Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 8 de 8
Filter
Add more filters










Database
Language
Publication year range
1.
J Phys Condens Matter ; 24(24): 244103, 2012 Jun 20.
Article in English | MEDLINE | ID: mdl-22595421

ABSTRACT

Recognizing the complexity of the fibrillogenesis process provides a solid ground for the development of therapeutic strategies aimed at preventing or inhibiting protein-protein aggregation. Under this perspective, it is meaningful to identify the possible aggregation pathways and their relative products. We found that Aß-peptide dissolved in a pH 7.4 solution at small peptide concentration and low ionic strength forms globular aggregates without typical amyloid ß-conformation. ThT binding kinetics was used to monitor aggregate formation. Circular dichroism spectroscopy, AFM imaging, static and dynamic light scattering were used for structural and morphological characterization of the aggregates. They appear stable or at least metastable with respect to fiber growth, therefore appearing as an incidental product in the pathway of fibrillogenesis.


Subject(s)
Amyloid beta-Peptides/chemistry , Peptide Fragments/chemistry , Protein Multimerization , Kinetics , Osmolar Concentration , Protein Stability , Protein Structure, Secondary , Spectrometry, Fluorescence , Temperature
2.
Biochim Biophys Acta ; 1820(2): 124-32, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22155633

ABSTRACT

BACKGROUND: α(s1)-Casein is one of the four types of caseins, the largest protein component of bovine milk. The lack of a compact folded conformation and the capability to form micelles suggest a relationship of α(s1)-casein with the class of the intrinsically disordered (or natively unfolded) proteins. These proteins are known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces. In the present work we focused on the effect of α(s1)-casein on the fibrillogenesis of 1-40 ß-amyloid peptide, involved in Alzheimer's disease. METHODS: The aggregation kinetics of ß-peptide in presence and absence of α(s1)-casein was followed under shear at 37°C by recording the Thioflavine fluorescence, usually taken as an indicator of fibers formation. Measurements of Static and Dynamic Light Scattering, Circular Dichroism, and AFM imaging were done to reveal the details of α(s1)-casein-Aß(1-40) interaction. RESULTS AND DISCUSSIONS: α(s1)-Casein addition sizably increases the lag-time of the nucleation phase and slows down the entire fibrillization process. α(s1)-Casein sequesters the amyloid peptide on its surface thus exerting a chaperone-like activity by means a colloidal inhibition mechanism. GENERAL SIGNIFICANCE: Insights on the working mechanism of natural chaperones in preventing or controlling the amyloid aggregation.


Subject(s)
Amyloid beta-Peptides/antagonists & inhibitors , Amyloid/drug effects , Caseins/pharmacology , Peptide Fragments/antagonists & inhibitors , Amyloid beta-Peptides/metabolism , Benzothiazoles , Circular Dichroism , Hydrodynamics , Light , Microscopy, Atomic Force , Particle Size , Peptide Fragments/metabolism , Protein Structure, Quaternary , Scattering, Radiation , Spectrometry, Fluorescence , Thiazoles/metabolism , Time Factors
3.
J Phys Chem B ; 115(12): 2691-8, 2011 Mar 31.
Article in English | MEDLINE | ID: mdl-21391626

ABSTRACT

Mechanisms of partial unfolding and aggregation of proteins are of extreme interest in view of the fact that several human pathologies are characterized by the formation and deposition of protein-insoluble material, mainly composed of amyloid fibrils. Here we report on an experimental study on the heat-induced aggregation mechanisms, at basic pH, of concanavalin A (ConA), used as a model system. Thioflavin T (ThT) fluorescence and multiangle light scattering allowed us to detect different intertwined steps in the formation of ConA aggregates. In particular, the ThT fluorescence increase, observed in the first phase of aggregation, reveals the formation of intermolecular ß-sheet structure which constitutes a rate-limiting step of the process. The intertwining between the formation of ß-aggregate structures and the whole aggregation process is discussed as a function of protein concentration: a coagulation process produces the same kind of aggregates at the different concentrations studied. Multiangle light-scattering data highlight the onset of the condensation process which gives rise to formation of compact fractal aggregates. AFM microscopy supports this conclusion showing thin fibrils of ConA, formed in the early stage of aggregation, which further interact to form larger structures with a netlike spatial organization.


