPRT from thermal inactivation. However, incubations with hypoxanthine rendered a less thermostable enzyme at any temperature tested. The irreversible inactivation of HGPRT proceeds in two exponential steps. The analysis of the apparent rate constants for the fast and the slow phases, λ1 and λ2 as per the Lumry and Eyring model suggests the existence of more than three states in the thermal denaturation pathway of the free enzyme. In the presence of PRPP:Mg the irreversible process follows a single exponential and proceeds very slowly below 70°C. PRPP:Mg also protects the enzyme from inactivation by NEM and pCMB, suggesting that -SH groups may be in the vicinity of the active site., The research was supported by the Fondo de Investigació Sanitaria (FISS), Grant 93/0802 and by the Dirección General de Investigación Científica y Técnica, Grant PB 93-0114.

The effects of several chemical modifications in the AMP molecule on its interaction with phosphorylase b are examined by microcalorimetry, equilibrium dialysis, light scattering and ultracentrifuge experiments. In this work we report the results obtained for eight AMP analogues corresponding to different substituents in the puric base or in the ribose, or to different positions of the phosphate. The thermodynamic properties of the interaction between the phosphorylase b and the above mentioned nucleotides are also reported. The following conclusions were obtained: a) Except for IMP and 2'dIMP all the nucleotides studied clearly show two types of binding sites in the enzyme, b) The interaction of the nucleotide with its weaker affinity binding site is highly dependent upon chemical alterations in the puric base. c) Both the amino group in C(6) and the N(1) of the adenine in the AMP seem to play an important role in the conformational transitions induced by the nucleotide on the enzyme, d) The tetramerization of phosphorylase b in the presence of 10 −2 M AMP and in the conditions of the ultracentrifuge experiments is drastically affected by modifications in the ribose-phosphate part of the AMP molecule.

The binding of AMP to activator site N and to inhibitor site I in glycogen phosphorylase b has been characterized by calorimetry, potentiometry and ultracentrifugation in the pH range 6.5-7.5 at 25 degrees C (mu = 0.1). Calorimetric titration data of phosphorylase b with adenosine 5'-phosphoramidate are also reported at pH 6.9 (T = 25 degrees C, mu = 0.1). Calorimetric curves have been analyzed on the basis of potentiometric and sedimentation velocity results to determine thermodynamic quantities for AMP binding to the enzyme. The comparison of calorimetric titration data of AMP and adenosine 5'-phosphoramidate at pH 6.9 supports the hypothesis previously suggested that the dianionic phosphate form of the nucleotide preferentially binds to the allosteric activator site. The thermodynamic parameters for AMP binding to site N are as follows: delta G0 = -22 kJ mol-1, delta H0 = -34 kJ mol-1 and delta S0 = -40 J mol-1 K-1. The binding of the nucleotide to site I was found to be strongly dependent on the pH. This behaviour may be explained in terms of coupled protonations of three groups having pKa values of 6.0, 6.0 and 6.1 in the unbound form and 7.0, 7.5 and 7.2 in the enzyme-nucleotide complex. The thermodynamic parameters for nucleotide binding to site I for the enzymatic form in which all the modified groups are completely deprotonated or protonated have been calculated to be: delta G0 = -7.7 kJ mol-1, delta H0 = -28 kJ mol-1 and delta S0 = -68 J mol-1 K-1 and delta G0 = -28 kJ mol-1, delta H0H = -10 kJ mol-1 and delta S0H = 61 J mol-1 K-1, respectively. These results suggest that attractive dispersion forces are of primary significance for AMP binding to activator site N, although electrostatic interactions act as a stabilizing factor in the nucleotide binding. The protonation states of those residues of which the pKa values are modified by AMP binding to site I highly influence the thermodynamic parameters for the nucleotide binding to this site.