Homology Modeling and Docking Analysis of the Interaction between Polyphenols and Mammalian 20S Proteasomes.

Molecular docking of small ligands to biologically active macromolecules has become a valuable strategy to predict the stability of complexes between potential partners and their binding modes. In this perspective, we applied this computational procedure to rationalize the reported role of polyphenols as inhibitors of the mammalian 20S proteasomes. In particular, polyphenols were shown to modulate each proteasomal activity at different extents both in the constitutive and the inducible enzyme. We performed a flexible molecular docking analysis between a set of polyphenols previously demonstrated to have the highest binding affinity and both the constitutive (from deposited PDB structures) and homology modeled active subunits of the IFN-gamma inducible proteasome, to provide insight into the possible mechanism of interaction. Among the tested polyphenols, (-)-epigallocatechin-3-gallate showed the highest affinity for the proteasome subunits, both in terms of intermolecular energy and predicted equilibrium constants, in particular for beta 5 and beta 5i subunits (E(Total)=-66 kcal/mol, K(i)=81.3 mu M and E(Total)=-83.9 kcal/mol, K(i)=0.29 mu M, respectively), known to be related to the chymotrypsin-like and BrAAP activities. Collectively, polyphenols showed a higher affinity for the inducible subunits, in agreement with previous in vitro studies. Additionally, different contributions to the interaction energy (van der Waals, electrostatic, H-bond) of proteasome-polyphenols complexes were dissected

Effect of polyphenolic compounds on the proteolytic activities of constitutive and immuno-proteasomes

The effect of several polyphenols on the 20S proteasomes, both the constitutive and the LMP proteasomes, isolated from bovine tissues, has been investigated. Polyphenolic compounds show many biological activities such as antiviral, antibacterial, antifungal, anti-inflammatory, antimutagenic, and antiallergic activities. However, the molecular mechanism underlying these effects has not been identified. It is well established that polyphenols possess inhibitory activities on several enzymes and among them the 20S proteasome. In the present work, the ChT-L, BrAAP, PGPH, and T-L activities of the isolated constitutive and immuno-proteasomes were assayed in order to get an overall information on the polyphenols binding to the complexes. The effects of the polyphenols on the proteasomal activities were analyzed, taking into account the different subunits composition of the two complexes. Furthermore the same activities were measured on whole extracts from cancer cells exposed to EGCG and gallic acid, evaluating, also, their antioxidant action under oxidative stress. EGCG and gallic acid are able to affect the 20S proteasomes functionality, depending on the complex subunit composition and, in cell extracts, they behave both as antioxidants and proteasome effectors

Binding of recombinant PrPc to human plasminogen: kinetic and thermodynamic study using a resonant mirror biosensor

Transmissible spongiform encephalopathies are a class of sporadic, genetic and transmissible neurodegenerative diseases that affect both humans and animals. Propagation of these diseases is thought to be due to the misfolding of a neuronal glyco-protein, PrP(c), into a pathological insoluble conformer, PrP(Sc). In earlier works, some serum components were identified as exclusive PrP(Sc)-interacting proteins (Fisher et al., Nature 2000;408:479), and thus those macromolecules were thought to represent a potential diagnostic endogenous factor discriminating between normal and pathological prion proteins. In contrast, in agreement with a recent work (Kornblatt et al., Biochem Biophys Res Commun 2003;305:518), in this paper we present a detailed thermodynamic and kinetic characterization of the interaction between recombinant bovine PrP(c 25-242) and the human serum component plasminogen, measured using a resonant mirror technique: our results reveal a high-affinity interaction between the two binding partners. For comparison, the complex obtained from the purified full-length PrP(c) and human plasminogen was also studied: both prion proteins (the recombinant bovine PrP(c 25-242) and the purified full-length PrP(c)) are able to bind human plasminogen. Both kinetic and thermodynamic parameters are affected by the modulation exerted by the H(+) ions in solution. Moreover, the analysis of binding, according to canonical linkage relationships, suggests the involvement of a His residue, consistent with the interaction between other serine (pro)enzymes and their ligands

Ginsenosides Rg1 and Rg2 Activate Autophagy and Attenuate Oxidative Stress in Neuroblastoma Cells Overexpressing Aβ(1-42)

Alzheimer’s disease is a neurodegeneration with protein deposits, altered proteolysis, and inflammatory and oxidative processes as major hallmarks. Despite the continuous search for potential therapeutic treatments, no cure is available to date. The use of natural molecules as adjuvants in the treatment of Alzheimer’s disease is a very promising strategy. In this regard, ginsenosides from ginseng root show a variety of biological effects. Here, we dissected the role of ginsenosides Rg1 and Rg2 in modulating autophagy and oxidative stress in neuroblastoma cells overexpressing Aβ(1-42). Key hallmarks of these cellular processes were detected through immunomethods and fluorometric assays. Our findings indicate that ginsenosides are able to upregulate autophagy in neuronal cells as demonstrated by increased levels of LC3II and Beclin-1 proteins and decreased amounts of p62. Simultaneously, an activation of lysosomal hydrolases was observed. Furthermore, autophagy activation promoted the clearance of Aβ(1-42). Rg1 and Rg2 also reduced oxidative stress sources and macromolecule oxidation, promoting NRF2 nuclear translocation and the expression of antioxidant enzymes. Our data further clarify the mechanisms of action of Rg1 and Rg2, indicating new insights into their role in the management of disorders like Alzheimer’s disease

Natural polyphenols as proteasome modulators and their role as anti-cancer compounds

