Biomedical subjects

G Rotilio

Publications and source records attributed to G Rotilio.

At least 91 records · Page 5Linked to original sources

PubMed1994

Impaired copper binding by the H46R mutant of human Cu,Zn superoxide dismutase, involved in amyotrophic lateral sclerosis.

Several point mutations in the gene coding for human Cu,Zn superoxide dismutase have been reported as being responsible for familial amyotrophic lateral sclerosis (FALS). However, no direct demonstration has been provided for a correlation between total superoxide dismutase activity and severity of the FALS pathology. In order to get a better insight into the mechanism(s) underlying the FALS phenotype, we have investigated the activity and the copper binding properties of the single mutant H46R, which is associated with a Japanese form of FALS. We have shown that this mutant is structurally stable but lacks significant enzyme activity and has impaired capability of binding catalytic copper. The mutant protein can be fully reconstituted with copper in vitro but its ESR spectrum displays an axial shape quite different from that of the wild-type.

Amyotrophic Lateral Sclerosis↗

PubMed1994

Purification and characterization of Ag,Zn-superoxide dismutase from Saccharomyces cerevisiae exposed to silver.

Cu,Zn-superoxide dismutase plays an important role in protecting cells from oxygen toxicity by catalyzing the dismutation of superoxide anion into hydrogen peroxide and oxygen. In Saccharomyces cerevisiae Cu,Zn-superoxide dismutase is coregulated with copper-thionein by copper via the transcription factor ACE 1. We demonstrate here that presence of AgNO3 in the culture medium leads to a five times increase of Cu,Zn-superoxide dismutase mRNA, with a concomitant six times decrease of the enzyme activity. Susceptibility of yeast to silver was apparently inversely related to Cu,Zn-superoxide dismutase activity. From silver-treated yeast a Cu,Zn-superoxide dismutase with impaired dismutase function was purified and was shown to contain silver, which was located to the copper site. These data suggest that Cu,Zn-superoxide dismutase may play an additional direct role in the defense of S. cerevisiae against metal stress by functioning as metal chelator.

Cell Division↗

PubMed1994

Mutation of Lys-120 and Lys-134 drastically reduces the catalytic rate of Cu,Zn superoxide dismutase.

Lys-120 and Lys-134, located at the edge of the active site channel in most Cu,Zn superoxide dismutases, have been suggested to play a major role in steering the anionic substrate towards the catalytic copper ion. In this study, mutants of Xenopus laevis Cu,Zn superoxide dismutase have been engineered, with Lys-120 and Lys-134 changed into leucine and threonine, respectively, and their catalytic properties have been investigated by pulse radiolysis. Results obtained demonstrate that both residues decrease the catalytic rate by about 40%, in partial disagreement with previous brownian dynamics calculations, carried out on bovine Cu,Zn superoxide dismutase.

Animals↗

PubMed1994

Differential decrease of copper content and of copper binding to superoxide dismutase in liver, heart and brain of copper-deficient rats.

Dietary copper-deficiency in rats produced a organ-specific decrease of copper content. This was paralleled by a decrease of the activity of the copper-enzyme superoxide dismutase. In liver such a decrease is partially due to the existence of an apo-form of superoxide dismutase, which can be reactivated by addition of exogenous copper to tissue extracts. These results demonstrate in vivo that superoxide dismutase is post-translationally modulated by copper in higher vertebrates as previously found for yeast and mammalian cells in culture.

Animals↗

PubMed1994

Copper-dependent metabolism of Cu,Zn-superoxide dismutase in human K562 cells. Lack of specific transcriptional activation and accumulation of a partially inactivated enzyme.

