Amphipathic alpha-helix AH2 is a major determinant for the oligomerization of hepatitis C virus nonstructural protein 4B.

Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. It induces a specific membrane rearrangement, designated membranous web, that serves as a scaffold for the HCV replication complex. However, the mechanisms underlying membranous web formation are poorly understood. Based on fluorescence resonance energy transfer (FRET) and confirmatory coimmunoprecipitation analyses, we provide evidence for an oligomerization of NS4B in the membrane environment of intact cells. Several conserved determinants were found to be involved in NS4B oligomerization, through homotypic and heterotypic interactions. N-terminal amphipathic ?-helix AH2, comprising amino acids 42 to 66, was identified as a major determinant for NS4B oligomerization. Mutations that affected the oligomerization of NS4B disrupted membranous web formation and HCV RNA replication, implying that oligomerization of NS4B is required for the creation of a functional replication complex. These findings enhance our understanding of the functional architecture of the HCV replication complex and may provide new angles for therapeutic intervention. At the same time, they expand the list of positive-strand RNA virus replicase components acting as oligomers.

A common BACE1 polymorphism is a risk factor for sporadic Creutzfeldt-Jakob disease

The β site APP cleaving enzyme 1 (BACE1) is the rate-limiting β-secretase enzyme in the amyloidogenic processing of APP and Aβ formation, and therefore it has a prominent role in Alzheimer"s disease (AD) pathology. Recent evidence suggests that the prion protein (PrP) interacts directly with BACE1 regulating its β-secretase activity. Moreover, PrP has been proposed as the cellular receptor involved in the impairment of synaptic plasticity and toxicity caused by Aβ oligomers. Provided that common pathophysiologic mechanisms are shared by Alzheimer"s and Creutzfeldt-Jakob (CJD) diseases, we investigated for the first time to the best of our knowledge a possible association of a common synonymous BACE1 polymorphism (rs638405) with sporadic CJD (sCJD). Our results indicate that BACE1 C-allele is associated with an increased risk for developing sCJD, mainly in PRNP M129M homozygous subjects with early onset. These results extend the very short list of genes (other than PRNP) involved in the development of human prion diseases; and support the notion that similar to AD, in sCJD several loci may contribute with modest overall effects to disease risk. These findings underscore the interplay in both pathologies of APP, Aβ oligomers, ApoE, PrP and BACE1, and suggest that aging and perhaps vascular risk factors may modulate disease pathologies in part through these key players

Protein loop compaction and the origin of the effect of arginine and glutamic acid mixtures on solubility, stability and transient oligomerization of proteins

Addition of a 50 mM mixture of l-arginine and l-glutamic acid (RE) is extensively used to improve protein solubility and stability, although the origin of the effect is not well understood. We present Small Angle X-ray Scattering (SAXS) and Nuclear Magnetic Resonance (NMR) results showing that RE induces protein compaction by collapsing flexible loops on the protein core. This is suggested to be a general mechanism preventing aggregation and improving resistance to proteases and to originate from the polyelectrolyte nature of RE. Molecular polyelectrolyte mixtures are expected to display long range correlation effects according to dressed interaction site theory. We hypothesize that perturbation of the RE solution by dissolved proteins is proportional to the volume occupied by the protein. As a consequence, loop collapse, minimizing the effective protein volume, is favored in the presence of RE.

The Human Acid-Sensing Ion Channel ASIC1a: Evidence for a Homotetrameric Assembly State at the Cell Surface.

The chicken acid-sensing ion channel ASIC1 has been crystallized as a homotrimer. We address here the oligomeric state of the functional ASIC1 in situ at the cell surface. The oligomeric states of functional ASIC1a and mutants with additional cysteines introduced in the extracellular pore vestibule were resolved on SDS-PAGE. The functional ASIC1 complexes were stabilized at the cell surface of Xenopus laevis oocytes or CHO cells either using the sulfhydryl crosslinker BMOE, or sodium tetrathionate (NaTT). Under these different crosslinking conditions ASIC1a migrates as four distinct oligomeric states that correspond by mass to multiples of a single ASIC1a subunit. The relative importance of each of the four ASIC1a oligomers was critically dependent on the availability of cysteines in the transmembrane domain for crosslinking, consistent with the presence of ASIC1a homo-oligomers. The expression of ASIC1a monomers, trimeric or tetrameric concatemeric cDNA constructs resulted in functional channels. The resulting ASIC1a complexes are resolved as a predominant tetramer over the other oligomeric forms, after stabilization with BMOE or NaTT and SDS-PAGE/western blot analysis. Our data identify a major ASIC1a homotetramer at the surface membrane of the cell expressing functional ASIC1a channel.

