Biomedical subjects

Ivano Eberini

Publications and source records attributed to Ivano Eberini.

27 records · Page 2Linked to original sources

PubMed2003

Subcellular localization of soybean 7S globulin in HepG2 cells and LDL receptor up-regulation by its alpha' constituent subunit.

The aims of this work were to monitor the subcellular localization of soybean 7S globulin in HepG2 cells and determine its interaction with cell protein components, by using laser-induced fluorescence capillary electrophoresis (LIF-CE). Furthermore, we evaluated in the same cell line the involvement of the alpha' constituent subunit from 7S globulin in the modulation of LDL catabolism. The results indicated a main fluorescein isothiocyanate-tagged 7S globulin (FITC-7S) component in the cytosolic fraction, that was not present in the nuclear compartment. The electrophoretic mobility of this tagged component suggested either a dissociation of the 7S oligomer or its partial intracellular degradation. Interactions of soybean 7S globulin with FITC-thioredoxin 1 and FITC-cyclophilin B, HepG2 cell membrane proteins, were demonstrated in in vitro assays. In a separate experiment with HepG2 cells, the ability of the alpha' subunit purified from soybean 7S globulin to modulate the activity of the LDL receptors was evaluated by tracking the uptake and degradation of labeled LDL. The up-regulation of LDL receptors by the alpha' subunit, as further confirmed by a LDL receptor promoter assay, was significantly greater than that found in the control cells. In conclusion, this study, while confirming our previous indirect evidence of the key role of alpha' subunit on the cell cholesterol homeostasis, reveals a potentially interesting association of soybean 7S globulin with proteins, such as thioredoxin 1 and cyclophilin B, that are involved in cell protection against oxidative stress.

Antigens, Plant↗

PubMed2002

pH and ionic strength dependence of protein (un)folding and ligand binding to bovine beta-lactoglobulins A and B.

Formation of complexes between bovine beta-lactoglobulins (BLG) and long-chain fatty acids (FAs), effect of complex formation on protein stability, and effects of pH and ionic strength on both complex formation and protein stability were investigated as a function of pH and ionic strength by electrophoretic techniques and NMR spectroscopy. The stability of BLG against unfolding is sharply affected by the pH of the medium: both A and B BLG variants are maximally stabilized against urea denaturation at acidic pH and against SDS denaturation at alkaline pH. The complexes of BLGB with oleic (OA) and palmitic acid (PA) appear more stable than the apoprotein at neutral pH whereas no differential behavior is observed in acidic and alkaline media. PA forms with BLG more stable complexes than OA. The difference between the denaturant concentration able to bring about protein unfolding in the holo versus the apo forms is larger for urea than for SDS treatment. This evidence disfavors the hypothesis of strong hydrophobic interactions being involved in complex formation. Conversely, a significant contribution to FA binding by ionic interactions is demonstrated by the effect of pH and of chloride ion concentration on the stoichiometry of FA.BLG complexes. At neutral pH in a low ionic strength buffer, one molecule of FA is bound per BLG monomer; this ratio decreases to ca. 0.5 per monomer in the presence of 200 mM NaCl. The polar heads of bound FA appear to be solvent accessible, and carboxyl resonances exhibit an NMR titration curve with an apparent pK(a) of 4.7(1).

Animals↗

PubMed2002

Size is a major determinant of dissociation and denaturation behaviour of reconstituted high-density lipoproteins.

Lipid-free apolipoprotein A-I (apoA-I) and A-I(Milano) (A-I(M)) were compared for their denaturation behaviour by running across transverse gradients of a chaotrope, urea, and of a ionic detergent, SDS. For both apo A-I and monomeric apoA-I(M) in the presence of increasing concentrations of urea the transition from high to low mobility had a sigmoidal course, whereas for dimeric A-I(M)/A-I(M) a non-sigmoidal shape was observed. The co-operativity of the unfolding process was lower for dimeric A-I(M)/A-I(M) than for apoA-I or for monomeric apoA-I(M). A slightly higher susceptibility to denaturation was observed for dimeric A-I(M)/A-I(M) than for monomeric apoA-I(M). A similar behaviour of A-I(M)/A-IM versus apoA-I(M) was observed in CD experiments. Large- (12.7/12.5 nm) and small- (7.8 nm) sized reconstituted high-density lipoproteins (rHDL) containing either apoA-I or A-I(M)/A-I(M) were compared with respect to their protein-lipid dissociation behaviour by subjecting them to electrophoresis in the presence of urea, of SDS and of a non-ionic detergent, Nonidet P40. A higher susceptibility to dissociation of small-sized versus large-sized rHDL, regardless of the apolipoprotein component, was observed in all three instances. Our data demonstrate that the differential plasticity of the various classes of rHDL is a function of their size; the higher stability of 12.5/12.7 nm rHDL is likely connected to the higher number of protein-lipid and lipid-lipid interactions in larger as compared with smaller rHDL.

