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del Rio, Jose A., Ferrer, Isidre, (2020). Potential of microfluidics and lab-on-chip platforms to improve understanding of “prion-like” protein assembly and behavior Frontiers in Bioengineering and Biotechnology 8, 570692

Human aging is accompanied by a relevant increase in age-associated chronic pathologies, including neurodegenerative and metabolic diseases. The appearance and evolution of numerous neurodegenerative diseases is paralleled by the appearance of intracellular and extracellular accumulation of misfolded proteins in affected brains. In addition, recent evidence suggests that most of these amyloid proteins can behave and propagate among neural cells similarly to infective prions. In order to improve understanding of the seeding and spreading processes of these “prion-like” amyloids, microfluidics and 3D lab-on-chip approaches have been developed as highly valuable tools. These techniques allow us to monitor changes in cellular and molecular processes responsible for amyloid seeding and cell spreading and their parallel effects in neural physiology. Their compatibility with new optical and biochemical techniques and their relative availability have increased interest in them and in their use in numerous laboratories. In addition, recent advances in stem cell research in combination with microfluidic platforms have opened new humanized in vitro models for myriad neurodegenerative diseases affecting different cellular targets of the vascular, muscular, and nervous systems, and glial cells. These new platforms help reduce the use of animal experimentation. They are more reproducible and represent a potential alternative to classical approaches to understanding neurodegeneration. In this review, we summarize recent progress in neurobiological research in “prion-like” protein using microfluidic and 3D lab-on-chip approaches. These approaches are driven by various fields, including chemistry, biochemistry, and cell biology, and they serve to facilitate the development of more precise human brain models for basic mechanistic studies of cell-to-cell interactions and drug discovery.

Keywords: Lab-On-Chip, Amyloid propagation, Microfluidics, Fibril, Seeding, Spreading, Prion-like, Prionoid

Beiert, T., Tiyerili, V., Knappe, V., Effelsberg, V., Linhart, M., Stöckigt, F., Klein, S., Schierwagen, R., Trebicka, J., Nickenig, G., Schrickel, J. W., Andrié, R. P., (2017). Relaxin reduces susceptibility to post-infarct atrial fibrillation in mice due to anti-fibrotic and anti-inflammatory properties Biochemical and Biophysical Research Communications , 490, (3), 643-649

Background Relaxin-2 (RLX) is a peptide hormone that exerts beneficial anti-fibrotic and anti-inflammatory effects in diverse models of cardiovascular disease. The goal of this study was to determine the effects of RLX treatment on the susceptibility to atrial fibrillation (AF) after myocardial infarction (MI). Methods Mice with cryoinfarction of the left anterior ventricular wall were treated for two weeks with either RLX (75 μg/kg/d) or vehicle (sodium acetate) delivered via subcutaneously implanted osmotic minipumps. Results RLX treatment significantly attenuated the increase in AF-inducibility following cryoinfarction and reduced the mean duration of AF episodes. Furthermore, epicardial mapping of both atria revealed an increase in conduction velocity. In addition to an attenuation of atrial hypertrophy, chronic application of RLX reduced atrial fibrosis, which was linked to a significant reduction in atrial mRNA expression of connective tissue growth factor. Transcript levels of the pro-inflammatory cytokines interleukin-6 and interleukin-1β were reduced in RLX treated mice, but macrophage infiltration into atrial myocardium was similar in the vehicle and RLX treated groups. Conclusion Treatment with RLX in mice after MI reduces susceptibility to AF due to anti-inflammatory and anti-fibrotic properties. Because to these favorable actions, RLX may become a new therapeutic option in the treatment of AF, even when complicating MI.