The regulation of Cu,Zn-superoxide dismutase by copper was investigated in human K562 cells. Copper ions caused a dose- and time-dependent increase, up to 3-fold, of the steady-state level of Cu,Zu-superoxide dismutase mRNA. A comparable increase was also observed for actin and ribosomal protein L32 mRNAs, but not for metallothionein mRNA which was augmented more than 50-fold and showed a different induction pattern. The copper-induced mRNAs were actively translated as judged from their enhanced loading on polysomes, the concomitantly increased cellular protein levels and an augmented incorporation of [3H]lysine into acid-precipitable material. Cu,Zn-superoxide dismutase protein followed this general trend, as demonstrated by dose- and time-dependent increases in immunoreactive and enzymically active protein. However, a specific accumulation of Cu,Zn-superoxide dismutase was noticed in cells grown in the presence of copper, that was not detectable for other proteins. Purification of the enzyme demonstrated that Cu,Zn-superoxide dismutase was present as a reconstitutable, copper-deficient protein with high specific activity (kcat./Cu = 0.89 x 10(9) M-1.s-1) in untreated K562 cells and as a fully metallated protein with low specific activity (kcat./Cu = 0.54 x 10(9) M-1.s-1) in copper-treated cells. Pulse-chase experiments using [3H]lysine indicated that turnover rates of Cu,Zn-superoxide dismutase in K562 cells were not affected by growth in copper-enriched medium, whereas turnover of total protein was significantly enhanced as a function of metal supplementation. From these results we conclude that: (i) unlike in yeast [Carrì, Galiazzo, Ciriolo and Rotilio (1991) FEBS Lett. 278, 263-266] Cu,Zn-superoxide dismutase is not specifically regulated by copper at the transcriptional level in human K562 cells, suggesting that this type of regulation has not been conserved during the evolution of higher eukaryotes; (ii) copper ions cause an inactivation of the enzyme in intact K562 cells; and (iii) the metabolic stability of Cu,Zn-superoxide dismutase results in its relative accumulation under conditions that lead to increased protein turnover.

Animals↗

PubMed1994

Crystal structure of the cyanide-inhibited Xenopus laevis Cu,Zn superoxide dismutase at 98 K.

The crystal structure of cyanide-inhibited X. laevis Cu,Zn superoxide dismutase has been studied and refined based on diffraction data collected at 98 K. The final R-factor for the 27,299 reflections in the 10.0-1.7 A resolution range is 0.170. The cyanide anion, which is a competitive inhibitor expected to mimic the superoxide binding mode, binds directly to the active site copper atom, replacing the coordinated water molecule. Moreover, the anion establishes a strong electrostatic interaction with the guanidinium group of the conserved active site residue Arg141. The coordination sphere of Cu2+ is partly altered with respect to the uninhibited enzyme: a displacement of 0.41 A in subunit A, and 0.27 A in subunit B of the dimeric enzyme is observed for the Cu2+ ions. Only two ligands in the Cu2+ coordination sphere (His46 and His118) are significantly affected by cyanide binding, whereas virtually no rearrangement of the Zn2+ ligands is reported.

Animals↗

PubMed1994

In vivo incorporation of cobalt into Propionibacterium shermanii superoxide dismutase.

Propionibacterium shermanii, an aerotolerant anaerobic bacterium, has already been shown to incorporate, depending on the metal supplementation to the medium, either iron or manganese or copper into the same superoxide dismutase protein. The in vivo incorporation of cobalt in the same superoxide dismutase was obtained in an iron-, manganese- and copper-depleted medium. The protein was isolated and characterized by NMR which offers the possibility to identify the amino acid residues at the active site exploiting isotropically shifted proton resonance.

Cobalt↗

PubMed1994

Crystallographic study of azide-inhibited bovine Cu,Zn superoxide dismutase.

The crystal structure of azide-inhibited bovine Cu,Zn superoxide dismutase has been studied and refined based on X-ray synchrotron radiation data, in conjunction with difference Fourier and restrained crystallographic refinement techniques. The final R-factor for the 20,756 reflections in the 10.0 to 2.1 A resolution range is 0.166. In both enzyme subunits, the azide anion, which is a competitive inhibitor expected to mimic the superoxide binding mode, is observed directly coordinated to the Cu2+ at the place of the metal-bound water molecule, forming an ion pair with the conserved active site residue Arg141. The coordination sphere of Cu2+ is partly altered with respect to the uninhibited enzyme: a displacement of 0.67 A in subunit A, and 0.37 A in subunit B of the dimeric enzyme is observed for the Cu2+. Only two ligands in the Cu2+ coordination sphere (His46 and His118) are affected by azide binding, whereas virtually no rearrangement of the Zn2+ ligands is reported.

