Evandro Fioretti

Peroxynitrite induced oxidation and its effects on isolated proteasomal systems

The proteasomes are the major intracellular proteolytic systems involved in the removal of altered proteins. In this study, we examined different susceptibilities of constitutive (XYZ) and interferon-gamma inducible (LMP) 20S proteasomes, isolated from bovine brain and thymus, respectively, to peroxynitrite-mediated oxidation. Exposure of XYZ and LMP proteasomes to increasing amounts of peroxynitrite resulted in different levels, in the two enzymes, of 3-nitrotyrosine groups and tryptophan residues oxidation. 1-Anilino-8-naphtalene-sulfonic acid binding studies and quenching of tryptophan residues indicated that the LMP complex was more sensitive to peroxynitrite. Regarding the proteolytic activities, the XYZ proteasome showed an overall activation (even if the trypsin-like (T-L) component was 20% inhibited), with the peptidyl-glutamyl peptide-hydrolyzing (PGPH) and branched-chain amino acid-preferring (BrAAP) activities being the most stimulated. On the other end, the LMP proteasome was inhibited, especially the BrAAP activity, whereas the T-L activity was not affected. Furthermore, exposure to increasing amounts of peroxynitrite induced a gradual decrease of beta-casein degrading rate by the LMP proteasome, whereas it did not influence the constitutive complex. Our results indicated that peroxynitrite caused a mild modification of the XYZ complex, leading to activation of its catalytic activities. Differently, the LMP proteasome showed a more significant conformational change resulting in the inhibition of the proteolytic functions.

Multimeric self-assembly equilibria involving the histone-like protein H-NS. A thermodynamic study

The thermodynamic parameters affecting protein-protein multimeric self-assembly equilibria of the histone-like protein H-NS were quantified by “large zone” gel-permeation chromatography. The abundance of the different association states (monomer, dimer, and tetramer) were found to be strictly dependent on the monomeric concentration and affected by physical (temperature) and chemical (cations) parameters. On the basis of the results obtained in this study and the available structural information concerning this protein, a mechanism is proposed to explain the association behavior also in relation to the functional properties of the protein.

Peroxynitrite-mediated oxidation of fibrinogen inhibits clot formation

The clotting activity of human fibrinogen was fully inhibited in vitro by peroxynitrite. The decrease of activity followed an exponential function and the concentration of peroxynitrite needed to inhibit 50% of fibrinogen clotting was 22 μM at 25°C. The oxidative modification(s) induced by the peroxynitrite system (i.e. ONOO−, ONOOH and ONOOH*) appeared specifically to affect fibrin clot formation (through the inhibition of fibrinogen polymerization) since the interaction of peroxynitrite‐modified fibrinogen with thrombin appeared to be unaffected. The addition of NaHCO3 decreased the peroxynitrite effect on fibrinogen clotting, suggesting that the reactive species formed by the reaction of CO2 with peroxynitrite are less efficient oxidants of peroxynitrite itself. Similar effects were observed after addition of bilirubin, which also exerted a significant protection against peroxynitrite‐mediated modification of fibrinogen.

Interaction of Hsp90 with 20s proteasome: Thermodynamic and kinetic characterization

The proteasome and heat shock proteins have been found in the centrosome. The evidence of their copurification reported by several studies suggests that they form stable complex. In addition, Hsp90 is involved in the loading of proteasome‐generated antigenic peptides to the class I major histocompatibility complex. In this article, we report a detailed thermodynamic and kinetic characterization of the Hsp90‐20S proteasome interaction, using a surface plasmon resonance technique. The modulation exerted by protons in solution has been investigated, and the results have been discussed, taking into account structural motifs characterizing the binding interface between the two macromolecules. Proteins 2002;48:169–177.

Effect of neurotoxic metal ions on the proteolytic activities of the 20S proteasome from bovine brain

Abstract. The effect of oxidative stress induced by neurotoxic metal ions on the properties of the brain 20S proteasome or multicatalytic proteinase complex (MPC) has been studied. Exposure of the 20S proteasome to increasing amounts of Fe(III), Fe(II), Cu(II) or Zn(II) affects its main hydrolytic activities: trypsin-like (T-L), chymotrypsin-like (ChT-L), peptidylglutamyl-peptide hydrolase (PGPH), branched-chain amino acid preferring (BrAAP) and caseinolytic activities, although in different ways. T-L activity showed gradual activation by both iron ions but inhibition by Cu(II) and Zn(II). ChT-L and PGPH activities were inhibited whereas BrAAP activity was widely activated by all the tested metal salts except for zinc ions. Moreover, the exposure to ferrous salt increased the degradation rate of casein. The functional effects appear to be linked to oxidation-induced modifications, as demonstrated by an increase of carbonyl groups following the exposure to metal ions. In addition, modifications induced by ferrous salt on the catalytic subunits were also supported by western blot analyses performed using anti-X, anti-Y and anti-Z antibodies. The results obtained clearly indicate that metal-catalyzed oxidation strongly affects the functions of the brain 20S proteasome, even though the catalytic subunits seem to be differently influenced by oxidative phenomena.

