RESUMO

The small soluble aggregates of Aß1-42 are broadly documented as potential targets for the development of new compounds with the capacity to inhibit the early stages of Alzheimer´s disease. Nevertheless, Aß1-42 peptides show an intrinsically disordered character with a high propensity for aggregation, which complicates the identification of conserved structural patterns. Because of this, experimental techniques find substantial difficulties in the characterization of such soluble oligomers. Theoretical techniques, such as molecular dynamics (MD) simulations, provide a possible workaround for this problem. However, the computational cost associated with comprehensively sampling the vast conformational space accessible to these peptides might become prohibitive. In this sense, coarse-grained (CG) simulations can effectively overcome that hurdle at a fraction of the computational cost. In this dataset, we furnish an extensive collection of Aß1-42 peptides in dimeric conformation generated with the SIRAH force field for CG MD simulation. It comprises 25 independent trajectories in .xtc (gromacs) format of Aß1-42 couples of peptides that evolve towards dimeric states along eleven µs-long unbiased simulations. Thanks to the backmapping capabilities of our force field, pseudo atomistic coordinates can be straightforwardly recovered from MD trajectories reported here and analyzed with popular molecular editing programs. This set of simulations performed at room conditions and physiological salt concentrations may furnish a complete collection of inter-peptide interfaces that can be used in high-throughput docking or as new starting states for peptide oligomerization seeding of Aß1-42 dimerization.

RESUMO

Pore forming toxins rely on oligomerization for membrane insertion to kill their targets. Bacillus thuringiensis produces insecticidal Cry-proteins composed of three domains that form pores that kill the insect larvae. Domain I is involved in oligomerization and membrane insertion, whereas Domains II and III participate in receptor binding and specificity. However, the structural changes involved in membrane insertion of these proteins remain unsolved. The most widely accepted model for membrane insertion, the 'umbrella model', proposed that the α-4/α-5 hairpin of Domain I swings away and is inserted into the membrane. To determine the topology of Cry1Ab in the membrane, disulfide bonds linking α-helices of Domain I were introduced to restrict their movement. Disulfide bonds between helices α-2/α-3 or α-3/α-4 lost oligomerization and toxicity, indicating that movement of these helices is needed for insecticidal activity. By contrast, disulfide bonds linking helices α-5/α-6 did not affect toxicity, which contradicts the 'umbrella model'. Additionally, Föster resonance energy transfer closest approach analyses measuring distances of different points in the toxin to the membrane plane and collisional quenching assays analysing the protection of specific fluorescent-labeled residues to the soluble potassium iodide quencher in the membrane inserted state were performed. Overall, the data show that Domain I from Cry1Ab may undergo a major conformational change during its membrane insertion, where the N-terminal region (helices α-1 to α-4) participates in oligomerization and toxicity, probably forming an extended helix. These data break a paradigm, showing a new 'folding white-cane model', which better explains the structural changes of Cry toxins during insertion into the membrane.

Assuntos

Bacillus thuringiensis , Inseticidas , Animais , Inseticidas/toxicidade , Bacillus thuringiensis/genética , Bacillus thuringiensis/química , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Endotoxinas/química , Proteínas Hemolisinas/metabolismo , Dissulfetos/metabolismo , Larva/metabolismo

RESUMO

The misfolding and aggregation of the protein α-synuclein (aSyn) into potentially neurotoxic oligomers is believed to play a pivotal role in the neuropathogenesis of Parkinson's disease (PD). Herein, we explore how apomorphine (Apo), a nonselective dopamine D1 and D2 receptor agonist utilized in the therapy for PD, affects the aggregation and toxicity of aSyn in vitro. Our data indicated that Apo inhibits aSyn fibrillation leading to the formation of large oligomeric species (Apo-aSyn-O), which exhibit remarkable toxicity in mesencephalic dopaminergic neurons in primary cultures. Interestingly, purified Apo-aSyn-O, even at very low concentrations, seems to be capable of converting unmodified aSyn monomer into neurotoxic species. Collectively, our findings warn for a possible dangerous effect of Apo on aSyn misfolding/aggregation pathway.

