Protofibril Formation of Amyloid β-Protein at Low pH via a Non-cooperative Elongation Mechanism

Deposition of the amyloid beta-protein (Abeta) in senile or diffuse plaques is a distinctive feature of Alzheimer's disease. The role of Abeta aggregates in the etiology of the disease is still controversial. The formation of linear aggregates, known as amyloid fibrils, has been proposed as the onset and the cause of pathological deposition. Yet, recent findings suggest that a more crucial role is played by prefibrillar oligomeric assemblies of Abeta that are highly toxic in the extracellular environment. In the present work, the mechanism of protofibril formation is studied at pH 3.1, starting from a solution of oligomeric precursors. By combining static light scattering and photon correlation spectroscopy, the growth of the mass and the size of aggregates are determined at different temperatures. Analysis and scaling of kinetic data reveal that under the studied conditions protofibrils are formed via a single non-cooperative elongation mechanism, not prompted by nucleation. This process is well described as a linear colloidal aggregation due to diffusion and coalescence of growing aggregates. The rate of elongation follows an Arrhenius law with an activation enthalpy of 15 kcal mol(-1). Such a value points to a conformational change of peptides or oligomers being involved in binding to protofibrils or in general to a local reorganization of each aggregate. These results contribute to establishing a clearer relation at the molecular level between the fibrillation mechanism and fibrillar precursors. The observation of a non-cooperative aggregation pathway supports the hypothesis that amyloid formation may represent an escape route from a dangerous condition, induced by the presence of toxic oligomeric species

Data concerning the proteolytic resistance and oxidative stress in LAN5 cells after treatment with BSA hydrogels

AbstractProteolytic resistance is a relevant aspect to be tested in the formulation of new nanoscale biomaterials. The action of proteolytic enzymes is a very fast process occurring in the range of few minutes. Here, we report data concerning the proteolytic resistance of a heat-set BSA hydrogel obtained after 20-hour incubation at 60°C prepared at the pH value of 3.9, pH at which the hydrogel presents the highest elastic character with respect to gel formed at pH 5.9 and 7.4 “Heat-and pH-induced BSA conformational changes, hydrogel formation and application as 3D cell scaffold” (G. Navarra, C. Peres, M. Contardi, P. Picone, P.L. San Biagio, M. Di Carlo, D. Giacomazza, V. Militello, 2016) [1]. We show that the BSA hydrogel produced by heating treatment is protected by the action of proteinase K enzyme. Moreover, we show that LAN5 cells cultured in presence of BSA hydrogels formed at pH 3.9, 5.9 and 7.4 did not exhibit any oxidative stress, one of the first and crucial events causing cell death “Are oxidative stress and mitochondrial dysfunction the key players in the neurodegenerative diseases?” (M. Di Carlo, D. Giacomazza, P. Picone, D. Nuzzo, P.L. San Biagio, 2012) [2] “Effect of zinc oxide nanomaterials induced oxidative stress on the p53 pathway” (M.I. Setyawati, C.Y. Tay, D.T. Leaong, 2013) [3]

Moringa oleifera Leaf Powder as Functional Additive in Cookies to Protect SH-SY5Y Cells

The aim of this work is the evaluation of the addition of Moringa leaf powder (MLP) in cookies in terms of antioxidant properties, dough processability and sensorial properties of the cookies. The total content of biophenols and flavonoids in MLP was detected and the identification of the bioactive molecules was performed by HPLC-ESI-TOF-MS measurements, before and after oven treatment at 180 ◦C for 20 min. After a preliminary evaluation of the MLP water soluble fraction (MLPsf) cytotoxicity, its protective effect against an oxidative injury induced in the SH-SY5Y cells was assessed. Data evidence that the bioactive molecules present in MLPsf are effective in preventing ROS production and in protecting neuronal cells against oxidative stress. Prototypes of cookies containing MLP in different concentrations were then produced and evaluated by a consumer panel. Selected doughs containing MLP were analysed to determine the total content of biophenols in the cookies after baking and their enrichment in terms of valuable chemical elements. The influence of MLP on the viscoelastic behaviour and morphology of the doughs was also assessed. Finally, the potential role in counteracting the insurgence of not treatable neurodegenerative pathologies of two main MLP components, glucomoringin and kaempferol derivatives, present also after the thermal treatment, was discussed

From Small Peptides to Large Proteins against Alzheimer'sDisease

Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly. The two cardinal neuropathological hallmarks of AD are the senile plaques, which are extracellular deposits mainly constituted by beta-amyloids, and neurofibrillary tangles formed by abnormally phosphorylated Tau (p-Tau) located in the cytoplasm of neurons. Although the research has made relevant progress in the management of the disease, the treatment is still lacking. Only symptomatic medications exist for the disease, and, in the meantime, laboratories worldwide are investigating disease-modifying treatments for AD. In the present review, results centered on the use of peptides of different sizes involved in AD are presented

