Vitor Hugo Moreau

Oxidative damage to sarcoplasmic reticulum Ca2+-ATPase at submicromolar iron concentrations: Evidence for metal-catalyzed oxidation

The sarcoplasmic reticulum (SR) calcium ATPase carries out active Ca2+ pumping at the expense of ATP hydrolysis. We have previously described the inhibition of SR ATPase by oxidative stress induced by the Fenton reaction (Fe2+ + H2O2 --> HO. + HO- + Fe3+). Inhibition was not related to peroxidation of the SR membrane nor to oxidation of ATPase thiols, and involved fragmentation of the ATPase polypeptide chain. The present study aims at further characterizing the mechanism of inhibition of the Ca2+-ATPase by oxygen reactive species at Fe2+ concentrations possibly found in pathological conditions of iron overload. ATP hydrolysis by SR vesicles was inhibited in a dose-dependent manner by micromolar concentrations of Fe2+, H2O2, and ascorbate. Measuring the rate constants of inactivation (k inact) at different Fe2+ concentrations in the presence of saturating concentrations of H2O2 and ascorbate (100 microM each) revealed a saturation profile with half-maximal inactivation rate at ca. 2 microM Fe2+. Inhibition was not affected by addition of 200 microM Ca2+ to the medium, indicating that it was not related to iron binding to the high affinity Ca2+ binding sites in the ATPase. Furthermore, inhibition was not prevented by the water-soluble hydroxyl radical scavengers mannitol or dimethylsulfoxide, nor by butylated hydroxytoluene (a lipid peroxidation blocker) or dithiothreitol (DTT). However, when Cu2+ was used instead of Fe2+ in the Fenton reaction, ATPase inhibition could be prevented by DTT. We propose that functional impairment of the Ca2+-pump may be related to oxidative protein fragmentation mediated by site-specific Fe2+ binding at submicromolar or low micromolar concentrations, which may occur in pathological conditions of iron overload.

Prediction of the amount of secondary structure of proteins using unassigned NMR spectra: a tool for target selection in structural proteomics

With the advent of structural genomics, the need for fast structural information about unknown proteins has increased. We describe a new methodology, based on 13C, 15N and 1H chemical shift dispersion to predict the amount of secondary structure of unassigned proteins from their 15N- and/or 13C-edited heteronuclear single quantum coherence (HSQC) spectra. This methodology has been coded into a software called PASSNMR (Prediction of the Amount of Secondary Structure by Nuclear Magnetic Resonance), which can be accessed directly from the Internet. PASSNMR program is a powerful tool for screening proteins for proteomic or structural genomic investigations when used with recent methodologies that take advantage of the use of the antibiotic rifampicin to selectively label the heterologous proteins expressed in E. coli. PASSNMR analysis can be useful as a first approach to predict the amount of secondary structure in proteins to structural genomics. Information about the secondary structure of proteins can be obtained even before protein purification, with small quantities of protein, just by performing two simple nuclear magnetic resonance (NMR) experiments and using PASSNMR program.