Methylglyoxal Produced by Amyloid- Peptide-Induced Nitrotyrosination of Triosephosphate Isomerase Triggers Neuronal Death in Alzheimer’s Disease

Amyloid-β peptide (Aβ) aggregates induce nitro-oxidative stress, contributing to the characteristic neurodegeneration found in Alzheimer's disease (AD). One of the most strongly nitrotyrosinated proteins in AD is the triosephosphate isomerase (TPI) enzyme which regulates glycolytic flow, and its efficiency decreased when it is nitrotyrosinated. The main aims of this study were to analyze the impact of TPI nitrotyrosination on cell viability and to identify the mechanism behind this effect. In human neuroblastoma cells (SH-SY5Y), we evaluated the effects of Aβ42 oligomers on TPI nitrotyrosination. We found an increased production of methylglyoxal (MG), a toxic byproduct of the inefficient nitro-TPI function. The proapoptotic effects of Aβ42 oligomers, such as decreasing the protective Bcl2 and increasing the proapoptotic caspase-3 and Bax, were prevented with a MG chelator. Moreover, we used a double mutant TPI (Y165F and Y209F) to mimic nitrosative modifications due to Aβ action. Neuroblastoma cells transfected with the double mutant TPI consistently triggered MG production and a decrease in cell viability due to apoptotic mechanisms. Our data show for the first time that MG is playing a key role in the neuronal death induced by Aβ oligomers. This occurs because of TPI nitrotyrosination, which affects both tyrosines associated with the catalytic center.

On the role of G protein-coupled receptors oligomerization

The existence of a supramolecular organization of the G protein-coupled receptor (GPCR) is now being widely accepted by the scientific community. Indeed, GPCR oligomers may enhance the diversity and performance by which extracellular signals are transferred to the G proteins in the process of receptor transduction, although the mechanism that underlies this phenomenon still remains unsolved. Recently, it has been proposed that a trans-conformational switching model could be the mechanism allowing direct inhibition/activation of receptor activation/inhibition, respectively. Thus, heterotropic receptor-receptor allosteric regulations are behind the GPCR oligomeric function. In this paper we want to revise how GPCR oligomerization impinges on several important receptor functions like biosynthesis, plasma membrane diffusion or velocity, pharmacology and signaling. In particular, the rationale of receptor oligomerization might lie in the need of sensing complex whole cell extracellular signals and translating them into a simple computational model.

BioSuper: A web tool for the superimposition of biomolecules and assemblies with rotational symmetry

Background Most of the proteins in the Protein Data Bank (PDB) are oligomeric complexes consisting of two or more subunits that associate by rotational or helical symmetries. Despite the myriad of superimposition tools in the literature, we could not find any able to account for rotational symmetry and display the graphical results in the web browser. Results BioSuper is a free web server that superimposes and calculates the root mean square deviation (RMSD) of protein complexes displaying rotational symmetry. To the best of our knowledge, BioSuper is the first tool of its kind that provides immediate interactive visualization of the graphical results in the browser, biomolecule generator capabilities, different levels of atom selection, sequence-dependent and structure-based superimposition types, and is the only web tool that takes into account the equivalence of atoms in side chains displaying symmetry ambiguity. BioSuper uses ICM program functionality as a core for the superimpositions and displays the results as text, HTML tables and 3D interactive molecular objects that can be visualized in the browser or in Android and iOS platforms with a free plugin. Conclusions BioSuper is a fast and functional tool that allows for pairwise superimposition of proteins and assemblies displaying rotational symmetry. The web server was created after our own frustration when attempting to superimpose flexible oligomers. We strongly believe that its user-friendly and functional design will be of great interest for structural and computational biologists who need to superimpose oligomeric proteins (or any protein). BioSuper web server is freely available to all users at http://ablab.ucsd.edu/BioSuper webcite.

