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The Italian Working Group on Nutrition and Stroke Prevention G. Rotilio 1,2, R. Berni Canani 2, G. Barba 3, E Branca 4,17, G. Cairella 5,17, B. Dilaghi 6, C. Fieschi 7, E Garbagnati 2, M.G. Gentile 8,17, G.E Gensini 6, A. Gualtieri 7, D. Inzitari 9, M. La Massa 9, M.L.E. Luisi 1°, 17, G. Mancia n, M. Marcelli 12,17, M.L. Masini 13, E Mastrilli 14, S. Paolucci 14 , L. Pratesi 14, P. Rubba 15 , M.L. Sacchetti 7 , A. Salvia 14 , L. Scalfi 16,17, U. Scognamiglio 2, A. Siani 3,17, p. Strazzul1015 1Department of Biology, Tor Vergata University of Rome, 2Centre of Research on Nutrition and Rehabilitation (CeSAR), IRCCS Fondazione S. Lucia, Rome, 3Institute of Food Sciences, CNR, Avellino, 4National Institute for Research on Food and Nutrition (1NRAN), Rome, 5Department of Prevention Nutrition Unit ASL RMB, Rome, 6Department of Medical and Surgical Critical Care, Section of Clinical Medicine and Cardiology, A.O.U. Careggi, Florence, 7Department of Neurology, University "La Sapienza", Rome, 8Clincal Nutrition Unit, Niguarda Hospital, Milan, 9Department of Neurological and Psychiatric Sciences, University of Florence, 1°Don Carlo Gnocchi Foundation, IRCCS Florence, 11Department of Clinical Medicine, Prevention and Applied Biotechnologies, University of Milano-Bicocca, Monza, 12Clinical Nutrition Unit A.O.S. Giovanni Addolorata, Rome, 13Dietetic Unit A.O.U. Careggi, Florence, 14IRCCS Fondazione S. Lucia, Rome, 15Department of Clinical and Experimental Medicine, Federico 1I University of Naples, 16Department of Food Science, Federico II University of Naples, and 17Italian Society of Human Nutrition (SINU)

The transcriptional co-activator PGC-1alpha and the NAD(+)-dependent deacetylase SIRT1 are considered important inducers of mitochondrial biogenesis because in the nucleus they regulate transcription of nucleus-encoded mitochondrial genes. We demonstrate that PGC-1alpha and SIRT1 are also present inside mitochondria and are in close proximity to mtDNA. They interact with mitochondrial transcription factor A (TFAM) as assessed by confocal microscopy analysis and by blue native-PAGE. Nucleoid purification allowed us to identify SIRT1 and PGC-1alpha as proteins associated with native and cross-linked nucleoids, respectively. After mtDNA immunoprecipitation analysis, carried out on mitochondrial extracts, we found that PGC-1alpha is present on the same D-loop region recognized by TFAM. Finally, by oligonucleotide pulldown assay, we found PGC-1alpha and SIRT1 associated with the TFAM consensus sequence (human mitochondrial transcription factor-binding site H). The results obtained suggest that in mitochondria PGC-1alpha and SIRT1 may function as their nuclear counterparts and represent the genuine factors mediating the cross-talk between nuclear and mitochondrial genome. Finally, this work adds new knowledge on the function of SIRT1 and PGC-1alpha and highlights the direct involvement of such proteins in regulation of mitochondrial biogenesis.

The H2O2 inactivation of the ''cambialistic'' superoxide dismutases from Propionibacterium shermanii, which is active with either iron or manganese at the active site, has been studied in the native and Val 73 --> Trp mutant enzymes. The wild-type iron-containing form of this enzyme is much more resistant to treatment with H2O2 with respect to the other metal-specific Fe superoxide dismutase isoenzymes. After incubation with high amounts of H2O2 the enzyme maintains more than 40% of the initial activity. The activity of the Val 73 --> Trp mutant drastically decreases 60 less than 5% of the initial activity after incubation with hydrogen peroxide. Amino acid analysis of the H2O2-treated mutant enzyme evidenced the loss of the Trp 73 residue which is shown to play a critical role in the stabilization of the monomer fold of the enzyme. On the other hand, the manganese-containing wild-type and mutant enzymes were completely resistant toward H2O2 demonstrating the specific role of iron in the inactivation process. (C) 1997 Academic Press.