Circular Dichroism↗

PubMed2002

Glutathionylation of human thioredoxin: a possible crosstalk between the glutathione and thioredoxin systems.

To identify proteins undergoing glutathionylation (formation of protein-glutathione mixed disulfides) in human T cell blasts, we radiolabeled the glutathione pool with (35)S, exposed cells to the oxidant diamide, and analyzed cellular proteins by two-dimensional electrophoresis. One of the proteins undergoing glutathionylation was identified by molecular weight, isoelectric point, and immunoblotting as thioredoxin (Trx). Incubation of recombinant human Trx with glutathione disulfide or S-nitrosoglutathione led to the formation of glutathionylated Trx, identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The glutathionylation site was identified as Cys-72. Glutathionylation of rhTrx abolished its enzymatic activity as insulin disulfide reductase in the presence of NADPH and Trx reductase. Activity was, however, regained with sigmoidal kinetics, indicating a process of autoactivation due to the ability of Trx to de-glutathionylate itself. These data suggest that the intracellular glutathione/glutathione disulfide ratio, an indicator of the redox state of the cell, can regulate Trx functions reversibly through thiol-disulfide exchange reactions.

Amino Acid Sequence↗

PubMed2002

Monitoring the effects of drug treatment in rat models of disease by serum protein analysis.

In this review we list from literature investigations on rat serum proteins using electrophoretic techniques in connection with drug testing. From our own research work, we provide annotated two-dimensional maps of rat serum proteins under control and experimental conditions. Emphasis is on species-specific components and on the effects of acute and chronic inflammation. We discuss our project of structural proteomics on rat serum as a minimally invasive approach to pharmacological investigation, and we outline a typical experimental plan for drug testing according to the above guidelines. We then report in detail on the results of our trials of anti-inflammatory drugs on adjuvant arthritis, an animal model of disease resembling in many aspects human rheumatoid arthritis. We demonstrate a correlation between biochemical parameters and therapeutic findings and outline the advantages of the chosen methodological approach, which proved also sensitive in revealing "side effects" of the test drugs. In an appendix we describe our experimental protocol when performing two-dimensional electrophoresis of rat serum.

Animals↗

PubMed2002

Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes.

Formation of mixed disulfides between glutathione and the cysteines of some proteins (glutathionylation) has been suggested as a mechanism through which protein functions can be regulated by the redox status. The aim of this study was to identify the proteins of T cell blasts that undergo glutathionylation under oxidative stress. To this purpose, we radiolabeled cellular glutathione with (35)S, exposed T cells to oxidants (diamide or hydrogen peroxide), and performed nonreducing, two-dimensional electrophoresis followed by detection of labeled proteins by phosphorimaging and their identification by mass spectrometry techniques. We detected several proteins previously not recognized to be glutathionylated, including cytoskeletal proteins (vimentin, myosin, tropomyosin, cofilin, profilin, and the already known actin), enzymes (enolase, aldolase, 6-phosphogluconolactonase, adenylate kinase, ubiquitin-conjugating enzyme, phosphoglycerate kinase, triosephosphate isomerase, and pyrophosphatase), redox enzymes (peroxiredoxin 1, protein disulfide isomerase, and cytochrome c oxidase), cyclophilin, stress proteins (HSP70 and HSP60), nucleophosmin, transgelin, galectin, and fatty acid binding protein. Based on the presence of several protein isoforms in control cells, we suggest that enolase and cyclophilin are heavily glutathionylated under basal conditions. We studied the effect of glutathionylation on some of the enzymes identified in the present study and found that some of them (enolase and 6-phosphogluconolactonase) are inhibited by glutathionylation, whereas the enzymatic activity of cyclophilin (peptidylprolyl isomerase) is not. These findings suggest that protein glutathionylation might be a common mechanism for the global regulation of protein functions.