Protected maleimide building blocks for the decoration of peptides, peptoids and peptide nucleic acids

Monomers allowing for the introduction of [2,5-dimethylfuran]-protected maleimides into polyamides such as peptides, peptide nucleic acids, and peptoids were prepared, as well as the corresponding oligomers. Suitable maleimide deprotection conditions were established in each case. The stability of the adducts generated by Michael-type maleimide-thiol reaction and Diels-Alder cycloaddition to maleimide deprotection conditions was exploited to prepare a variety of conjugates from peptide and PNA scaffolds incorporating one free and one protected maleimide. The target molecules were synthesized by using two subsequent maleimide-involving click reactions separated by a maleimide deprotection step. Carrying out maleimide deprotection and conjugation simultaneously gave better results than performing the two reactions subsequently.

Síntese de membranas cerâmicas via método sol-gel utilizando TEOS e N,N-dimetilformamida

In this work we obtained microporous and mesoporous silica membranes by sol-gel processing. Tetraethylortosilicate (TEOS) was used as precursor. Nitric acid was used as catalyst. In order to study the affect of N,N-dimethylformamide (NDF) as drying additive, we used a molar ratio TEOS/NDF of 1/3. The performance of N,N-dimethylformamide was evaluated through monolithicity measurements. The structural evolutions occurring during the sol-gel transition and in the interconnected network of the membranes during thermal treatment were monitored by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analyses and nitrogen sorption. We noted that in the presence of N,N-dimethylformamide, polymerization goes through a temporary stabilization of oligomers. The Si-O(H) bonds are stronger and belong to a more cross-linked structure for the N,N-dimethylformamide containing sol. The membranes obtained in the presence of N,N-dimethylformamide have larger surface area and its pore structure is in the range of mesoporous. The membranes obtained without additive have pore structure in the range of microporous.

Identification of acetates in elasmopalpulus lignosellus pheromone glands using a newly created mass spectral database and kóvats retention indices

Based on a specially created mass spectral database utilizing 23 tetradecenyl and 22 hexadecenyl acetate standards along with Kóvats retention indices obtained on a very polar stationary phase [poly (biscyanopropyl siloxane)] (SP 2340), (Z)-9-hexadecenyl acetate, (Z)-11-hexadecenyl acetate and (E)-8-hexadecenyl acetate were identified in active pheromone extracts of Elasmopalpus lignosellus. This identification was more efficient than our previous study using gas chromatography/mass spectrometry with a dimethyl disulfide derivative where we could only identify the first two acetates. The acetate composition of the pheromone gland differed from region to region in Brazil and from that from the Tifton (GA, USA) population, suggesting polymorphism or a different sub-species.

Desenvolvimento de sistemas líquido-cristalinos empregando silicone fluido de co-polímero glicol e poliéter funcional siloxano

For the construction of the phase diagrams, the method of the aqueous titration was used. There were prepared 5 ternary diagrams, varying the surfactant and the oil phase. The liquid-crystalline phases were identified by polarized light microscopy. The formulations prepared with silicon glycol copolymer, polyether functional siloxane (PFS) and water (S1) and with diisopropyl adipate, PFS and water (S4) presented liquid-crystalline phases with lamellar arrangement. Moreover, after 15 days in hot oven (37 ºC), the formulations presented hexagonal arrangement, evidencing the influence of the temperature in the organization of the system.

Polimerização do glicerol: uma reação simples e versátil para produzir diferentes materiais a partir do coproduto do biodiesel

In this work, it is proposed a simple experiment in polymer science for undergraduates, involving the glycerol polymerization catalyzed by H2SO4 to produce different materials, e.g. from oligomers to crosslinked polymer. The students can investigate the reaction conditions such as time, temperature and catalyst concentration, thus controlling the extent of polymerization and its kinetics. This experiment stimulates students to see polymer science in a broader context, involving diverse topics, such as biodiesel, and processing of a co-product of low value in polymeric materials with potential industrial application.