Keywords: Atrial fibrillation, Atrial fibrosis, Myocardial infarction, Relaxin-2

Beun, L. H., Albertazzi, L., Van Der Zwaag, D., De Vries, R., Cohen Stuart, M. A., (2016). Unidirectional living growth of self-assembled protein nanofibrils revealed by super-resolution microscopy ACS Nano 10, (5), 4973-4980

Protein-based nanofibrils are emerging as a promising class of materials that provide unique properties for applications such as biomedical and food engineering. Here, we use atomic force microscopy and stochastic optical reconstruction microscopy imaging to elucidate the growth dynamics, exchange kinetics, and polymerization mechanism for fibrils composed of a de novo designed recombinant triblock protein polymer. This macromolecule features a silk-inspired self-assembling central block composed of GAGAGAGH repeats, which are known to fold into a β roll with turns at each histidine and, once folded, to stack, forming a long, ribbon-like structure. We find several properties that allow the growth of patterned protein nanofibrils: the self-assembly takes place on only one side of the growing fibrils by the essentially irreversible addition of protein polymer subunits, and these fibril ends remain reactive indefinitely in the absence of monomer ("living ends"). Exploiting these characteristics, we can grow stable diblock protein nanofibrils by the sequential addition of differently labeled proteins. We establish control over the block length ratio by simply varying monomer feed conditions. Our results demonstrate the use of engineered protein polymers in creating precisely patterned protein nanofibrils and open perspectives for the hierarchical self-assembly of functional biomaterials.

Keywords: Nanofibrils, Protein polymers, Self-assembly, STORM microscopy

Arimon, M., Sanz, F., Giralt, E., Carulla, N., (2012). Template-assisted lateral growth of amyloid-β42 fibrils studied by differential labeling with gold nanoparticles Bioconjugate Chemistry , 23, (1), 27-32

Amyloid-β protein (Aβ) aggregation into amyloid fibrils is central to the origin and development of Alzheimer’s disease (AD), yet this highly complex process is poorly understood at the molecular level. Extensive studies have shown that Aβ fibril growth occurs through fibril elongation, whereby soluble molecules add to the fibril ends. Nevertheless, fibril morphology strongly depends on aggregation conditions. For example, at high ionic strength, Aβ fibrils laterally associate into bundles. To further study the mechanisms leading to fibril growth, we developed a single-fibril growth assay based on differential labeling of two Aβ42 variants with gold nanoparticles. We used this assay to study Aβ42 fibril growth under different conditions and observed that bundle formation is preceded by lateral interaction of soluble Aβ42 molecules with pre-existing fibrils. Based on this data, we propose template-assisted lateral fibril growth as an additional mechanism to elongation for Aβ42 fibril growth.

Keywords: AFM, Beta-Amyloid Fibrils, Polymorphism, Association, Elongation, Dynamics, State

Valle-Delgado, J. J., Liepina, I., Lapidus, D., Sabaté, R., Ventura, S., Samitier, J., Fernàndez-Busquets, X., (2012). Self-assembly of human amylin-derived peptides studied by atomic force microscopy and single molecule force spectroscopy Soft Matter 8, (4), 1234-1242

The self-assembly of peptides and proteins into amyloid fibrils of nanometric thickness and up to several micrometres in length, a phenomenon widely observed in biological systems, has recently aroused a growing interest in nanotechnology and nanomedicine. Here we have applied atomic force microscopy and single molecule force spectroscopy to study the amyloidogenesis of a peptide derived from human amylin and of its reverse sequence. The spontaneous formation of protofibrils and their orientation along well-defined directions on graphite and DMSO-coated graphite substrates make the studied peptides interesting candidates for nanotechnological applications. The measured binding forces between peptides correlate with the number of hydrogen bonds between individual peptides inside the fibril structure according to molecular dynamics simulations.

Keywords: Amyloid fibril, Amyloidogenesis, Binding forces, Fibril structure, Graphite substrate, Molecular dynamics simulations, Nanometrics, Protofibrils, Single molecule force spectroscopy, Spontaneous formation, Atomic force microscopy, Atomic spectroscopy, Graphite, Hydrogen bonds, Medical nanotechnology, Molecular dynamics, Molecular physics, Self assembly, Thickness measurement, Peptides

Villar-Pique, A., De Groot, N. S., Sabaté, R., Acebrón, S. P., Celaya, G., Fernàndez-Busquets, X., Muga, A., Ventura, S., (2012). The effect of amyloidogenic peptides on bacterial aging correlates with their intrinsic aggregation propensity Journal of Molecular Biology , 421, (2-3), 270-281

