Search

Allium roseum is an important medicinal and aromatic plant, specific to the North African flora and a rich source of important nutrients and bioactive molecules including flavonoids and organosulfur compounds whose biological activities and pharmacological properties are well known. In the present study, the inhibition of amyloid beta protein toxicity by the ethanolic extract of this plant is investigated for the first time. Preliminary biochemical analyses identified kæmpferol and luteolin-7-o-glucoside as the more abundant phenolic compounds. The effects of A. roseum extract (ARE) on aggregation and aggregate cytotoxicity of amyloid beta-42 (Aβ42), whose brain aggregates are a hallmark of Alzheimer's disease, were investigated by biophysical (ThT assay, Dynamic light scattering and transmission electron microscopy) and cellular assays (cytotoxicity, aggregate immunolocalization, ROS measurement and intracellular Ca2+ imaging). The biophysical data suggest that ARE affects the structure of the Aβ42 peptide, inhibits its polymerization, and interferes with the path of fibrillogenesis. The data with cultured cells shows that ARE reduces Aß42 aggregate toxicity by inhibiting aggregate binding to the cell membrane and by decreasing both oxidative stress and intracellular Ca2+. Accordingly, ARE could act as a neuroprotective factor against Aβ aggregate toxicity in Alzheimer's disease., Competing Interests: The authors have declared that no competing interests exist.

Oleuropein aglycone (OleA), the most abundant polyphenol in extra virgin olive oil (EVOO), and Hydroxythyrosol (HT), the OleA main metabolite, have attracted our interest due to their multitarget effects, including the interference with amyloid aggregation path. However, the mechanistic details of their anti-amyloid effect are not known yet. We report here a broad biophysical approach and cell biology techniques that enabled us to characterize the different molecular mechanisms by which OleA and HT modulate the Aβ 1-42 fibrillation, a main histopathological feature of Alzheimer's disease (AD). In particular, OleA prevents the growth of toxic Aβ 1-42 oligomers and blocks their successive growth into mature fibrils following its interaction with the peptide N-terminus, while HT speeds up harmless fibril formation. Our data demonstrate that, by stabilizing oligomers and fibrils, both polyphenols reduce their seeding activity and aggregate/membrane interaction on human neuroblastoma SH-SY5Y cells. These findings highlight the great potential of EVOO polyphenols and offer the possibility to validate and to optimize their use for possible AD prevention and therapy., (Copyright © 2019. Published by Elsevier Ltd.)

Mounting evidence indicates soluble Aβ 42 oligomers as the most toxic species causing neuronal death which leads to the onset and progression of Alzheimer disease (AD). Recently, it has been found that neurotoxic Aβ 42 oligomers grow from monomeric species or arise following secondary nucleation by preformed mature fibrils. Thus, the use of natural compounds such as polyphenols to hinder the growth or to remodel Aβ 42 fibrils is one of the most promising strategies for AD treatment. In our previous study, we showed that 1, 2, 4-trihydroxynaphthalene-2-O-β-d-glucopyranoside (THNG) inhibits Aβ 42 aggregation during the early steps of the aggregation process, inhibits its conformational change to a β-sheet-rich structure, decreases its polymerization, inhibits its fibrillogenisis and reduces oxidative stress and aggregate cytotoxicity. Here, we used different spectroscopic and cell culture methods to check the effect of THNG on fibrils disaggregation. We showed that THNG binds to mature Aβ 42 fibrils, rearrange their secondary structure, and remodels them into non-amyloid, less toxic, species by inhibiting their interaction with the plasma membrane. Our findings reveal that THNG is a good agent to remodel amyloid fibrils and could be used as a starting molecular scaffold to design new anti-AD drugs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Presently, misfolding and aggregation of amyloid-β 42 (Aβ 42 ) are considered early events in Alzheimer's disease (AD) pathogenesis. The use of natural products to inhibit the aggregation process and to protect cells from cytotoxicity of early aggregate grown at the onset of the aggregation path is one of the promising strategies against AD. Recently, we have purified a new powerful antioxidant and inhibitor of Aβ 42 aggregation from the leaves of Lawsonia inermis. The new compound was identified as a new Lawsoniaside; 1,2,4-trihydroxynaphthalene-2-O-β-D-glucopyranoside (THNG). Herein, we show that THNG interferes with Aβ 42 aggregation, inhibits its conformational change to a β-sheet-rich structure, decreases its polymerization into large fibrillar species, reduces oxidative stress, and aggregate cytotoxicity. These results indicate that THNG has great potential as a neuroprotective and therapeutic agent against AD. © 2018 BioFactors, 44(3):272-280, 2018., (© 2018 International Union of Biochemistry and Molecular Biology.)

