Ivano Eberini

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 35S, 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.

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 35S, 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.

The molecular basis of lecithin:cholesterol acyltransferase deficiency syndromes: a comprehensive study of molecular and biochemical findings in 13 unrelated Italian families.

Objective—To better understand the role of lecithin:cholesterol acyltransferase (LCAT) in lipoprotein metabolism through the genetic and biochemical characterization of families carrying mutations in the LCAT gene. Methods and Results—Thirteen families carrying 17 different mutations in the LCAT gene were identified by Lipid Clinics and Departments of Nephrology throughout Italy. DNA analysis of 82 family members identified 15 carriers of 2 mutant LCAT alleles, 11 with familial LCAT deficiency (FLD) and 4 with fish-eye disease (FED). Forty-four individuals carried 1 mutant LCAT allele, and 23 had a normal genotype. Plasma unesterified cholesterol, unesterified/total cholesterol ratio, triglycerides, very-low-density lipoprotein cholesterol, and pre-&bgr; high-density lipoprotein (LDL) were elevated, and high-density lipoprotein (HDL) cholesterol, apolipoprotein A-I, apolipoprotein A-II, apolipoprotein B, LpA-I, LpA-I:A-II, cholesterol esterification rate, LCAT activity and concentration, and LDL and HDL3 particle size were reduced in a gene–dose-dependent manner in carriers of mutant LCAT alleles. No differences were found in the lipid/lipoprotein profile of FLD and FED cases, except for higher plasma unesterified cholesterol and unesterified/total cholesterol ratio in the former. Conclusion—In a large series of subjects carrying mutations in the LCAT gene, the inheritance of a mutated LCAT genotype causes a gene–dose-dependent alteration in the plasma lipid/lipoprotein profile, which is remarkably similar between subjects classified as FLD or FED.

New aspects of altitude adaptation in Tibetans: a proteomic approach

A prolonged sojourn above 5500 m induces muscle deterioration and accumulation of lipofuscin in Caucasians, probably because of overproduction of reactive oxygen species (ROS). Because Sherpas, who live at high altitude, have very limited muscle damage, it was hypothesized that Himalayan natives possess intrinsic mechanisms protecting them from oxidative damage. This possibility was investigated by comparing the muscle proteomes of native Tibetans permanently residing at high altitude, second‐generation Tibetans born and living at low altitude, and Nepali control subjects permanently residing at low altitude, using 2D gel electrophoresis and mass spectrometry. Seven differentially regulated proteins were identified: glutathione‐S‐transferase P1‐1, which was 380% and 50% overexpressed in Tibetans born and living at high and low altitude, respectively; Δ2‐enoyl‐CoA‐hydratase, which was up‐regulated in both Tibetan groups; glyceraldehyde‐3‐phosphate dehydrogenase and lactate dehydrogenase, which were both slightly down‐regulated in Tibetans born and living at high altitude; phosphoglycerate mutase, which was 50% up‐regulated in the native Tibetans; NADH‐ubiquinone oxidoreductase, slightly overexpressed in Tibetans born and living at high altitude; and myoglobin, which was overexpressed in both Tibetan groups. We concluded that Tibetans at high altitude, and to some extent, those born and living at low altitude, are protected from ROS‐induced tissue damage and possess specific metabolic adaptations.

Urea and Guanidinium Chloride Denature Protein L in Different Ways in Molecular Dynamics Simulations

In performing protein-denaturation experiments, it is common to employ different kinds of denaturants interchangeably. We make use of molecular dynamics simulations of Protein L in water, in urea, and in guanidinium chloride (GdmCl) to ascertain if there are any structural differences in the associated unfolding processes. The simulation of proteins in solutions of GdmCl is complicated by the large number of charges involved, making it difficult to set up a realistic force field. Furthermore, at high concentrations of this denaturant, the motion of the solvent slows considerably. The simulations show that the unfolding mechanism depends on the denaturing agent: in urea the beta-sheet is destabilized first, whereas in GdmCl, it is the alpha-helix. Moreover, whereas urea interacts with the protein accumulating in the first solvation shell, GdmCl displays a longer-range electrostatic effect that does not perturb the structure of the solvent close to the protein.

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.

Proteins of rat serum, urine, and cerebrospinal fluid : VI. Further protein identifications and interstrain comparison

We have investigated the biological fluids – serum, cerebrospinal fluid, and urine – of three strains of rats; the present data extend our database (also available on‐line) and may be of interest for pharmacological and toxicological investigation. Specifically, we have defined reference maps of the major protein components in cerebrospinal fluid and urine. Compartment‐specific isoforms were recognized for transferrin and transthyretin. Mass spectrometric data established the cleavage site of the signal peptide and identified the N‐terminal blocking group of prostaglandin D synthase from rat cerebrospinal fluid. A previously undescribed member of the family of low molecular mass rat urinary proteins was characterized as containing a sequence similar, but not identical, to the N‐terminal region of rat urinary protein‐2 (RUP‐2), and divergent from RUP‐1.

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.

Acute-Phase Proteins Before Cerebral Ischemia in Stroke-Prone Rats Identification by Proteomics

Background and Purpose— A high degree of proteinuria has been reported in stroke-prone spontaneously hypertensive rats (SHRSP). We studied the effect of salt loading on the detailed protein pattern of serum and urine in 3 rat strains: Wistar-Kyoto, spontaneously hypertensive rats, and SHRSP, an inbred animal model for a complex form of cerebrovascular disorder resembling the human disease. Methods— Rats were given a permissive diet and received 1% NaCl in drinking water. The protein pattern in body fluids was assessed over time by 2-dimensional electrophoretic analysis. Brain alterations were monitored by MRI and histology. Results— Several proteins were excreted in urine after weeks of treatment and in advance of stroke: transferrin, hemopexin, albumin, &agr;2-HS-glycoprotein, kallikrein-binding protein, &agr;1-antitrypsin, Gc-globulin, and transthyretin. Markers of an inflammatory response, including very high levels of thiostatin, were detected in the serum of SHRSP at least 4 weeks before a stroke occurred. Conclusions— In SHRSP subjected to salt loading, an atypical inflammatory condition and widespread alterations of vascular permeability developed before the appearance of anomalous features in the brain detected by MRI. Urinary concentrations of each of the excreted serum proteins correlated positively with time before stroke occurred.

The oxysterol–CXCR2 axis plays a key role in the recruitment of tumor-promoting neutrophils

Tumor-derived oxysterols recruit protumor neutrophils in an LXR-independent, CXCR2-dependent manner, thus favoring tumor growth by promoting neoangiogenesis and immunosuppression.