RESUMO
Peroxynitrite, via post-translational modifications to target proteins, contributes to cardiovascular injury and cancer. Since tissue inhibitor of metalloproteinase-4 (TIMP-4), the activity of which is impaired in both pathological conditions, has several amino acid residues susceptible to peroxynitrite, we investigated its role as a potential target of peroxynitrite. Peroxynitrite-induced nitration and oligomerization of TIMP-4 attenuated its inhibitory activity against MMP-2 activity and endothelial or tumor cell invasiveness. Moreover, cell treatment with peroxynitrite promoted the nitration of endogenous TIMP-4. HPLC/ESI-MS/MS analysis of peroxynitrite-treated TIMP-4 showed modifications at Y114, Y195, Y188 and Y190. In conclusion, TIMP-4 nitration might be a potential mechanism contributing to cardiovascular disease and cancer.
Assuntos
Ácido Peroxinitroso/farmacologia , Inibidores Teciduais de Metaloproteinases/química , Animais , Bovinos , Linhagem Celular Tumoral , Movimento Celular , Células Cultivadas , Endotélio Vascular/citologia , Humanos , Inibidores Teciduais de Metaloproteinases/antagonistas & inibidores , Inibidores Teciduais de Metaloproteinases/metabolismo , Tirosina/análogos & derivados , Tirosina/análise , Inibidor Tecidual 4 de MetaloproteinaseRESUMO
The present study describes the pattern of protein modification undergone by human holo-myoglobin by reactive fluoroquinones enzymatically produced by oxidation of 3-fluorophenol in mild conditions (pH 7.4, 25 degrees C). The fluoroquinones react with a number of histidine residues. Surface residues H24, H36, H48, and H82 and the heme distal histidine H64 were all found to be modified to a significant extent. In contrast, cysteine C110 is not appreciably affected, possibly because it is not accessible to the fluoroquinones. The sites of protein modification were assessed by mass spectrometry analysis of the peptide fragments resulting from controlled proteolysis of the apoprotein. As a consequence of the reaction with quinones, the globular structure of myoglobin becomes more prone to denaturation by the partial loss of its secondary structure. As a more intriguing consequence, the fluoroquinones promote the formation of structured aggregates of moderate size that lack the typical morphology of fibrillar structures.