RESUMO
Serine protease inhibitors (serpins) regulate the activities of circulating proteases. Serpins inhibit proteases by acylating the serine hydroxyl at their active sites. Before deacylation and complete proteolysis of the serpin can occur, massive conformational changes are triggered in the serpin while maintaining the covalent linkage between the protease and serpin. Here we report the structure of a serpin-trypsin Michaelis complex, which we visualized by using the S195A trypsin mutant to prevent covalent complex formation. This encounter complex reveals a more extensive interaction surface than that present in small inhibitor-protease complexes and is a template for modeling other serpin-protease pairs. Mutations of several serpin residues at the interface reduced the inhibitory activity of the serpin. The serine residue C-terminal to the scissile peptide bond is found in a closer than usual interaction with His 57 at the active site of trypsin.
Assuntos
Manduca/química , Serpinas/química , Serpinas/metabolismo , Inibidores da Tripsina/química , Inibidores da Tripsina/metabolismo , Tripsina/química , Tripsina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Ligação de Hidrogênio , Manduca/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Conformação Proteica , Mapeamento de Interação de Proteínas , Ratos , Alinhamento de Sequência , Serpinas/genética , Tripsina/genética , Inibidores da Tripsina/genéticaRESUMO
Mexicali dust (MD) is a mixture of particles of potassium aluminum silicates (98%) and sodium dioxide (2%) that induces pulmonary damage under experimental conditions, and is capable of inducing in vitro chromosomal alterations in exposed lymphocytes. It has been proposed as an atmospheric contaminant with pathogenic potential. Among the chromosomal alterations observed, numeric alterations were predominant. The present study was designed to evaluate the capacity of MD to induce anaphasic changes in the Balb c 3T3 cell line. Chrysotile asbestos was used as a positive control. MD was found to induce abnormal anaphases, and the percentage of abnormalities increased as the dose increased (27.41% with 20 mg/mL, 29.60% with 40 mg/mL and 37.10% with 80 mg/mL). Multipolar anaphases constituted the most frequent alteration (69.1-78.8%), followed by lagging chromosomes (18.2-29.5%) and anaphasic bridges (1.51-5.9%). The anaphasic alterations induced by MD showed differences in comparison to those observed with asbestos, especially for anaphasic bridges (10.4% vs. 1.51%, p<0.05). The capacity of MD to induce alterations reported in the process of chromosomal disjunction could explain the numeric aberrations reported previously by the authors of this paper. Therefore, these data support that MD could act as a clastogenic agent.