DEGRADAÇÃO MICROBIOLÓGICA E ENZIMÁTICA DE POLÍMEROS: UMA REVISÃO

Polymer recycling has been one of the most important trend in the petrochemical area. Among different technologies, biotechnological (enzymatic and/or microbial) degradation of polymers for the recovery of monomers and oligomers is environmentally-friendly and meet some green chemistry principles. In this work, conditions for the biotechnological degradation of some industrially-relevant polymers (e.g. poly(ethylene terephthalate) and polyethylene) were revised, and the main biocatalysts were identified. In most cases, biodegradation mechanisms are still unclear, thus being necessary more studies to unravel these promising bioprocesses. Polymer biodegradation studies also present considerable importance for other fields, including biomedical and agricultural.

Extraction of polymeric galactoglucomannans from spruce wood by pressurized hot water

The major type of non-cellulosic polysaccharides (hemicelluloses) in softwoods, the partly acetylated galactoglucomannans (GGMs), which comprise about 15% of spruce wood, have attracted growing interest because of their potential to become high-value products with applications in many areas. The main objective of this work was to explore the possibilities to extract galactoglucomannans in native, polymeric form in high yield from spruce wood with pressurised hot-water, and to obtain a deeper understanding of the process chemistry involved. Spruce (Picea abies) chips and ground wood particles were extracted using an accelerated solvent extractor (ASE) in the temperature range 160 – 180°C. Detailed chemical analyses were done on both the water extracts and the wood residues. As much as 80 – 90% of the GGMs in spruce wood, i.e. about 13% based on the original wood, could be extracted from ground spruce wood with pure water at 170 – 180°C with an extraction time of 60 min. GGMs comprised about 75% of the extracted carbohydrates and about 60% of the total dissolved solids. Other substances in the water extracts were xylans, arabinogalactans, pectins, lignin and acetic acid. The yields from chips were only about 60% of that from ground wood. Both the GGMs and other non-cellulosic polysaccharides were extensively hydrolysed at severe extraction conditions when pH dropped to the level of 3.5. Addition of sodium bicarbonate increased the yields of polymeric GGMs at low additions, 2.5 – 5 mM, where the end pH remained around 3.9. However, at higher addition levels the yields decreased, mainly because the acetyl groups in GGMs were split off, leading to a low solubility of GGMs. Extraction with buffered water in the pH range 3.8 – 4.4 gave similar yields as with plain water, but gave a higher yield of polymeric GGMs. Moreover, at these pH levels the hydrolysis of acetyl groups in GGMs was significantly inhibited. It was concluded that hot-water extraction of polymeric GGMs in good yields (up to 8% of wood) demands appropriate control of pH, in a narrow range about 4. These results were supported by a study of hydrolysis of GGM at constant pH in the range of 3.8 – 4.2 where a kinetic model for degradation of GGM was developed. The influence of wood particle size on hot-water extraction was studied with particles in the range of 0.1 – 2 mm. The smallest particles (< 0.1 mm) gave 20 – 40% higher total yield than the coarsest particles (1.25 – 2 mm). The difference was greatest at short extraction times. The results indicated that extraction of GGMs and other polysaccharides is limited mainly by the mass transfer in the fibre wall, and for coarse wood particles also in the wood matrix. Spruce sapwood, heartwood and thermomechnical pulp were also compared, but only small differences in yields and composition of extracts were found. Two methods for isolation and purification of polymeric GGMs, i.e. membrane filtration and precipitation in ethanol-water, were compared. Filtration through a series of membranes with different pore sizes separated GGMs of different molar masses, from polymers to oligomers. Polysaccharides with molar mass higher than 4 kDa were precipitated in ethanol-water. GGMs comprised about 80% of the precipitated polysaccharides. Other polysaccharides were mainly arabinoglucuronoxylans and pectins. The ethanol-precipitated GGMs were by 13C NMR spectroscopy verified to be very similar to GGMs extracted from spruce wood in low yield at a much lower temperature, 90°C. The obtained large body of experimental data could be utilised for further kinetic and economic calculations to optimise technical hot-water extractionof softwoods.