The formation of aggregates by misfolded proteins is thought to be inherently toxic, affecting cell fitness. This observation has led to the suggestion that selection against protein aggregation might be a major constraint on protein evolution. The precise fitness cost associated with protein aggregation has been traditionally difficult to evaluate. Moreover, it is not known if the detrimental effect of aggregates on cell physiology is generic or depends on the specific structural features of the protein deposit. In bacteria, the accumulation of intracellular protein aggregates reduces cell reproductive ability, promoting cellular aging. Here, we exploit the cell division defects promoted by the intracellular aggregation of Alzheimer's-disease-related amyloid β peptide in bacteria to demonstrate that the fitness cost associated with protein misfolding and aggregation is connected to the protein sequence, which controls both the in vivo aggregation rates and the conformational properties of the aggregates. We also show that the deleterious impact of protein aggregation on bacterial division can be buffered by molecular chaperones, likely broadening the sequential space on which natural selection can act. Overall, the results in the present work have potential implications for the evolution of proteins and provide a robust system to experimentally model and quantify the impact of protein aggregation on cell fitness.

Keywords: Amyloid fibrils, Chaperones, Escherichia coli, Inclusion bodies, Protein aggregation

Valle-Delgado, J. J., Alfonso-Prieto, M., de Groot, N. S., Ventura, S., Samitier, J., Rovira, C., Fernàndez-Busquets, X., (2010). Modulation of A beta(42) fibrillogenesis by glycosaminoglycan structure FASEB Journal , 24, (11), 4250-4261

The role of amyloid beta (A beta) peptide in the onset and progression of Alzheimer's disease is linked to the presence of soluble A beta species. Sulfated glycosaminoglycans (GAGs) promote A beta fibrillogenesis and reduce the toxicity of the peptide in neuronal cell cultures, but a satisfactory rationale to explain these effects at the molecular level has not been provided yet. We have used circular dichroism, Fourier transform infrared spectroscopy, fluorescence microscopy and spectroscopy, protease digestion, atomic force microscopy (AFM), and molecular dynamics simulations to characterize the association of the 42-residue fragment A beta(42) with sulfated GAGs, hyaluronan, chitosan, and poly(vinyl sulfate) (PVS). Our results indicate that the formation of stable A beta(42) fibrils is promoted by polymeric GAGs with negative charges placed in-frame with the 4.8-angstrom separating A beta(42) monomers within protofibrillar beta-sheets. Incubation of A beta(42) with excess sulfated GAGs and hyaluronan increased amyloid fibril content and resistance to proteolysis 2- to 5-fold, whereas in the presence of the cationic polysaccharide chitosan, A beta(42) fibrillar species were reduced by 25% and sensitivity to protease degradation increased similar to 3-fold. Fibrils of intermediate stability were obtained in the presence of PVS, an anionic polymer with more tightly packed charges than GAGs. Important structural differences between A beta(42) fibrils induced by PVS and A beta(42) fibrils obtained in the presence of GAGs and hyaluronan were observed by AFM, whereas mainly precursor protofibrillar forms were detected after incubation with chitosan. Computed binding energies per peptide from -11.2 to -13.5 kcal/mol were calculated for GAGs and PVS, whereas a significantly lower value of -7.4 kcal/mol was obtained for chitosan. Taken together, our data suggest a simple and straightforward mechanism to explain the role of GAGs as enhancers of the formation of insoluble A beta(42) fibrils trapping soluble toxic forms.

Keywords: Alzheimer's disease, Amyloid fibril structure, Fibrillogenesis enhancers and inhibitors, Polysaccharides

Fernàndez-Busquets, X., Ponce, J., Bravo, R., Arimon, M., Martianez, T., Gella, A., Cladera, J., Durany, N., (2010). Modulation of amyloid beta peptide(1-42) cytotoxicity and aggregation in vitro by glucose and chondroitin sulfate Current Alzheimer Research , 7, (5), 428-438