Assuntos

alfa-Sinucleína

RESUMO

Transthyretin (TTR) amyloidogenesis involves the formation, aggregation, and deposition of amyloid fibrils from tetrameric TTR in different organs and tissues. While the result of amyloidoses is the accumulation of amyloid fibrils resulting in end-organ damage, the nature, and sequence of the molecular causes leading to amyloidosis may differ between the different variants. In addition, fibril accumulation and toxicity vary between different mutations. Structural changes in amyloidogenic TTR have been difficult to identify through X-ray crystallography; but nuclear magnetic resonance spectroscopy has revealed different chemical shifts in the backbone structure of mutated and wild-type TTR, resulting in diverse responses to the cellular conditions or proteolytic stress. Toxic mechanisms of TTR amyloidosis have different effects on different tissues. Therapeutic approaches have evolved from orthotopic liver transplants to novel disease-modifying therapies that stabilize TTR tetramers and gene-silencing agents like small interfering RNA and antisense oligonucleotide therapies. The underlying molecular mechanisms of the different TTR variants could be responsible for the tropisms to specific organs, the age at onset, treatment responses, or disparities in the prognosis.

Assuntos

Neuropatias Amiloides Familiares/patologia , Amiloide/metabolismo , Mutação , Pré-Albumina/genética , Neuropatias Amiloides Familiares/etiologia , Neuropatias Amiloides Familiares/metabolismo , Animais , Humanos

RESUMO

In order to form functional filaments, human septins must assemble into hetero-oligomeric rod-like particles which polymerize end-to-end. The rules governing the assembly of these particles and the subsequent filaments are incompletely understood. Although crystallographic approaches have been successful in studying the separate components of the system, there has been difficulty in obtaining high resolution structures of the full particle. Here we report a first cryo-EM structure for a hexameric rod composed of human septins 2, 6 and 7 with a global resolution of ~3.6 Å and a local resolution of between ~3.0 Å and ~5.0 Å. By fitting the previously determined high-resolution crystal structures of the component subunits into the cryo-EM map, we are able to provide an essentially complete model for the particle. This exposes SEPT2 NC-interfaces at the termini of the hexamer and leaves internal cavities between the SEPT6-SEPT7 pairs. The floor of the cavity is formed by the two α0 helices including their polybasic regions. These are locked into place between the two subunits by interactions made with the α5 and α6 helices of the neighbouring monomer together with its polyacidic region. The cavity may serve to provide space allowing the subunits to move with respect to one another. The elongated particle shows a tendency to bend at its centre where two copies of SEPT7 form a homodimeric G-interface. Such bending is almost certainly related to the ability of septin filaments to recognize and even induce membrane curvature.

Assuntos

Proteínas de Ciclo Celular/química , Septinas/química , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Septinas/metabolismo

RESUMO

This dataset contains a collection of molecular dynamics (MD) simulations of polyglutamine (polyQ) and glutamine-rich (Q-rich) peptides in the multi-microsecond timescale. Primary data from coarse-grained simulations performed using the SIRAH force field has been processed to provide fully atomistic coordinates. The dataset encloses MD trajectories of polyQs of 4 (Q4), 11 (Q11), and 36 (Q36) amino acids long. In the case of Q11, simulations in presence of Q5 and QEQQQ peptides, which modulate aggregation, are also included. The dataset also comprises MD trajectories of the gliadin related p31-43 peptide, and Insulin's C-peptide at pH=7 and pH=3.2, which constitute examples of Q-rich and Q-poor aggregating peptides. The dataset grants molecular insights on the role of glutamines in spontaneous and unbiased ab-initio aggregation of a series of peptides using a homogeneous set of simulations [1]. The trajectory files are provided in Protein Data Bank (PDB) format containing the Cartesian coordinates of all heavy atoms in the aggregating peptides. Further analyses of the trajectories can be performed directly using any molecular visualization/analysis software suites.

