RESUMO
The reactions of proteins with biologically relevant oxidants have been widely studied, although most of the work has been performed in diluted homogenous solutions conditions that differ from those in intracellular environments. Cellular compartments represent highly crowded milieu in which high concentrations of biomolecules are present, unspecific intermolecular interactions are promoted, and physicochemical properties of constituents are modified. In this work, we propose that the high concentration at which proteins are present inside cells favours radical oxidative reactions between polypeptides which propagate in an oxygen-dependent process similar to membrane lipid peroxidation. The results presented herein show that highly concentrated solutions of bovine serum albumin (BSA) exposed to peroxynitrite, or metmyoglobin/H2O2, initiate the formation and propagation of protein peroxyl radicals, as evidenced by oxygen consumption, fluorescence spectroscopy, chemiluminescence, and electron paramagnetic resonance studies. Moreover, peroxyl radicals are capable of converting nitrite to nitrogen dioxide, which can oxidise amino acid residues to further assist radical-mediated protein oxidation. In addition, we also show that nitrone spin traps stop these propagation reactions in proteins, in line with the previously reported antioxidant role of these compounds in vivo. In summary, our results suggest that in crowded environments such as cellular compartments radical chain reactions propagate protein oxidative damage, highlighting a previously under recognised mechanism of cellular nitroxidative stress.
Assuntos
Radicais Livres/química , Soroalbumina Bovina/química , Peróxido de Hidrogênio/química , Ácido Peroxinitroso/química , SoluçõesRESUMO
Protein disulfide isomerase (PDI) is a dithiol-disulfide oxidoreductase that has essential roles in redox protein folding. PDI has been associated with protective roles against protein aggregation, a hallmark of neurodegenerative diseases. Intriguingly, PDI has been detected in the protein inclusions found in the central nervous system of patients of neurodegenerative diseases. Oxidized proteins are also consistently detected in such patients, but the agents that promote these oxidations remain undefined. A potential trigger of protein oxidation is the bicarbonate-dependent peroxidase activity of the human enzyme superoxide dismutase 1 (hSOD1). Therefore, we examined the effects of this activity on PDI structure and activity. The results showed that PDI was oxidized to radicals that lead to PDI inactivation and aggregation. The aggregates are huge and apparently produced by covalent cross-links. Spin trapping experiments coupled with MS analysis indicated that at least 3 residues of PDI are oxidized to tyrosyl radicals (Y(63), Y(116) and Y(327)). Parallel experiments showed that PDI is also oxidized to radicals, inactivated and aggregated by the action of photolytically generated carbonate radical and by UV light. PDI is prone to inactivation and aggregation by one-electron oxidants and UV light probably because of its high content of aromatic amino acids.