One mechanism leading to neurodegeneration during Alzheimer's Disease (AD) is amyloid beta peptide (A beta)-induced neurotoxicity. Among the factors proposed to potentiate A beta toxicity is its covalent modification through carbohydrate-derived advanced glycation endproducts (AGEs). Other experimental evidence, though, indicates that certain polymeric carbohydrates like the glycosaminoglycan (GAG) chains found in proteoglycan molecules attenuate the neurotoxic effect of A beta in primary neuronal cultures. Pretreatment of the 42-residue A beta fragment (A beta(1-42)) with the ubiquitous brain carbohydrates, glucose, fructose, and the GAG chondroitin sulfate B (CSB) inhibits A beta beta(1-42)-induced apoptosis and reduces the peptide neurotoxicity on neuroblastoma cells, a cytoprotective effect that is partially reverted by AGE inhibitors such as pyridoxamine and L-carnosine. Thioflavin T fluorescence measurements indicate that at concentrations close to physiological, only CSB promotes the formation of A beta amyloid fibril structure. Atomic force microscopy imaging and Western blot analysis suggest that glucose favours the formation of globular oligomeric structures derived from aggregated species. Our data suggest that at short times carbohydrates reduce A beta(1-42) toxicity through different mechanisms both dependent and independent of AGE formation.

Keywords: Alzheimer's disease, Advanced glycation endproducts, Amyloid fibrils, Amyloid beta peptide, Apoptosis, Carbohydrates, Glycosaminoglycans

Rico, P., Rodriguez Hernandez, J. C., Moratal, D., Altankov, G., Monleon Pradas, M., Salmeron-Sanchez, M., (2009). Substrate-induced assembly of fibronectin into networks. Influence of surface chemistry and effect on osteoblast adhesion Tissue Engineering Part A , 15, (00), 1-11

The influence of surface chemistry -substrates with controlled surface density of -OH groups- on fibronectin conformation and distribution is directly observed by Atomic Force Microscopy (AFM). FN fibrillogenesis, which is known to be a process triggered by interaction with integrins, is shown in our case to be induced by the substrate (in absence of cells), which is able to enhance FN-FN interactions leading to the formation of a protein network on the material surface. This phenomenon depends both on surface chemistry and protein concentration. The level of the FN fibrillogenesis was quantified by calculating the fractal dimension of the adsorbed protein from image analysis of the AFM results. The total amount of adsorbed FN is obtained by making use of a methodology which employs western-blotting combined with image analysis of the corresponding protein bands, with the lowest sensitivity threshold equal to 15 ng of adsorbed protein. Furthermore, FN adsorption is correlated to human osteoblast adhesion through morphology and actin cytoskeleton formation. Actin polymerization is in need of the formation of the protein network on the substrate's surface. Cell morphology is more rounded (as quantified by calculating the circularity of the cells by image analysis) the lower the degree of FN fibrillogenesis on the substrate.

Keywords: Cell-adhesion, Conformational-changes, Electron-microscopy, Protein adsorption, Fractal dimension, Integrin binding, Biocompatibility, Monolayers, Matrix, Fibrillogenesis

Morell, M., Bravo, R., Espargaro, A., Sisquella, X., Aviles, F. X., Fernàndez-Busquets, X., Ventura, S., (2008). Inclusion bodies: Specificity in their aggregation process and amyloid-like structure Biochimica et Biophysica Acta - Molecular Cell Research , 1783, (10), 1815-1825

The accumulation of aggregated protein in the cell is associated with the pathology of many diseases and constitutes a major concern in protein production. Intracellular aggregates have been traditionally regarded as nonspecific associations of misfolded polypeptides. This view is challenged by studies demonstrating that, in vitro, aggregation often involves specific interactions. However, little is known about the specificity of in vivo protein deposition. Here, we investigate the degree of in vivo co-aggregation between two self-aggregating proteins, A beta A2 amyloid peptide and foot-and-mouth disease virus VP1 capsid protein, in prokaryotic cells. In addition, the ultrastructure of intracellular aggregates is explored to decipher whether amyloid fibrils and intracellular protein inclusions share structural properties. The data indicate that in vivo protein aggregation exhibits a remarkable specificity that depends on the establishment of selective interactions and results in the formation of oligomeric and fibrillar structures displaying amyloid-like properties. These features allow prokaryotic A beta A2 intracellular aggregates to act as effective seeds in the formation of A beta A2 amyloid fibrils. overall, our results suggest that conserved mechanisms underlie protein aggregation in different organisms. They also have important implications for biotechnological and biomedical applications of recombinant polypeptides.

Keywords: Protein aggregation, Inclusion bodies, Conformational diseases, Amyloid fibrils, Protein folding