RESUMO

The accumulation of amyloid protein aggregates in tissues is the basis for the onset of diseases known as amyloidoses. Intriguingly, many amyloidoses impact the central nervous system (CNS) and usually are devastating diseases. It is increasingly apparent that neurotoxic soluble oligomers formed by amyloidogenic proteins are the primary molecular drivers of these diseases, making them lucrative diagnostic and therapeutic targets. One promising diagnostic/therapeutic strategy has been the development of antibody fragments against amyloid oligomers. Antibody fragments, such as fragment antigen-binding (Fab), scFv (single chain variable fragments), and VHH (heavy chain variable domain or single-domain antibodies) are an alternative to full-length IgGs as diagnostics and therapeutics for a variety of diseases, mainly because of their increased tissue penetration (lower MW compared to IgG), decreased inflammatory potential (lack of Fc domain), and facile production (low structural complexity). Furthermore, through the use of in vitro-based ligand selection, it has been possible to identify antibody fragments presenting marked conformational selectivity. In this review, we summarize significant reports on antibody fragments selective for oligomers associated with prevalent CNS amyloidoses. We discuss promising results obtained using antibody fragments as both diagnostic and therapeutic agents against these diseases. In addition, the use of antibody fragments, particularly scFv and VHH, in the isolation of unique oligomeric assemblies is discussed as a strategy to unravel conformational moieties responsible for neurotoxicity. We envision that advances in this field may lead to the development of novel oligomer-selective antibody fragments with superior selectivity and, hopefully, good clinical outcomes.

Assuntos

Amiloide/imunologia , Amiloidose/diagnóstico , Síndromes Neurotóxicas/diagnóstico , Agregação Patológica de Proteínas/diagnóstico , Amiloide/antagonistas & inibidores , Amiloidose/imunologia , Amiloidose/patologia , Animais , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/imunologia , Síndromes Neurotóxicas/imunologia , Síndromes Neurotóxicas/patologia , Fragmentos de Peptídeos/imunologia , Agregação Patológica de Proteínas/imunologia , Anticorpos de Domínio Único , Relação Estrutura-Atividade

RESUMO

Cry proteins produced by Bacillus thuringiensis are pore-forming toxins that disrupt the membrane integrity of insect midgut cells. The structure of such pore is unknown, but it has been shown that domain I is responsible for oligomerization, membrane insertion and pore formation activity. Specifically, it was proposed that some N-terminal α-helices are lost, leading to conformational changes that trigger oligomerization. We designed a series of mutants to further analyze the molecular rearrangements at the N-terminal region of Cry1Ab toxin that lead to oligomer assembly. For this purpose, we introduced Cys residues at specific positions within α-helices of domain I for their specific labeling with extrinsic fluorophores to perform Föster resonance energy transfer analysis to fluorescent labeled Lys residues located in Domains II-III, or for disulfide bridges formation to restrict mobility of conformational changes. Our data support that helix α-1 of domain I is cleaved out and swings away from the toxin core upon binding with Manduca sexta brush border membrane vesicles. That movement of helix α-2b is also required for the conformational changes involved in oligomerization. These observations are consistent with a model proposing that helices α-2b and α-3 form an extended helix α-3 necessary for oligomer assembly of Cry toxins.

Assuntos

Bacillus cereus/metabolismo , Toxinas de Bacillus thuringiensis/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Manduca/efeitos dos fármacos , Controle Biológico de Vetores , Animais , Bacillus cereus/genética , Toxinas de Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis/metabolismo , Endotoxinas/química , Endotoxinas/genética , Endotoxinas/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Manduca/metabolismo , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Mutação , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Relação Estrutura-Atividade

RESUMO

Many studies have shown that the degree of organization and repetitiveness of an antigen correlates with its efficiency to induce a B-cell response and production of neutralizing antibodies. Here we describe the design of a chimeric protein based on the hexamer form of the highly immunogenic Fasciola hepatica leucine aminopeptidase as a carrier system of small peptides with potential use as a multiepitope vaccine.

