RESUMEN
Tyrosine nitration is an oxidative post-translational modification that can occur in proteins associated to hydrophobic bio-structures such as membranes and lipoproteins. In this work, we have studied tyrosine nitration in membranes using a model system consisting of phosphatidylcholine liposomes with pre-incorporated tyrosine-containing 23 amino acid transmembrane peptides. Tyrosine residues were located at positions 4, 8 or 12 of the amino terminal, resulting in different depths in the bilayer. Tyrosine nitration was accomplished by exposure to peroxynitrite and a peroxyl radical donor or hemin in the presence of nitrite. In egg yolk phosphatidylcholine liposomes, nitration was highest for the peptide with tyrosine at position 8 and dramatically increased as a function of oxygen levels. Molecular dynamics studies support that the proximity of the tyrosine phenolic ring to the linoleic acid peroxyl radicals contributes to the efficiency of tyrosine oxidation. In turn, α-tocopherol inhibited both lipid peroxidation and tyrosine nitration. The mechanism of tyrosine nitration involves a "connecting reaction" by which lipid peroxyl radicals oxidize tyrosine to tyrosyl radical and was fully recapitulated by computer-assisted kinetic simulations. Altogether, this work underscores unique characteristics of the tyrosine oxidation and nitration process in lipid-rich milieu that is fueled via the lipid peroxidation process.
Asunto(s)
Membrana Celular/metabolismo , Radicales Libres/metabolismo , Peroxidación de Lípido , Péptidos/metabolismo , Ácido Peroxinitroso/metabolismo , Tirosina/metabolismo , Amidinas/metabolismo , Secuencia de Aminoácidos , Membrana Celular/química , Hemina/metabolismo , Liposomas/química , Liposomas/metabolismo , Simulación de Dinámica Molecular , Oxidación-Reducción , Oxígeno/metabolismo , Péptidos/química , Tirosina/químicaRESUMEN
Hb S-São Paulo (SP) [HBB:c.20A>T p.Glu6Val; c.196A>G p.Lys65Glu] is a new double-mutant hemoglobin that was found in heterozygosis in an 18-month-old Brazilian male with moderate anemia. It behaves like Hb S in acid electrophoresis, isoelectric focusing and solubility testing but shows different behavior in alkaline electrophoresis, cation-exchange HPLC and RP-HPLC. The variant is slightly unstable, showed reduced oxygen affinity and also appeared to form polymers more stable than the Hb S. Molecular dynamics simulation suggests that the polymerization is favored by interfacial electrostatic interactions. This provides a plausible explanation for some of the reported experimental observations.
Asunto(s)
Anemia de Células Falciformes/genética , Hemoglobina Falciforme/metabolismo , Oxígeno/metabolismo , Globinas beta/metabolismo , Sustitución de Aminoácidos , Anemia de Células Falciformes/metabolismo , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Electroforesis , Hemoglobina Falciforme/química , Hemoglobina Falciforme/genética , Heterocigoto , Humanos , Lactante , Focalización Isoeléctrica , Masculino , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Polímeros , Estabilidad Proteica , Solubilidad , Electricidad Estática , Globinas beta/química , Globinas beta/genéticaRESUMEN
A better understanding of the structural effects induced by thyroid hormones in model membranes is attained by Raman spectroscopy. The interactions of T3 and T4 with multilamellar vesicles of dipalmytoylphosphatidylcholine (DPPC) in the gel phase are characterized by analyzing the spectral behavior of the C-H and C-C stretching vibrations of the acyl chains. The spectra evidence an increase in the relative number of gauche conformation, which indicates the hormones are able to penetrate into the hydrophobic region of the bilayer and partially alter the lipid structure. In addition, the density packing of the acyl chains appears increased and the rotational mobility of the terminal methylene groups is slightly reduced in the iodothyronine/DPPC mixtures. These effects are interpreted in terms of the transition to an interdigitated phase due to the hormone incorporation to the membrane. The polar heads of the lipids also interact with the hormone, as evidenced by the PO2(-) symmetric stretching band.