The molecular anatomy of human Hsp60 and its effects on Amyloid-β peptide

Heat Shock Protein 60 (HSP60) is ubiquitous and highly conserved, being present in eukaryotes and prokaryotes, including pathogens. This chaperonin is typically considered a mitochondrial protein but it is also found in other intracellular sites, extracellularly and in circulation. HSP60 is an indispensable component of the Chaperoning System and plays a key role in protein quality control, preventing off-pathway folding events and refolding misfolded proteins. This makes HSP60 a putative therapeutic agent for neurodegenerative diseases associated with aggregation of misfolded proteins, for example, Alzheimer’s Disease. We produced and purified recombinant human HSP60 and investigated the effects of its monomeric and tetradecameric forms onAmyloid-β aggregation. In addition, we induced oligomerization of HSP60 monomers by means of ATP. We measuredHSP60 stability in relation to degree of oligomerization. The structural stability of the HSP60 forms were also investigated by differential scanning calorimetry and isothermal titration calorimetry. The protein purified mainly appears in multimeric forms with a large fraction in dimers and monomers. We observed that Hsp60 is less stable in its monomeric form, but is more active in inhibiting the fibrillogenesis of beta amyloid peptide

Clinical anatomic, immunomorphologic and molecular anatomic data suggest interplay of thyroidal molecules, autoantibodies and Hsp60 in Hashimoto’s disease

Hsp60 is, typically, a mitochondrial protein, but it also occurs in the cytosol, vesicles, and plasma membrane, and in the intercellular space and biological fluids, e.g., blood. Changes in the levels and distribution of Hsp60 are linked to several pathologies, including cancer and chronic inflammatory and autoimmune disorders. What is the histopathological pattern of Hsp60 in the thyroid of Hashimoto’s patients? Are there indications of a pathogenic role of Hsp60 that may make Hashimoto’s thyroiditis a chaperonopathy? Experiments reported here provide information regarding those questions. We found by various immunomorphological techniques increased levels of Hsp60 in the thyroid from HT patients, localized to thyrocytes of small and degenerated follicles and to oncocytes (Hurtle cells). Immunofluorescence showed the chaperonin both inside the cells and also in the plasma membrane, especially in oncocytes. We also found that Hsp60 levels in the blood of HT patients were increased compared to controls and correlated with those of autoantibodies against two distinctive thyroidal proteins, thyroglobulin (TG) and thyroid peroxidase (TPO) (r=0.379, p=0.0103; r=0.484, p=0.0008; respectively). Molecular analysis of these two proteins in comparison with Hsp60 demonstrated various regions of high structural similarity shared by them, which could very well be immunologically crossreactive epitopes. Thus, it is likely that the three proteins potentiate each other as immunogens to elicit autoantibodies and, as antigens, to cause antigen-antibody reactions at those sites in which Hsp60 is exposed, for example the surface of oncocytes. This would lead to inflammation and oncocyte lysis with destruction of thyroidal tissue. The cytometric bead assay revealed that recombinant Hsp60 did not induce increment of cytokine production by peripheral blood mononuclear cells from HT patients. Consequently, we propose that Hsp60 is implicated in the pathogenesis of Hashimoto’s thyroiditis as autoantigen, via a participation of autoantibodies that also recognize TG and TPO, whereas participation of inflammatory cytokines induced by the chaperonin is unlikely. Supported by IEMEST (FC and AJLM)

The Interplay between PolyQ and Protein Context Delays Aggregation by Forming a Reservoir of Protofibrils

Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by the expansion of CAG codon repeats, which code for polyQ in the corresponding gene products. These diseases are associated with the presence of amyloid-like protein aggregates, induced by polyQ expansion. It has been suggested that the soluble aggregates rather than the mature fibrillar aggregates are the toxic species, and that the aggregation properties of polyQ can be strongly modulated by the surrounding protein context. To assess the importance of the protein carrier in polyQ aggregation, we have studied the misfolding pathway and the kinetics of aggregation of polyQ of lengths above (Q41) and below (Q22) the pathological threshold fused to the well-characterized protein carrier glutathione S-transferase (GST). This protein, chosen as a model system, is per se able to misfold and aggregate irreversibly, thus mimicking the behaviour of domains of naturally occurring polyQ proteins. We prove that, while it is generally accepted that the aggregation kinetics of polyQ depend on its length and are faster for longer polyQ tracts, the presence of GST alters the polyQ aggregation pathway and reverses this trend. Aggregation occurs through formation of a reservoir of soluble intermediates whose populations and kinetic stabilities increase with polyQ length. Our results provide a new model that explains the toxicity of expanded polyQ proteins, in which the interplay between polyQ regions and other aggregation-prone domains plays a key role in determining the aggregation pathway

Intrinsic Disorder and Chaperon-Like Activity of Different Caseins

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Data concerning the rheological behavior of high methoxyl pectin during gelation process

The present data concern the structuring kinetics of aqueous high methoxyl pectin (HMP) solutions at acid pH (3.1), constant pectin concentration (0.2% w/w) and sucrose concentrations ranging from 56 to 65% w/w. Consecutive frequency sweep was applied to samples immediately after their preparation. The generalized Maxwell (gM) model was used to describe the change of the mechanical spectra for each different sucrose concentration and to determine the viscoelastic parameters controlling the gelation of the HMP solutions. The viscosities in the sol region are explored in the range 0 to 55% 0 to 40% (w/w) sucrose concentration