Assuntos

Fasciola hepatica/imunologia , Proteínas de Helminto/imunologia , Leucil Aminopeptidase/imunologia , Peptídeos/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Linfócitos B/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/parasitologia , Epitopos de Linfócito B/imunologia , Fasciolíase/imunologia , Fasciolíase/parasitologia

RESUMO

Control of amylin agglomeration is of interest for both the study of pathophysiology and the design of amylin-based pharmaceutical products. Here we report the effects of a large set of common buffering agents, aminoacids and nucleoside phosphates over the amylin amyloid aggregation. Circular dichroism showed no apparent effects of the co-solutes over the secondary-structure of soluble amylin. Instead, we found a large dependence of the fibrillation process on the total amount of co-solute charged groups. The amyloid nature of the aggregates was confirmed by transmission electron microscopy, X-ray diffraction and infrared spectroscopy. While acidic pH and low-ionic co-solutes shows the largest size effect in hampering aggregation, no further effect was observed that could identify a single compound as a major direct heterotropic determinants of the amyloid process. These data suggest a more physico-chemical effect of co-solutes over the modulation of amylin instead of a chemical entity-related causal factor.

Assuntos

Amiloide/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Agregação Patológica de Proteínas , Soluções Tampão , Dicroísmo Circular/métodos , Diabetes Mellitus/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Transmissão/métodos , Estrutura Secundária de Proteína , Espectrofotometria Infravermelho/métodos , Difração de Raios X/métodos

RESUMO

Butantan Institute produces a whole cell pertussis vaccine through the fermentation, purification and inactivation processes of Bordetella pertussis cells. B. pertussis is a gram- negative cocobacillus that produces several virulence factors, including adhesins and toxins. Pertussis toxin (PT) is a major virulence factor of B. pertussis and it is considered a key component of the protective immune response against B. pertussis. It has been suggested that the protective efficacy of the whole-cell vaccine may in part be related to residual active PT, and the B oligomer of PT has already been demonstrated to act as adjuvant. The present work aimed to produce the B oligomer and evaluate it as an adjuvant for vaccines produced at Butantan Institute. To obtain oligomer B, the bacterium B. pertussis was grown in a solid medium Bordet & Gengou followed by cultivation in liquid medium to be inoculated in the horizontal Wave bioreactor. The production cultures were performed with 100 mL of the above inoculum in a total of 1 L fermentation, in the Wave horizontal bioreactor for at least 20 hours with controlled temperature and agitation. During the upstream steps, we performed the in-process control, evaluating the following parameters: pH, dissolved oxygen, opacity, optical density at 590 nm (OD590nm), microscopy (Gram staining) and presence of agglutinogens. After cultivation, in the downstream process, centrifugation was performed followed by sterile filtration of the supernatant. To check the production of PT toxin, SDS- PAGE electrophoresis and Western Blotting analysis were performed using a NIBSC reference PT, JNIH-5 (10 mg/mL) and the primary antibody NIBSC Anti-Pertussis PT JNIH- 12. For purification of the pertussis toxin, we performed ion exchange chromatography, using HiTrap CM Fast Flow column. The sample was applied to the column in 50 mM sodium phosphate buffer containing 2 M urea (equilibration buffer), pH 6.0. Then, PT was eluted from the column with Buffer A (pH 7.4) and after elution of PT, FHA was eluted in a gradient of 0-50% buffer A and B (50 mM sodium phosphate pH 7.4 containing Urea 2 M and 1 M NaCl) in a flow of 3 mL/min. After the samples were eluted, the column was washed with 100% buffer B (5 column volumes). Growth in the Wave horizontal bioreactor resulted in cultures with an opacity of 50 UOp/mL, O.D (590nm) of 2.47 and a change in pH between 6.5 and 7.8 during the time of cultivation. The agglutinogen test showed the presence of Agg1, Agg2 and Agg3. Microscopic analysis revealed characteristic gram-negative cells of B. pertussis. The analysis by SDS-PAGE and Western blotting showed bands compatible with those of the positive control, corresponding to the PT subunits. In chromatography, the proteins were eluted separately from the same column and the SDS-PAGE and Western blotting showed separate bands of PT and FHA. As a perspective, after the purification of oligomer B, we will evaluate its potential as a vaccine adjuvant.