Animals↗

PubMed1994

Role of zinc-coordination and of the glutathione redox couple in the redox susceptibility of human transcription factor Sp1.

We show that thiol-groups confer redox-susceptibility to the zinc-finger transcription factor Sp1 and that this redox-susceptibility is prevented by DNA-binding and depends on zinc-coordination of the protein. Apo-Sp1 contained in metal depleted nuclear extracts of human K562 cells exhibited a markedly increased susceptibility towards oxidizing and alkylating agents, as compared to holo-Sp 1. Moreover, DNA binding of apo-Sp1, but not of the holo-protein, was dramatically decreased in the presence of GSH/GSSG ratios within the physiological range. We compared these results with the redox behaviour of two other transcription factors, OTF-1 and NF1, which was found to be different in several aspects from that of Sp1.

Apoproteins↗

PubMed1994

Role of hydroxyl radicals in the activation of human platelets.

Platelets primed by exposure to subthreshold concentrations of arachidonic acid or collagen are known to be activated by nanomolar levels of hydrogen peroxide. We here demonstrate that this effect is mediated by hydroxyl radicals (OHzero) formed in an extracellular Fenton-like reaction. H2O2-induced platelet aggregation, serotonin release and thromboxane A2 productions were inhibited by OHzero scavengers and by the iron chelator desferrioxamine; hydroxyl radicals were detected directly by ESR measurements of the spin-trapped OHzero adduct. The role of OHzero was confirmed in experiments with exogenously added iron; free or EDTA-bound ferrous iron activated platelets in a process blocked by deoxyribose, mannitol or catalase, whereas ferric iron was without effect unless reductants were included. The activation by OHzero depended on concomitant release of arachidonic acid and was blocked by the phospholipase A2 inhibitors mepacrine and aristolochic acid, and by the Na+/K+ antiporter inhibitor ethylisopropylamiloride. In contrast, neomycin and staurosporin were without effects, indicating that phospholipase C and protein kinase C were not involved in the initial phase of activation. Neither radical formation nor arachidonic acid release was blocked by aspirin. In whole blood aggregation of platelets could be induced by H2O2 generated upon specific stimulation of neutrophils by N-formyl-methionyl-leucyl-phenylalanine; platelet activation and radical formation were blocked by the NADPH oxidase inhibitor diphenyliodonium as well as by catalase and mannitol. These results suggest that reactive oxygen species act as 'second messengers' during the initial phase of the platelet activation process.

Arachidonic Acid↗

PubMed1994

Molecular modeling and electrostatic potential calculations on chemically modified Cu,Zn superoxide dismutases from Bos taurus and shark Prionace glauca: role of Lys134 in electrostatically steering the substrate to the active site.

The three-dimensional structure of Cu,Zn superoxide dismutase (SOD) from the shark Prionace glauca was homology modeled on the structure of the bovine enzyme used as a template. Shark SOD displays the conservative substitution of one of the residues involved in the electrostatic guidance of superoxide toward the active site of the enzyme, i.e., Lys134, which is replaced by arginine. The association rate, calculated by brownian dynamics simulations incorporating electrostatic terms, and the enzyme reaction rate, measured by the pulse radiolysis method, were found to be identical for both bovine and shark SODs. In order to quantify the role of Lys134 in electrostatically steering the superoxide to the active site, chemical modification of the lysine residues by carbamoylation was carried out on both bovine and shark SODs. Experimental determination of the enzymatic reaction rate indicated that the charge located on the residue 134 gives 19% contribution to the guidance of superoxide anion at low ionic strength. This value matches the association rates predicted by brownian dynamics.