The role of electrostatic factors in the enzyme-substrate encounter process of manganese and iron containing superoxide dismutases has been studied in the enzyme from Propionibacterium shermanii by chemical neutralization of lysine residues and site-directed mutagenesis of the highly conserved residue Lys175. Lysine residues have been neutralized by carbamoylation and Lys175 has been selectively replaced by isoleucine and arginine, Catalytic measurements show a dramatic decrease of the activity in the chemically modified enzyme, Electrostatic potential calculations evidence in the modified enzyme a large contraction of the positive potential areas which surround the active sites in the-native enzyme, indicating that electrostatic factors are critical in the enzyme-substrate encounter process of Mn- and Fe-superoxide dismutases. The activity drastically decreases also in Lys175-->Ile but not in the Lys175-->Arg mutant. Brownian dynamics simulations indicate that the decrease of activity in the Lys175-->Ile mutant cannot be due only to a decrease of the enzyme-substrate association rate, suggesting that Lys175 plays a relevant role also in the structural stabilization of the active site.

The thermal stability of two single (K3R, K67R) and one double (K3R-K67R) mutants of Xenopus laevis B Cu,Zn superoxide dismutase has been studied to test Lys-->Arg substitution as an 'electrostatically conservative' strategy to increase protein stability, The K3R mutant displays an increased thermostability with respect to the wild-type enzyme, whilst a decreased stability was observed in the case of the K67R and K3R-K67R mutants, Concentration dependence of the apparent inactivation constant (k(app)) of the latter mutants, as compared to that of the wild type enzyme and K3R mutant, indicates that their higher sensitivity to heat inactivation is due to a perturbation of the dimer association. These results are confirmed also by fluorescence anisotropy measurements of the internal probe Tyr149. The possible role of Arg67 in perturbing the dimer dissociation equilibrium toward the monomeric form is discussed.

The catalytic rate of four single and three double mutants of Xenopus laevis Cu,Zn superoxide dismutase B, neutralized at Lys120, Asp130, Glu131, and Lys134, has been determined by pulse radiolysis as a function of ionic strength. Neutralization of Glu131 increases the catalytic rate by 80% at low ionic strength, but the effect is reduced to 50% at physiological ionic strength. The rate is unperturbed upon neutralization of Asp130, while neutralization of either of the two lysines drastically decreases the enzyme activity. The Lys 120Leu-Lys134Thr and Lys134Thr-Asp130Gln double mutations have an additive and a compensative effect, respectively, on the activity values, while neutralization of the Glu131-Lys134 pair, which also has a compensative effect, gives rise to a faster enzyme at any ionic strength value. The effects observed in the single Asp130Gln and Lys120Leu mutants differ from those reported on human or bovine enzymes [Getzoff et al. (1992) Nature (London) 358, 347-351; Sines et al. (1990) Biochemistry 29, 9403-9412], indicating that some residues occupying the same position in the linear sequence of different Cu,Zn superoxide dismutases have a different functional weight. Our results also suggest that the strategy of multiple charge mutation may be a promising approach in order to increase the catalytic rate of Cu,Zn SODs independently of ionic strength.

The pH dependence of the activity of Cu,Zn superoxide dismutases from bovine erythrocytes and shark liver was studied by pulse radiolysis in both the native enzymes and those chemically modified at lysine side chains. The study was aimed at identifying the residues responsible for the activity decrease at pH > 9, observed in all native Cu,Zn superoxide dismutases, and is based on the Lys --> Arg substitution present in the shark protein at position 134, which has been established to be critical for the catalytic efficiency of the enzyme. Both native enzymes display a pH dependence that can be deconvoluted by three deprotonation equilibria, at pH 9-9.5 (pK(1)), at pH 10.2 (pK(2)), and at pH 11.5 (pR(3)). pK(1) is lacking in both the modified enzymes and thus can be assigned to activity-linked lysine residues. pK(2) is clearly dominated by Arg134 in the modified shark enzyme and can be assigned to surface arginine residues. pK(3) is shared by all four enzyme forms and is likely to be due to the invariant Arg141. (C) 1995 Academic Press, Inc.

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 Angstrom 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 Arg(141). The coordination sphere of Cu2+ is partly altered with respect to the uninhibited enzyme: a displacement of 0.41 Angstrom in subunit A, and 0.27 Angstrom in subunit B of the dimeric enzyme is observed for the Cu2+ ions. Only two ligands in the Cu2+ coordination sphere (His(46)and His(118)) are significantly affected by cyanide binding, whereas virtually no rearrangement of the Zn2+ ligands is reported.

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. (C) 1994 Academic Press, Inc.

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.