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O Instituto Butantan produz uma vacina contra pertussis de células inteiras através dos processos de fermentação, purificação e inativação das células de Bordetella pertussis. B. pertussis é um cocobacilo gram-negativo que produz vários fatores de virulência, incluindo adesinas e toxinas. A toxina pertussis (PT) é um fator de virulência importante de B. pertussis e é considerada um componente-chave da resposta imune protetora contra B. pertussis. Foi sugerido que a eficácia protetora da vacina de células inteiras pode, em parte, estar relacionada à PT ativa residual, e já foi demonstrado que o oligômero B da PT atua como adjuvante. O presente trabalho teve como objetivo produzir o oligômero B purificado a partir de cultivos de B. pertussis a fim de avaliá-lo como adjuvante de vacinas. Para obtenção do oligômero B, a bactéria B. pertussis foi cultivada em meio sólido Bordet & Gengou seguida por cultivos em meio de cultura líquido e por produção em biorreator horizontal Wave. O cultivo de produção em Wave foi realizado num volume total de 1 L de fermentação por 20 horas, com temperatura e agitação controladas. Durante as etapas de upstream, realizamos os controles em processo, avaliando os seguintes parâmetros: pH, oxigênio dissolvido, opacidade, densidade óptica a 590 nm (D.O 590nm), microscopia (coloração de Gram) e presença de aglutinógenos. Após o cultivo, as células foram separadas do sobrenadante através de centrifugação. O sobrenadante foi filtrado estéril em membranas de 0,22 um. Para verificar a presença da toxina PT, foram realizadas eletroforese por SDS-PAGE e análise Western Blotting usando uma PT de referência NIBSC, JNIH-5 (10 mg/mL) e o anticorpo primário NIBSC Anti-Pertussis PT JNIH-12. Para a purificação da toxina pertussis foi usada cromatografia de troca iônica. A amostra foi aplicada à coluna HiTrap CM Fast Flow em tampão 50 mM fosfato de sódio contendo ureia 2 M (tampão de equilíbrio), pH 6,0. Em seguida, a PT foi eluída da coluna com Tampão A (pH 7,4) e após a eluição da PT, a FHA foi eluída em gradiente de 0-50% tampão A e B (50 mM fosfato de sódio pH 7,4, contendo Ureia 2 M e NaCl 1 M), num fluxo de 3 mL/min. Após a eluição das amostras, a coluna foi lavada com 100% tampão B (5 volumes de coluna).O crescimento no biorreator horizontal Wave resultou em cultivos com opacidade de 50 UOp/mL, D.O (590nm) de 2,47 e alteração no pH entre 6,5 e 7,8 durante o tempo de cultivo. O teste de aglutinógenos mostrou a presença de Agg1, Agg2 e Agg3. A análise microscópica revelou células gram-negativas características de B. pertussis. A análise por SDS-PAGE e Western blotting mostrou bandas compatíveis com as do controle positivo, correspondentes às subunidades de PT. Na cromatografia, as proteínas foram eluídas separadamente da mesma coluna, e o SDS–PAGE e o Western blotting mostraram as bandas separadas de PT e FHA. Como perspectiva, após a purificação do oligômero B, avaliaremos seu potencial como adjuvante de vacinas.