Amino Acid Sequence↗

PubMed1993

Transduction of reducing power across the plasma membrane by reduced glutathione. A 1H-NMR spin-echo study of intact human erythrocytes.

The NMR signal of reduced glutathione (GSH) was monitored in intact human erythrocytes by the 1H spin-echo Carr-Purcell-Meiboom-Gill pulse sequence. Addition of GSH, which was unable to cross the erythrocyte membrane, produced an approximate twofold increase of the GSH signal in glucose-depleted cells. Addition of oxidised glutathione (GSSG), did not affect the signal, and addition of GSH to hemolysates gave a much smaller increase. Reduction of internal GSSG by NADPH-dependent enzymes was excluded by experiments with glucose-supplied or glucose-6-phosphate dehydrogenase deficient cells. Involvement of external thiol groups of the erythrocyte membrane was shown by the lack of effect in cells treated with an impermeable thiol-blocking compound. Involvement of spectrin was indicated by the proportional loss of the effect in erythrocytes with variable genetic deficiency of spectrin. Protein-glutathione mixed disulfides appeared to be the source of the NMR response since an increase of their content, by diamide treatment or aging procedures, produced a higher GSH signal, while their reduction by permeable reductants gave the opposite effect. It is concluded that GSH can transduce its reducing power by a thiol/disulfide exchange mechanism that sequentially involves sulfur-rich proteins spanning across the erythrocyte membrane.

Adult↗

PubMed1993

Copper(I) transfer into metallothionein mediated by glutathione.

Rabbit liver metallothionein depleted of Cd(II) and Zn(II) was fully reconstituted using a Cu(I)-GSH complex under strictly anaerobic conditions. Anaerobic fluorescence titration, using an emission band at 625 nm which is diagnostic of the correct insertion of Cu(I) into the thiolate clusters of metallothionein, showed that the fluorescence maximum was obtained on addition of as many Cu(I) equivalents as the available Cu(I)-binding sites in the protein (i.e. 12). Binding was nearly complete within 1 min, and Cu(I)-GSH was much more efficient than Cu(I)-thiourea or Cu(I)-acetonitrile in metallothionein reconstitution. In air, full reconstitution was obtained with stoichiometric copper only when an excess of GSH was present in the reaction mixture. Cu(I)-GSH was also able to displace Zn(II) and Cd(II) from natural metallized thionein. It is concluded that: (a) Cu(I)-GSH is a potential physiological Cu(I) carrier, not only for Cu2+/Zn2+ superoxide dismutase [Ciriolo, Desideri, Paci and Rotilio (1990) J. Biol. Chem. 265, 11030-11034] but also for metallothionein; (b) in the case of metallothionein, physiological concentrations of GSH protect the protein from autoxidation in air and facilitate Cu(I)-thiolate exchange; (c) the natural metal composition of metallothionein may be related to metal bioavailability rather than to evolutionary changes in protein structure.

Anaerobiosis↗

PubMed1993

Saturated dicarboxylic acids as products of unsaturated fatty acid oxidation.

Upon chemical, radiation-induced or enzymatic oxidation, cis-polyunsaturated fatty acids, i.e., C18:2(n-6), C18:3(n-3), C20:2(n-6), C20:3(n-6), C20:3(n-3), C20:4(n-6), C20:5(n-3), C22:2(n-3), C22:4(n-6), C22:6(n-3), were found to generate saturated short and medium-chain length dicarboxylic acids, which can be regarded as a distinctive feature of the particular double bonds positions in the polyunsaturated fatty acid molecule. Two different dicarboxylic acids, which were unambiguously quantified by GC-MS, were produced from a single fatty acid: one deriving from the oxidative splitting at the level of the first double bond in the molecule, the other being two-carbon-atoms lower homologous. Formation of dicarboxylic acids occurred also from triacylglycerols and phospholipids containing cis-polyunsaturated fatty acids. In this case, following oxidation, the diacids remained covalently bound to the starting molecule and transesterification was necessary for identification. Being extremely stable and easily detectable compounds, dicarboxylic acids may be considered potential markers of oxidative attack to both free and esterified unsaturated fatty acids.