Subject(s)
Amyloid/chemistry , Concanavalin A/chemistry , Protein Multimerization , Benzothiazoles , Hydrogen-Ion Concentration , Light , Protein Structure, Secondary , Scattering, Small Angle , Spectrometry, Fluorescence , Temperature , Thiazoles/chemistry
4.
J Biomed Biotechnol ; 2010: 147835, 2010.
Article in English | MEDLINE | ID: mdl-20490276

ABSTRACT

Alzheimer's disease (AD) and type 2 diabetes are connected in a way that is still not completely understood, but insulin resistance has been implicated as a risk factor for developing AD. Here we show an evidence that insulin is capable of reducing cytotoxicity induced by Amyloid-beta peptides (A-beta) in its oligomeric form in a dose-dependent manner. By TUNEL and biochemical assays we demonstrate that the recovery of the cell viability is obtained by inhibition of intrinsic apoptotic program, triggered by A-beta and involving caspase 9 and 3 activation. A protective role of insulin on mitochondrial damage is also shown by using Mito-red vital dye. Furthermore, A-beta activates the stress inducible Hsp70 protein in LAN5 cells and an overexpression is detectable after the addition of insulin, suggesting that this major induction is the necessary condition to activate a cell survival program. Together, these results may provide opportunities for the design of preventive and therapeutic strategies against AD.


Subject(s)
Amyloid beta-Peptides/antagonists & inhibitors , Caspase Inhibitors , HSP70 Heat-Shock Proteins/metabolism , Insulin/pharmacology , Neurons/drug effects , Peptide Fragments/antagonists & inhibitors , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/toxicity , Analysis of Variance , Apoptosis/drug effects , Blotting, Western , Caspase 3/metabolism , Caspase 9/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Enzyme Activation , Humans , Mitochondria/drug effects , Mitochondria/metabolism , Neuroblastoma , Neurons/metabolism , Peptide Fragments/metabolism , Peptide Fragments/toxicity , Protein Multimerization , Recombinant Proteins/metabolism , Recombinant Proteins/toxicity , Up-Regulation
5.
Arch Biochem Biophys ; 483(1): 120-6, 2009 Mar 01.
Article in English | MEDLINE | ID: mdl-19135026

ABSTRACT

Alzheimer's disease (AD) is the most common form of dementia. The cause of AD is closely related to the accumulation of amyloid beta peptide in the neuritic plaques. The use of animal model systems represents a good strategy to elucidate the molecular mechanism behind the development of this pathology. Here we use the Paracentrotus lividus embryo to identify molecules and pathways that can be involved in the degenerative process. As a first step, we identified the presence of an antigen related to the human APP, called PlAPP. This antigen, after gastrula stage, is processed producing a polypeptide of about 10kDa. By immunohistochemistry we localized the PlAPP antigen in some serotonin expressing cells. Similarly, after 48 or 96h incubation, a recombinant beta-amyloid peptide, rAbeta42, accumulates around the intestinal tube and oesophagus. In addition, incubation of sea urchin embryos with two different solutions rich in oligomers and fibrillar aggregates of rAbeta42 induce activation of apoptosis as detected by TUNEL assay. Moreover, we demonstrate that aggregates induce apoptosis by extrinsic pathway activation, whereas oligomers induce apoptosis both by extrinsic and intrinsic pathway activation. Utilizing an apoptotic inhibitor, caspases activation was offset and morphological damage rescued. Taken together all these observations suggest that the sea urchin may be a simple and suitable model to characterize the mechanism underlining the cytotoxicity of Abeta42.


Subject(s)
Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/toxicity , Paracentrotus/drug effects , Paracentrotus/metabolism , Alzheimer Disease/etiology , Alzheimer Disease/metabolism , Animals , Antigens/metabolism , Apoptosis/drug effects , Caspases/metabolism , Humans , Models, Animal , Nervous System/embryology , Nervous System/metabolism , Paracentrotus/embryology , Peptide Fragments/metabolism , Peptide Fragments/toxicity , Recombinant Proteins/metabolism , Recombinant Proteins/toxicity
6.
FASEB J ; 20(11): 1916-7, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16818470