RESUMO

Calreticulin (CRT) is a calcium-binding protein that participates in several cellular processes including the control of protein folding and homeostasis of Ca2+. Its folding, stability and functions are strongly controlled by the presence of Ca2+. The oligomerization state of CRT is also relevant for its functions. We studied the thermal transitions of monomers and oligomers of CRT by differential scanning calorimetry (DSC), circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) in the presence and absence of Ca2+. We found three and two components for the calorimetric transition in the presence and absence of Ca2+ respectively. The presence of several components was also supported by CD and FTIR spectra acquired as a function of the temperature. The difference between the heat capacity of the native and the unfolded state strongly suggests that interactions between protein domains also contribute to the heat uptake in a calorimetry experiment. We found that once unfolded at high temperature the process is reversible and the native state can be recovered upon cooling only in the absence of Ca2+. We also propose a new simple method to obtain pure CRT oligomers.

Assuntos

Calreticulina/química , Cálcio/química , Varredura Diferencial de Calorimetria , Calreticulina/genética , Dicroísmo Circular , Conformação Proteica , Desdobramento de Proteína , Proteínas Recombinantes/química , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Termodinâmica

RESUMO

The human amylin is a pancreatic peptide hormone found in hyperhormonemic state along with insulin in subclinical diabetes. Amylin has been associated with the pathology of type 2 diabetes, particularly due to its ability to assembly into toxic oligomers and amyloid specimens. On the other hand, some variants such as murine amylin has been described as non-amyloidogenic, either in vitro or in vivo. Recent data have demonstrated the amyloid propensity of murine amylin and the therapeutic analogue pramlintide, suggesting a universality for amylin amyloidosis. Here, we report the amyloidogenesis of murine amylin, which showed lower responsivity to the fluorescent probe thioflavin T compared to human amylin, but presented highly organized fibrilar amyloid material. The aggregation of murine amylin also resulted in the formation of cytotoxic specimens, as evaluated in vitro in INS-1 cells. The aggregation product from murine amylin was responsive to a specific antibody raised against amyloid oligomers, the A11 oligomer antibody. Pancreatic islets of wild-type Swiss male mice have also shown responsivity for the anti-oligomer, indicating the natural abundance of such specimen in rodents. These data provide for the first time evidences for the toxic nature of oligomeric assemblies of murine amylin and its existence in wild-type, non-transgenic mice.

Assuntos

Amiloide/imunologia , Anticorpos/farmacologia , Células Secretoras de Insulina/imunologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/imunologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/toxicidade , Agregação Patológica de Proteínas/imunologia , Animais , Anticorpos/imunologia , Humanos , Células Secretoras de Insulina/patologia , Masculino , Camundongos , Agregação Patológica de Proteínas/patologia

RESUMO

Xylella fastidiosa is a xylem-limited bacterium that infects a wide variety of plants. Stationary phase survival protein E is classified as a nucleotidase, which is expressed when bacterial cells are in the stationary growth phase and subjected to environmental stresses. Here, we report four refined X-ray structures of this protein from X. fastidiosa in four different crystal forms in the presence and/or absence of the substrate 3'-AMP. In all chains, the conserved loop verified in family members assumes a closed conformation in either condition. Therefore, the enzymatic mechanism for the target protein might be different of its homologs. Two crystal forms exhibit two monomers whereas the other two show four monomers in the asymmetric unit. While the biological unit has been characterized as a tetramer, differences of their sizes and symmetry are remarkable. Four conformers identified by Small-Angle X-ray Scattering (SAXS) in a ligand-free solution are related to the low frequency normal modes of the crystallographic structures associated with rigid body-like protomer arrangements responsible for the longitudinal and symmetric adjustments between tetramers. When the substrate is present in solution, only two conformers are selected. The most prominent conformer for each case is associated to a normal mode able to elongate the protein by moving apart two dimers. To our knowledge, this work was the first investigation based on the normal modes that analyzed the quaternary structure variability for an enzyme of the SurE family followed by crystallography and SAXS validation. The combined results raise new directions to study allosteric features of XfSurE protein.

