Crosstalk between the ubiquitin-proteasome system and autophagy in a human cellular model of Alzheimer's disease

Alzheimer's disease is the most common progressive neurodegenerative disorder characterized by the abnormal deposition of amyloid plaques, likely as a consequence of an incorrect processing of the amyloid-β precursor protein (AβPP). Dysfunctions in both the ubiquitin-proteasome system and autophagy have also been observed. Recently, an extensive cross-talk between these two degradation pathways has emerged, but the exact implicated processes are yet to be clarified. In this work, we gained insight into such interplay by analyzing human SH-SY5Y neuroblastoma cells stably transfected either with wild-type AβPP gene or 717 valine-to-glycine AβPP-mutated gene. The over-expression of the AβPP mutant isoform correlates with an increase in oxidative stress and a remodeled pattern of protein degradation, with both marked inhibition of proteasome activities and impairment in the autophagic flux. To compensate for this altered scenario, cells try to promote the autophagy activation in a HDAC6-dependent manner. The treatment with amyloid-β(42) oligomers further compromises proteasome activity and also contributes to the inhibition of cathepsin-mediated proteolysis, finally favoring the neuronal degeneration and suggesting the existence of an Aβ(42) threshold level beyond which proteasome-dependent proteolysis becomes definitely dysfunctional

Modulation of Gut Microbiota and Neuroprotective Effect of a Yeast‐Enriched Beer

Beer is the most consumed alcoholic beverage worldwide. It is rich in nutrients, and with its microbial component it could play a role in gut microbiota modulation. Conflicting data are currently available regarding the consequences of alcohol and alcohol‐containing beverages on dementia and age‐associated disorders including Alzheimer’s disease (AD), a neurodegeneration characterized by protein aggregation, inflammatory processes and alterations of components of the gut–brain axis. The effects of an unfiltered and unpasteurized craft beer on AD molecular hallmarks, levels of gut hormones and composition of micro/mycobiota were dissected using 3xTg‐AD mice. In addition, to better assess the role of yeasts, beer was enriched with the same Saccharomyces cerevisiae strain used for brewing. The treatment with the yeast‐enriched beer ameliorated cognition and favored the reduction of A(1‐42) and pro‐inflammatory molecules, also contributing to an increase in the concentration of anti‐inflammatory cytokines. A significant improvement in the richness and presence of beneficial taxa in the gut bacterial population of the 3xTg‐AD animals was observed. In addition, the fungal order, Sordariomycetes, associated with gut inflammatory conditions, noticeably decreased with beer treatments. These data demonstrate, for the first time, the beneficial effects of a yeast‐enriched beer on AD signs, suggesting gut microbiota modulation as a mechanism of action

Structure/activity virtual screening and in vitro testing of small molecule inhibitors of 8-hydroxy-5-deazaflavin:NADPH oxidoreductase from gut methanogenic bacteria

Virtual screening techniques and in vitro binding/inhibitory assays were used to search within a set of more than 8,000 naturally occurring small ligands for candidate inhibitors of 8-hydroxy-5-deazaflavin:NADPH oxidoreductase (FNO) from Methanobrevibacter smithii, the enzyme that catalyses the bidirectional electron transfer between NADP+ and F420H2 during the intestinal production of CH4 from CO2. In silico screening using molecular docking classified the ligand-enzyme complexes in the range between − 4.9 and − 10.5 kcal/mol. Molecular flexibility, the number of H-bond acceptors and donors, the extent of hydrophobic interactions, and the exposure to the solvent were the major discriminants in determining the affinity of the ligands for FNO. In vitro studies on a group of these ligands selected from the most populated/representative clusters provided quantitative kinetic, equilibrium, and structural information on ligands’ behaviour, in optimal agreement with the predictive computational results

50 Hz Extremely Low Frequency Electromagnetic Fields Enhance Protein Carbonyl Groups Content in Cancer Cells: Effects on Proteasomal Systems

Electromagnetic fields are an assessed cause of prolonging free radicals lifespan. This study was carried out to investigate the influence of extremely low frequency electromagnetic fields on protein oxidation and on the 20S proteasome functionality, the complex responsible for the degradation of oxidized proteins. Caco 2 cells were exposed, for 24–72 hours, to 1 mT, 50 Hz electromagnetic fields. The treatment induced a time-dependent increase both in cell growth and in protein oxidation, more evident in the presence of TPA, while no changes in cell viability were detected. Exposing the cells to 50 Hz electromagnetic fields caused a global activation of the 20S proteasome catalytic components, particularly evident at 72 hours exposure and in the presence of TPA. The finding that EGCG, a natural antioxidant compound, counteracted the field-related pro-oxidant effects demonstrates that the increased proteasome activity was due to an enhancement in intracellular free radicals

Wheat sprout extract induces changes on 20S proteasomes functionality.