The purpose of this review is to discuss the effect of natural antioxidantcompounds as modulators of the 20S proteasome, a multi-enzymatic multicatalytic complex present in the cytoplasm and nucleus of eukaryotic cells and involved in several cellular activities such as cell-cycle progression, proliferation and the degradation of oxidized and damaged proteins. From this perspective, proteasome inhibition is a promising approach to anticancer therapy and such natural antioxidant effectors can be considered as potential relevant adjuvants and pharmacological models in the study of new drugs

Optimizing the Heavy Metal Ion Sensing Properties of Functionalized Silver Nanoparticles: The Role of Surface Coating Density

We present a colorimetric sensor based on functionalized silver nanoparticles for the detection of metal ions in aqueous solutions. The interaction between the target metal ion and the functionalizing agent triggers the aggregation of these nanoparticles, and the consequent change in optical properties allows the detection/quantification of the analyte. In detail, this work describes the synthesis of AgNPs by a chemical reduction method, and the production of mercaptoundecanoic acid functionalized NPs with different surface densities (multi-, full-, and two partial layers). UV-Vis spectroscopy was used to monitor the functionalization processes, and to investigate the aggregation behavior of each AgNPs@11MUA sensor upon titration with the metal ions of interest, namely Ni2+, Zn2+, Co2+, Cd2+, Mn2+, and Cu2+. The resulting UV-Vis raw data obtained for each layer density were submitted to principal component analysis to dissect the role of the metal ions in NP aggregation and in establishing the sensitivity and selectivity of the AgNPs@11MUA sensor. Interestingly, we observed an increase in sensor sensitivity and selectivity at a lower density of the functionalizing agent on the AgNPs’ surface, which results in characteristic colors of the NP suspension upon titration with each metal ion

Mechanism of inhibition of wt-dihydrofolate reductase from E. coli by tea epigallocatechin-gallate.

Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is the rate-controlling enzyme of cholesterol synthesis, and owing to its biological and pharmacological relevance, researchers have investigated several compounds capable of modulating its activity with the hope of developing new hypocholesterolemic drugs. In particular, polyphenol-rich extracts were extensively tested for their cholesterol-lowering effect as alternatives, or adjuvants, to the conventional statin therapies, but a full understanding of the mechanism of their action has yet to be reached. Our work reports on a detailed kinetic and equilibrium study on the modulation of HMGR by the most-abundant catechin in green tea, epigallocatechin-3-gallate (EGCG). Using a concerted approach involving spectrophotometric, optical biosensor, and chromatographic analyses, molecular docking, and site-directed mutagenesis on the cofactor site of HMGR, we have demonstrated that EGCG potently inhibits the in vitro activity of HMGR (K(i) in the nanomolar range) by competitively binding to the cofactor site of the reductase. Finally, we evaluated the effect of combined EGCG-statin administration

SLAB51 Probiotic Formulation Activates SIRT1 Pathway Promoting Antioxidant and Neuroprotective Effects in an AD Mouse Model

The gut-brain axis is a bidirectional communication network functionally linking the gut and the central nervous system (CNS). Based on this, the rational manipulation of intestinal microbiota represents a novel attractive therapeutic strategy for the treatment of CNS-associated disorders. In this study, we explored the properties of a probiotic formulation (namely SLAB51) in counteracting brain oxidative damages associated with Alzheimer's disease (AD). Specifically, transgenic AD mice (3xTg-AD) were treated with SLAB51 and the effects on protein oxidation, neuronal antioxidant defence and repair systems were monitored, with the particular focus on the role of SIRT1-related pathways. We demonstrated that SLAB51 markedly reduced oxidative stress in AD mice brain by activating SIRT1-dependent mechanisms, thus representing a promising therapeutic adjuvant in AD treatment

Ghrelin induces apoptosis in colon adenocarcinoma cells via proteasome inhibition and autophagy induction.

Ghrelin is a metabolism-regulating hormone recently investigated for its role in cancer survival and progression. Controversially, ghrelin may act as either anti-apoptotic or pro-apoptotic factor in different cancer cells, suggesting that the effects are cell type dependent. Limited data are currently available on the effects exerted by ghrelin on intracellular proteolytic pathways in cancer. Both the lysosomal and the proteasomal systems are fundamental in cellular proliferation and apoptosis regulation. With the aim of exploring if the proteasome and autophagy may be possible targets of ghrelin in cancer, we exposed human colorectal adenocarcinoma cells to ghrelin. Preliminary in vitro fluorimetric assays evidenced for the first time a direct inhibition of 20S proteasomes by ghrelin, particularly evident for the trypsin-like activity. Moreover, 1 μM ghrelin induced apoptosis in colorectal adenocarcinoma cells by inhibiting the ubiquitin-proteasome system and by activating autophagy, with p53 having an "interactive" role

Interplay between 20S proteasomes and prion proteins in scrapie disease.

Scrapie is a transmissible spongiform encephalopathy affecting the central nervous system in sheep. The key event in such neurodegeneration is the conversion of the normal prion protein (PrPC) into the pathological isoform (PrPSc). Misfolded prion proteins are normally degraded by the proteasome. This work, analyzing models of scrapie disease, describes the in vivo relationship between the proteasome and prions. We report that the disease is associated with an increase of proteasome functionality, most likely as a means of counteracting the increased levels of oxidative stress. Here, we show that prions coprecipitate with the 20S proteasome and that they colocalize within the same neuron, thus raising the possibility that PrP interacts with the proteasome in both normal and diseased brain, affecting substrate trafficking and proteasome functionality. This interaction, inducing proteasome activation, leads to different neuronal alterations and triggers apoptosis. Furthermore, testing the effects of isolated PrPC on purified 20S proteasomes, we obtain a concentration- and proteasome composition-dependent decrease in the complex activity. (C) 2009 Wiley-Liss, Inc