Assuntos

Proteínas de Bactérias/química , Plantas/microbiologia , Xylella/química , Cristalografia por Raios X , Espalhamento a Baixo Ângulo , Xylella/patogenicidade

RESUMO

In this work we study the effect of solution ionic strength on the structural evolution of amidated amyloid beta peptide Aß (1-40) oligomers at the early stages of fibril formation. By light scattering, we follow the time evolution of the structure and short-time dynamics of peptide structures at low ionic strengths. Our results allow identifying initial oligomer structures as the effective building blocks in the amyloid fibrils formation and indicate that the oligomers growth pathway, from compact structures to flexible chain-like structures, becomes faster as the solution ionic strength is increased. Furthermore, we find no evidence of structural branching what suggests that elongation of amyloid fibrils is dominated by linear association. To describe our results we adapt a phenomenological model based on population balance equations and linear polymer growth, where the parameters required are obtained from the experiments. Model calculations are in good agreement with experimentally-obtained estimates for the radius of gyration of Aß (1-40) oligomers, thus further supporting our findings. Additionally, we introduce a model for the effective interaction among initial Aß structures that captures the dependence of the effective association rates on solution ionic strength.

Assuntos

Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Peptídeos beta-Amiloides/síntese química , Cinética , Luz , Microscopia Eletrônica de Transmissão , Simulação de Dinâmica Molecular , Concentração Osmolar , Fragmentos de Peptídeos/síntese química , Estabilidade Proteica , Espalhamento de Radiação , Água/química

RESUMO

The characterization of conformational and oligomeric distribution of proteins is of paramount importance for the understanding of the correlation between structure and function. Among the bioanalytical approaches currently available, the electrospray ionization-mass spectrometry (ESI-MS) coupled to ion mobility spectrometry (IMS) is the best suited for high resolution identification with high sensitivity, allowing the in situ separation of oligomeric and conformational species. We tested the performance of the ESI-MS technique along with the IMS separation approach on a broad variety of insulin and insulin analogues with distinct oligomeric distribution pattern. The measurement of commercial insulin allowed the identification of species ranging from monomers to hexamers and their complexes with zinc ions. Dissimilar distribution profile for regular insulin as a function of formulation component and among the insulin analogues were observed by ESI-IMS-MS but not by ESI-MS along, crystallographic assays or size-exclusion chromatography. These data suggest the additional suitability of ESI-IMS-MS in conformational and oligomeric profiling of biomacromolecules and biopharmaceuticals. The easiness of the technique provides further motivation for its application in the characterization of both raw and formulated protein biopharmaceuticals in routine and comparability exercises.

Assuntos

Insulina/química , Espectrometria de Massas/métodos , Proteínas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Cromatografia em Gel/métodos , Dicroísmo Circular , Cristalização , Cristalografia por Raios X , Bases de Dados de Proteínas , Humanos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Soluções , Zinco/química

RESUMO

The secretory granule of the pancreatic ß-cells is a zinc-rich environment copopulated with the hormones amylin and insulin. The human amylin is shown to interact with zinc ions with major contribution from the single histidine residue, which is absent in amylin from other species such as cat, rhesus and rodents. We report here the interaction of murine amylin with zinc ions in vitro. The self-assembly of murine amylin is tightly regulated by zinc and pH. Ion mobility mass spectrometry revealed zinc interaction with monomers and oligomers. Nuclear magnetic resonance confirms the binding of zinc to murine amylin. The aggregation process of murine amylin into amyloid fibrils is accelerated by zinc. Collectively these data suggest a general role of zinc in the modulation of amylin variants oligomerization and amyloid fibril formation.