Amyloid aggregation starts with the initial misfolding of peptide/protein precursors, with subsequent structural rearrangement into oligomers and protofibrils; the latter eventually organize into fibrils with shared basic structural features, found deposited in amyloid diseases. Mounting evidence indicates early oligomers as the most toxic amyloid species; accordingly, the search of inhibitors of their growth is considered a promising target to prevent amyloid toxicity. We recently showed that oleuropein aglycon, a polyphenol abundant in the extra virgin olive oil, interferes with the aggregation of amylin (involved in type-2 diabetes), eliminating its cytotoxicity. Here we report that oleuropein aglycon also hinders amyloid aggregation of Aβ(1-42) and its cytotoxicity, suggesting a general effect of such polyphenol. In particular, by using a wide panel of different spectroscopic, immunologic, cell viability and imaging techniques we provide a more detailed description of Aβ(1-42) structural modifications arising in the presence of the inhibitor and the resulting cytotoxicity. We here report that the polyphenol eliminates the appearance of early toxic oligomers favouring the formation of stable harmless protofibrils, structurally different from the typical Aβ(1-42) fibrils. We also show that oleuropein aglycon is maximally effective when is present at the beginning of the aggregation process; furthermore, when added to preformed fibrils, it does not induce the release of toxic oligomers but, rather, neutralizes any residual toxicity possibly arising from the residual presence of traces of soluble oligomers and other toxic aggregates. The possible use of this polyphenol as anti-aggregation molecule is discussed in the light of these data.

p28 is a 28-amino acid peptide fragment of the cupredoxin azurin derived from Pseudomonas aeruginosa that preferentially penetrates cancerous cells and arrests their proliferation in vitro and in vivo. Its antitumor activity reportedly arises from post-translational stabilization of the tumor suppressor p53 normally downregulated by the binding of several ubiquitin ligases. This would require p28 to specifically bind to p53 to inhibit specific ligases from initiating proteosome-mediated degradation. In this study, atomic force spectroscopy, a nanotechnological approach, was used to investigate the interaction of p28 with full-length p53 and its isolated domains at the single molecule level. Analysis of the unbinding forces and the dissociation rate constant suggest that p28 forms a stable complex with the DNA-binding domain of p53, inhibiting the binding of ubiquitin ligases other than Mdm2 to reduce proteasomal degradation of p53.

The transcriptional regulator p53 plays an essential role in tumor suppression. Accordingly, it is found mutated, and its activity is reduced, in many human cancers. Recent reports show that some cancers are characterized by a loss of function of wild-type p53, which, in several cases, accumulates in intracellular aggregates. Although the nature of such aggregates is still unclear, recent evidence indicates that the p53 C-terminal and core domains can undergo amyloid aggregation in vitro under mild denaturing conditions, although no information is available on the largely unstructured N-terminal transactivation domain. We therefore decided to investigate the amyloid propensity of the acidic unfolded 1-63 fragment of the transactivation domain, cloned, expressed, and purified from a bacterial strain. Here we show that, when exposed to acidic pH, the 1-63 fragment forms thioflavine T-positive aggregates whose amyloid nature was confirmed by Fourier transform infrared spectroscopy analysis, atomic force microscopy, and x-ray diffraction. These aggregates were shown to be cytotoxic to human SH-SY5Y cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, lactate dehydrogenase release, and caspase-3 activity assays. These results add new significant details to the picture describing the propensity of single domains of p53 to aggregate, further suggesting that, under suitable destabilizing conditions, the whole protein may aggregate into amyloid assemblies in vivo.

Five phosphotyrosine-containing peptides have been synthesized by FMOC solid-phase peptide synthesis. These peptides correspond to the 411-419 sequence of the Xenopus src oncogene, to the 1191-1220 sequence of the human EGF receptor precursor, to the 1146-1158 sequence of the human insulin receptor, to the 856-865 sequence of the human beta-PDGF receptor, and to the 5-16 sequence of the erythrocyte human band 3. The peptides were used as substrates for activity assay of two isoforms (AcP1 and AcP2) of a low molecular weight cytosolic PTPase. The assay, performed in microtiter EIA plates using Malachite green to determine the released phosphate, was rapid, reproducible, and sensitive. Both PTPase isoforms were able to hydrolyze all synthesized peptides, though with different affinity and rate. The main kinetic parameters were compared and discussed with respect to the role of the two enzymes in the cell.