Animals↗

PubMed1993

An X-ray absorption study of the reconstitution process of bovine Cu,Zn superoxide dismutase by Cu(I)-glutathione complex.

The Cu(I)GSH complex has recently been shown to be a good candidate for delivering copper to the active site of Cu-free Cu,Zn superoxide dismutase both in vivo and in vitro. In this work X-ray absorption spectroscopy has been used to characterize the Cu(I)GSH complex and to follow in vitro the reconstitution of Cu,Zn superoxide dismutase from the copper-free protein and this complex. The results obtained indicate that the copper is directly transferred as Cu(I) from the GSH complex into the empty copper binding site. No evidence has been obtained for a ternary complex in which the metal is bound to both GSH and the protein.

Animals↗

PubMed1993

Chaperonins dependent increase of Cu,Zn superoxide dismutase production in Escherichia coli.

Over-expression of the chaperonins GroEL and GroES significantly suppressed the temperature-dependent pattern of expression of Cu,Zn superoxide dismutases in Escherichia coli and increased the yield of active enzyme. The results obtained indicate that chaperonins prevent degradation of metal-deficient enzyme molecules. GroEL was shown to form a complex with unfolded Cu,Zn superoxide dismutase in vitro, confirming that GroEL can interact with beta-stranded proteins.

Animals↗

PubMed1993

Two-dimensional NMR assignment of hyperfine-shifted resonances of very fast relaxing metal binding sites of proteins by NOE spectroscopy. The case of Cu, Co superoxide dismutase.

The isotropically shifted NMR resonances of bovine Cu, Co superoxide dismutase, arising from the imidazole protons of the metal binding site of the native Cu, Zn enzyme when Zn(II) is replaced by Co(II), were investigated by two-dimensional NMR. NOE spectra have been measured at different mixing times in order to obtain a good set of interproton dipolar connectivities. A number of NOEs were obtained for very fast relaxing resonances close to the paramagnetic copper ion and new isotropically shifted resonances were detected in the proximity of the diamagnetic region. The recently obtained original X-ray coordinates of the Cu, Co enzyme were used in order to give the appropriate values to the interproton distances of the imidazole rings of the copper-coordinated histidines. The data allowed an unequivocal and definitive assignment of the isotropically shifted resonances to be done, with respect to previous results obtained using the coordinates of the Cu, Zn enzyme and monodimensional NMR techniques, which contain a potential source of artifacts in the selective excitation required prior to acquisition of spectra.

Animals↗

PubMed1993

Electrostatic recognition in redox copper proteins: a 1H NMR study of the protonation behavior of His 19 in oxidized and reduced Cu,Zn superoxide dismutase.

The pK shift of the His 19 residue in bovine Cu,Zn superoxide dismutase was carefully measured by nuclear magnetic resonance spectroscopy as a function of the change of the copper oxidation state. The measured pK values were identical within experimental error, at variance with results obtained with other copper proteins under similar conditions. The DelPhi program, based on a macroscopic dielectric model for the electrostatic interactions in proteins, was used to calculate pK shifts, as a function of charge perturbation introduced by metal oxidation, between the oxidized and the reduced enzyme, which are assumed to have the same structure on the basis of previous spectroscopic data. A nice fit with the experimental pK values was obtained protonating the imidazole of the copper-zinc bridging His 61, which is known to release the copper in the reduced enzyme. Protonation of His 61 in the reduced state gives rise to an electrostatic potential distribution around the protein almost identical to that observed in the oxidized one. These results suggest that a major role for the bridging histidine in Cu,Zn superoxide dismutase is to provide identical electrostatic steering of the substrate in the two oxidation states of the enzyme by redox-linked protonation-deprotonation processes. This property is discussed in comparison with the effect of copper reduction on the distribution of the electric field in small blue copper proteins.

Acids↗