Wheat sprouts contain a very high level of organic phosphates and a powerful cocktail of different molecules such as enzymes, reducing glycosides and polyphenols. The antioxidant properties of wheat sprouts have been widely documented and it has been shown that they are able to protect DNA against free-radicals mediated oxidative damage. Furthermore, we have recently reported on the effects of several polyphenols on 20S proteasomes, underlying the dual role of epigallocatechin-3-gallate as an antioxidant and a proteasome effector in cancer cells. The aim of this study was to investigate the effects of wheat sprout extracts on 20S proteasome functionality. Wheat sprout extracts have been analysed and characterized for their polyphenolic content using the Folin-Ciocalteau reagent and RP-HPLC technique. Comparing our data with a polyphenol standard mixture we identified five different polyphenols: gallic acid, epigallocatechin-3-gallate, epigallocatechin, epicatechin and catechin. The treatment of isolated 20S proteasomes with the extract induced a gradual inhibition of all the tested components, ChT-L, T-L, PGPH and BrAAP, in both the complexes. At low extract concentration a slight activation of the enzyme was evident only for the BrAAP component of the constitutive enzyme and the ChT-L activity of the immunoproteasome. b-casein degradation rate decreased, particularly with the immunoproteasome. Human Colon adenocarcinoma (Caco) cells, stimulated with 12-O-tetradecanoylphorbol-13-acetate, showed activation of the 20S proteasome activities at short incubation times and an increase in intracellular oxidative proteins. Cells treatment with wheat sprout extract led to proteasome inhibition in unstimulated cells and attenuated the effects mediated by TPA. Finally, exposure to the extract affected the expression levels of pro-apoptotic proteins

Sanguisorba minor extract suppresses plasmin-mediated mechanisms of cancer cell migration

Background: Sanguisorba minor, as well as several other edible herbs and vegetables, has been used extensively in traditional medicine. The observed beneficial effects can be attributed at least in part to the direct modulation of several enzymatic activities by its polyphenolic constituents. Methods: The ethanol extract of Sanguisorba minor was characterized by reversed-phase liquid chromatography, and most relevant analytes were identified by multiple stage mass spectrometry. The whole extract and the most relevant isolated constituents were tested for their ability to modulate the activity of human plasmin both toward a synthetic substrate and in human breast cancer cell culture models. Kinetic and equilibrium parameters were obtained by a concerted spectrophotometric and biosensor-based approach. Results: Quercetin-3- glucuronide was recognized as the compound mainly responsible for the in vitro plasmin inhibition by S. minor extract, with an inhibition constant in the high nanomolar range; in detail, our approach based on bioinformatic, enzymatic and binding analyses classified the inhibition as competitive. Most interestingly, cell-based assays showed that this flavonoid was effective in suppressing plasmin-induced loss of cancer cell adhesion. General significance: Our results show that the extract from Sanguisorba minor limits plasmin-mediated tumor cell motility in vitro, mostly due to quercetin-3-glucuronide. This glucuronated flavonoid is a promising template for rational designing of anticancer drugs to be used in the treatment of pathological states involving the unregulated activity of plasmin. © 2012 Elsevier B.V. All rights reserved

Identification of an EGCG oxidation derivative with proteasome modulatory activity.

(-)-epigallocatechin-3-gallate (EGCG) has been shown to possess chemopreventative properties and the ability to inhibit proteasome, a multicatalytic protease involved in the removal of oxidized and misfolded proteins and in the turnover of important checkpoint proteins. The stability of EGCG under neutral-alkaline and cellular physiological conditions was evaluated, identifying a biologically active ring-fission oxidative product. This derivative differentially affected proteasome activities with respect to EGCG in vitro, whereas, in cervical carcinoma cells, both compounds inhibited proteasome functionality to a similar extent, promoting a significant accumulation of ubiquitinated proteins and apoptotic markers. Despite of EGCG high instability, an equally active metabolite, able to modulate both proteasome functionality and apoptotic pathways, is generated. Interestingly this derivative protracts both the EGCG antioxidant and proteasome modulating efficacy, irrespective of the catechin short half-life