Manila Amici

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.

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

The purpose of this review is to discuss the effect of natural antioxidant compounds as modulators of the 20S proteasome, a multi‐enzymatic multi‐catalytic 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.

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.

Amyloid peptides in different assembly states and related effects on isolated and cellular proteasomes

The role of amyloid-beta protein (Abeta) in the pathogenesis of Alzheimers disease (AD) has been widely investigated and amyloid aggregates are considered a major cause of neuronal dysfunction. Increasing evidence has identified a correlation between this protein and the proteasome, the cellular proteolytic machinery, in particular the ubiquitin-proteasome system. The 20S proteasome is the catalytic core of a complex, known as 26S proteasome, and is the main responsible for the clearance of misfolded and oxidized proteins. In this work we have investigated the effects of different assembly states of two major amyloid peptides, Abeta (1-40) and Abeta (1-42) on the 20S proteasome functionality and on the ubiquitin-dependent pathway of protein degradation. In particular, we have tested proteasome activities after Abeta treatment on purified 20S complexes and on lysates of a human neuroblastoma cell line. Our findings show a significant decrease in proteasome activity, more evident in cell lysates than in isolated complexes, and an increased amount of ubiquitin-protein conjugates and of a known proteasome substrate (p27). Furthermore, the altered proteasome functionality is not associated with a decrease in cell viability, but is linked with increased levels of protein oxidation.

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.

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, PrPc, into a pathological insoluble conformer, PrPSc. In earlier works, some serum components were identified as exclusive PrPSc‐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 PrPc 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 PrPc and human plasminogen was also studied: both prion proteins (the recombinant bovine PrPc 25–242 and the purified full‐length PrPc) 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. Proteins 2005.

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.

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.

Co-chaperonin GroES as a modulator of proteasomal activity

The proteasome has a crucial part in the degradation of normal, damaged, mutant or misfolded proteins within both the ubiquitin ATP‐dependent and the ubiquitin ATP‐independent pathways. Proteasome‐mediated proteolysis is modulated by diverse factors, and in this regard, chaperonins have been attracting great interest. The investigation on the role of a co‐chaperonin, namely GroES, in the modulation of proteasomal activity was the focus of this work. Our study reports on an analytical approach based on combined fluorimetric, chromatographic (applied to the enzymatic activity evaluation), surface plasmon resonance techniques and molecular modelling, addressed to the assessment and characterization of the interaction. Globally, we described a high affinity interaction between GroES and two different 20 S (immuno‐ and constitutive) proteasomes, uncovering new scenarios on their possible physio‐pathological role, specifically on the ability of proteasomes to interact both with unfolding and folding‐ assisting macromolecules. Copyright