ABSTRACT

A distinctive feature of Alzheimer's disease is the deposition of amyloid beta-protein (Abeta) in senile or diffuse plaques. The 42 residue beta-peptide (Abeta42) is the predominant form found in plaques. In the present work we report a high-yield expression and purification method of production of a recombinant Abeta42. The purified recombinant peptide shows characteristics similar to the synthetic human peptide. Different size aggregates, either small oligomers or larger aggregates, were obtained upon dissolving the recombinant Abeta42 peptide under different conditions at pH 7.2 or pH 3, respectively. We report a new toxicity assay on the morphogenic development of the sea urchin Paracentrotus lividus and study the toxicity of the two kinds of aggregates. Despite the difference between the ionic strength of human extracellular fluid (0.154 mol/l) and artificial sea water (0.48 mol/l), toxicity data collected in this system have an intrinsic relevance. The different ionic strength, in fact, could change the kinetics of oligomer formation, but the effect of morphogenic development reported here is related to the final oligomer sizes. Results of the toxicity assay of Abeta42 on sea urchin development also show a dose-dependent effect. After only 4 h of embryo development, one can note morphological defects in the cell membrane. Retardation of the embryo's development, along with cellular disorders visible inside the blastocoele, can be observed after 1 day of development. Cellular degeneration in two different pathological phenotypes-the occluded blastulae and the occluded prism-is present after 48 h of development. Results show that a greater effect on cell death is induced by the small oligomers stabilized under physiological conditions than at acid pH. In this case only occluded blastulae are found after 48 h of development.


Subject(s)
Amyloid beta-Peptides/toxicity , Embryo, Nonmammalian/drug effects , Paracentrotus/embryology , Peptide Fragments/toxicity , Animals , Base Sequence , Cloning, Molecular , DNA Primers , Humans , Morphogenesis/drug effects , Paracentrotus/drug effects , Recombinant Proteins/toxicity , Restriction Mapping , Reverse Transcriptase Polymerase Chain Reaction
7.
Protein Sci ; 10(7): 1312-8, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11420433

ABSTRACT

In one of the first studies of isolated intermediates in protein aggregation, we have used circular dichroism and fluorescence spectroscopy to characterize metastable oligomers that are formed in the early steps of beta-lactoglobulin heat aggregation. The intermediates show typical molten globule characteristics (secondary structure content similar to the native and less tight packing of the side chains), in agreement with the belief that partly folded states play a key role in protein aggregation. The far-UV CD signal bears strong resemblance to that of a known folding intermediate. Cryo-transmission electron microscopy of the aggregates reveals spherical particles with a diameter of about 50 nm and an internal threadlike structure. Isolated oligomers as well as larger aggregates bind the dye thioflavin T, usually a signature of the amyloid superstructures found in many protein aggregates. This result suggests that the structural motif recognized by thioflavin T can be formed in small oligomers.


Subject(s)
Lactoglobulins/metabolism , Oligopeptides/chemistry , Animals , Benzothiazoles , Circular Dichroism , Dimerization , Hot Temperature , Humans , Lactoglobulins/chemistry , Microscopy, Electron , Particle Size , Protein Conformation , Protein Folding , Spectrometry, Fluorescence , Thiazoles
8.
Biophys J ; 79(2): 1030-8, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10920033

ABSTRACT

The early stages of heat induced aggregation at 67.5 degrees C of beta-lactoglobulin were studied by combined static light scattering and size exclusion chromatography. At all conditions studied (pH 8.7 without salt and pH 6.7 with or without 60 mM NaCl) we observe metastable heat-modified dimers, trimers, and tetramers. These oligomers reach a maximum in concentration at about the time when large aggregates (1000-4000 kg/mol) appear, after which they decline in concentration. By isolating the oligomers it was demonstrated that they rapidly form aggregates upon heating in the absence of monomeric protein, showing that these species are central to the aggregation process. To our knowledge this is the first time that intermediates in protein aggregation have been isolated. At all stages of aggregation the dominant oligomer was the heat-modified dimer. Whereas the heat-modified oligomers are formed at a higher rate at pH 8.7 than at pH 6.7, the opposite is the case for the formation of aggregates from the metastable oligomers indicating cross-linking via disulfide bridges for the oligomers and noncovalent interaction in the formation of the aggregates. The data suggest that an aggregate nucleus is formed from four oligomers. For protein concentrations of 10 or 20 g/l a heat-modified monomer can be observed until about the time when the maximum in concentration appears of the heat-modified dimer. The disappearance of this heat-modified monomer correlates to the formation of dimers (trimers and tetramers).


Subject(s)
Hydrogen-Ion Concentration , Lactoglobulins/chemistry , Dimerization , Hot Temperature , Light , Macromolecular Substances , Protein Binding , Scattering, Radiation , Thermodynamics
SELECTION OF CITATIONS
SEARCH DETAIL
...