Cells, Cultured↗

PubMed2002

Macrophage metalloproteinases degrade high-density-lipoprotein-associated apolipoprotein A-I at both the N- and C-termini.

Atheromatous plaques contain various cell types, including macrophages, endothelial cells and smooth-muscle cells. To investigate the possible interactions between secreted matrix metalloproteinases and high-density lipoprotein (HDL) components, we tested the above cell types by culturing them for 24 h. HDL(3) (HDL subfractions with average sizes of between 8.44 nm for HDL(3A) and 7.62 nm for HDL(3C)) were then incubated in their cell-free conditioned media. Proteolytic degradation of apolipoprotein A-I was observed with macrophages, but not with endothelial-cell- or muscle-cell-conditioned supernatant. Absence of calcium or addition of EDTA to incubation media prevented all proteolytic processes. The identified apolipoprotein A-I fragments had sizes of 26, 22, 14 and 9 kDa. Two-dimensional electrophoresis and MS resolved the 26 and the 22 kDa components and identified peptides resulting from both N- and C-terminal cleavage of apolipoprotein A-I. The higher abundance of C- than N-terminally cleaved peptides agrees with data in the literature for a fully structured alpha-helix around Tyr(18) compared with an unstructured region around Gly(185) and Gly(186). The flexibility in the latter region of apolipoprotein A-I may explain its susceptibility to proteolysis. In our experimental set-up, HDL(3C) was more extensively degraded than the other HDL(3) subclasses (HDL(3A) and HDL(3B)). Proteolytic fragments produced by metalloproteinase action were shown by gel filtration and electrophoresis to be neither associated with lipids nor self-associated.

Animals↗

PubMed2002

Strategies for proteomics with incompletely characterized genomes: the proteome of Bos taurus serum.

A reference map for Bos taurus serum was obtained using proteomic tools: 21 proteins, plus several serum albumin fragments, have been identified in 47 spots. One of the major acute-phase reactants, haptoglobin, was also detected in a pathological serum. A number of technical problems had to be solved. (i) Spot resolution in two-dimensional electrophoresis (2-DE) is not easily optimized, as several proteins have similar molecular mass; different polyacrylamide concentration gradients were used for the analysis of various size ranges. (ii) Identification of proteins through mass spectrometry (MS) procedures is also difficult as the genome of Bos taurus is incompletely characterized. The program FASTS proved particularly useful, since it allows simultaneous searching of several unordered sequence fragments, which may be individually too short to provide a statistically valid match using BLAST.

Amine Oxidase (Copper-Containing)↗

PubMed2002

Protein glutathionylation: coupling and uncoupling of glutathione to protein thiol groups in lymphocytes under oxidative stress and HIV infection.

We show here that exposure to oxidative stress induces glutathione (GSH) modification of protein cysteinyl residues (glutathionylation) in T cell blasts. Treating the cells with the oxidant diamide induces thiolation of a series of proteins that can be detected by 2D electrophoresis when 35S-cysteine is used to label the intracellular GSH pool. This thiolation is reversible, proteins are rapidly dethiolated and GSH is released from proteins once the oxidants are washed and the cells are allowed to recover. Dethiolation is dependent on the availability of GSH and thiols, since it is inhibited by GSH-depleting agents and improved by N-acetyl-L-cysteine (NAC). The capacity of these agents to reverse glutathionylation is diminished in T cell blasts infected in vitro with HIV, which is known to cause oxidative stress. Consistent with these findings, the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme known to be inhibited by glutathionylation, is inhibited in diamide-treated cells and recovers rapidly when cells are allowed to dethiolate. Further, GAPDH activity is diminished by GSH-depleting agents and augmented by NAC. Thus, reversible glutathionylation of proteins can rapidly shift the activity of a key metabolic enzyme and thereby result in dramatic, reversible changes in cellular metabolism.

Acetylcysteine↗