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. beta-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.

Wheat sprout extract-induced apoptosis in human cancer cells by proteasomes modulation

Natural occurring modulators of proteasome functionality are extensively investigated for their implication in cancer therapy. On the basis of our previous evidences both on proteasomal inhibition by monomeric polyphenols, and on the characterization of wheat sprout hydroalcoholic extract, herein we thoroughly report on a comparative study of the effect of wheat sprout extract on both normal and tumour cells. Treatment of isolated 20S proteasomes with wheat sprout extracts induced a gradual inhibition of all proteasome activities. Next, two wheat sprout extract components were separated: a polyphenol and a protein fraction. Both components exerted an in vitro inhibitory effect on proteasome activity. HeLa tumour cells and FHs 74 Int normal cells were exposed to both fractions, resulting in different rates of proteasome inhibition, with tumour cells showing a significantly higher degree of proteasome impairment and apoptosis induction. Furthermore, a decrease in proteasome activities and in cell survival of the human plasmacytoma RPMI 8226 cell line, upon the same treatments, was observed. Collectively, our results provide additional evidences supporting the possible use of natural extracts as coadjuvants in cancer treatments.

Pomegranate fruit components modulate human thrombin

Pomegranate (Punica granatum) is an important source of polyphenols with assessed antioxidant properties. The aims of this study were: (i) the characterization of the monomeric phenolic variability on each isolated fruit component (endocarp, mesocarp, aril); (ii) the study on the effect of pomegranate fruit components on human thrombin amidolytic activity. Collectively, our data show that pomegranate components contain bioactive metabolites (mainly ellagic acid) and suggest a potential role for the pomegranate extract in the regulation of a number of physio-pathological processes involving thrombin (or thrombin-like proteinase).

Binding of aflatoxins to the 20S proteasome: effects on enzyme functionality and implications for oxidative stress and apoptosis.

Abstract Aflatoxins (AF) are contaminants of improperly stored foods; they are potent genotoxic and carcinogenic compounds, exerting their effects through damage to DNA. They can also induce mutations that increase oxidative damage. The goal of this study was to evaluate the possibility that a third mechanism could be involved in the carcinogenic action of aflatoxins, namely, direct binding to key enzymes involved in the regulatory pathways of the cell cycle, thereby modulating enzyme functionality. The 20S constitutive and immunoproteasome peptidase and proteolytic activities were assayed in the presence of aflatoxins B1, G1 and M1. All three toxins activated multiple peptidase activities of the proteasome. Aflatoxin (AF) M1 was the most potent activator of proteasome activity, while the constitutive 20S proteasome was specifically stimulated by AFG1. Furthermore, the effects of AFB1 on cultured hepatoma cells were investigated and the various proteasomal activities determined with cell lysates were differently affected. Taking into account the key role of the proteasome in cellular defense against oxidative stress, the carbonyl group content and the activities of antioxidant enzymes in cell lysates were analyzed. The proapoptotic effect of AFB1 was also investigated by measuring caspase-3 activity and cellular levels of p27 and IκBα.

Aspirin modulates LPS-induced nitric oxide release in rat glial cells.

Nitric oxide and prostaglandins are among the numerous substances released by activated glial cells. The aim of this study was to evaluate the effect of high-level aspirin on iNOS expression in cultured rat glial cells treated with lipopolysaccharide (LPS) as pathological stimulator. Using Western Blotting, we verified that aspirin enhanced LPS-induced iNOS expression and the presence of 15-deoxy-Delta(12,14)-prostaglandin (15d-PGJ2) suppressed this aspirin effect. However, the exposure of LPS-treated glial cells to aspirin resulted in a decrease of NO production. These results suggest that aspirin interferes with the cross-talk of prostaglandins and NO, blocking the endogenous negative control exerted by COX products on iNOS expression. On the other side, aspirin seems to act directly on iNOS reducing its activity, even if it does not completely block NO release by LPS-stimulated glial cells. Then aspirin could maintain homeostatic functions of NO, while it prevents toxic effects, corresponding to high NO concentrations.