Assuntos

Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Zinco/farmacologia , Amiloide/biossíntese , Amiloide/efeitos dos fármacos , Animais , Concentração de Íons de Hidrogênio , Polipeptídeo Amiloide das Ilhotas Pancreáticas/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Camundongos , Agregados Proteicos/efeitos dos fármacos

RESUMO

A molecule with a π conjugated backbone built from aromatic thiophene and dialkoxyphenylene units and substituted imidazolium groups (TPO) is designed to obtain ultra-stable single walled carbon nanotube (SWCNT) dispersion in aqueous medium. The proposed mechanism of non-covalent interaction is accompanied by individualization of SWCNT and comprises of dominant nondisruptive π-π and cation-π interaction between them and the TPO conjugated oligomer. The individualization of SWCNT and dispersibility and stability of the ultra-stable suspensions were estimated using high resolution transmission electron microscopy, UV-Visible-NIR absorption spectroscopy, Raman spectroscopy, photoluminescence and zeta potential measurement. Nuclear magnetic resonance data provides direct evidence toward possible cation-π interaction.

RESUMO

Recent studies suggest that the toxic effects of Aß can be attributed to its capability to insert in membranes and form pore-like structures, which are permeable to cations and molecules such as ATP. Our working hypothesis is that Aß increases extracellular ATP causing activation of P2X receptors and potentiating excitatory synaptic activity. We found that soluble oligomers of ß-amyloid peptide increased cytosolic Ca(2+) 4-fold above control (415 ± 28% of control). Also, ATP leakage (157 ± 10% of control) was independent of extracellular Ca(2+), suggesting that ATP traveled from the cytosol through an Aß pore-mediated efflux and not from exocytotic mechanisms. The subsequent activation of P2XR by ATP can contribute to the cytosolic Ca(2+) increase observed with Aß. Additionally, we found that ß-amyloid oligomers bind preferentially to excitatory neurons inducing an increase in excitatory synaptic current frequency (248.1 ± 32.7%) that was blocked by the use of P2XR antagonists such as PPADS (Aß + PPADS: 110.9 ± 18.35%) or Apyrase plus DPCPX (Aß + inhibitors: 98.97 ± 17.4%). Taken together, we suggest that Aß induces excitotoxicity by binding preferentially to excitatory neuron membranes forming a non-selective pore and by increasing intracellular calcium by itself and through P2XR activation by extracellular ATP leading to an augmention in mEPSC activity. All these effects were blocked with a non-specific P2XR antagonist, indicating that part of the neurotoxicity of Aß is mediated by P2XR activation and facilitation of excitatory neurotransmitter release. These findings suggest that P2XR can be considered as a potential new target for the development of drugs or pharmacological tools to treat Alzheimer's disease. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'.

Assuntos

Trifosfato de Adenosina/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/toxicidade , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Hipocampo/metabolismo , Neurônios/metabolismo , Receptores Purinérgicos P2X/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos Sprague-Dawley

RESUMO

ABSTRACT Thiophene oligomer has been investigated using DFT/TDDFT calculations with an aim to check its suitability for opto electronic applications and also to analyse the influence of π-bridge. Our results revealed that thiophene oligomers have excellent π-conjugation throughout. FMO analysis give an estimate of band gap of thiophene oligomer and further revealed HOMO are localized on π - bridge, donor group and LUMO are localized on π - bridge and acceptor group. A TDDFT calculation has been performed to understand the absorption properties of them in gas phase and solvent phase. PCM calculations convey that absorption maxima show positive solvatochromism. Among the designed candidates, the one with more π - bridge show higher wavelength of absorption maxima and would be a choice for better optoelectronic materials. NBO analysis provides support for complete delocalization in these systems. It is interesting to note that oligomer with more π-bridge display an enhanced optoelectronic properties than with less π - bridge.