Construção de híbridos dos interferons humanos beta e alfa2: papel das alfa-helices e alças na atividade biológica

Abstract

Interferons are proteins involved in the organism defense processes, derived from cellular responses to microorganisms, tumors and antigens and are classified in type I, comprehending the IFN-, IFN-, IFN-, IFN-, IFN- and IFN-, type II (IFN-) and a new family described as IFN--1, IFN--2 and IFN--3. These proteins bind to specific cells receptors, activating cytoplasmatic signals that are directed to the nucleus to stimulate genes that codifies proteins responsible by the cellular defense mechanism. The described cellular surface receptors are three types: a receptor that interacts with IFN type I, one with IFN- and another with IFN-. Despite all the research and knowledge about these interactions, between interferons type I with your or yours receptors, these mechanisms have not been clarified satisfactorily. In relation to its structure, the interferons are part of the helicoidally cytokines family, presenting a three-dimensional structure in -helixes. The molecule is constituted by 5 helixes (A, B, C, D and E), joined to each other by grips (ab, bc, cd and de). The threedimensional structure of the human IFN type 1 has been described and presents a significant similarity between the HuIFN-2 and HuIFN-. This similarity is of extreme importance in the understanding of the interactions of the molecules with their cellular receptor and allows the mapping of regions involved in this process, as a better understanding in the trigged mechanisms, after interaction with their cellular receptors. Studies with the humans IFN (HuIFN-2 and HuIFN-) shows significant changes in their activity when their aminoacids are modified or deleted, what can cause a substantial reduction in the activity and binding to their receptor, as demonstrated in bovine cells. The strategy used in this work was the introduction of restrictions sites between the 5 alfahelixes and grips of the humans IFN type I (-2 and -), in order to substitute the -helixes B, C, D, E and grips bc, cd e de, whose participations has been sugested in biological functions of the IFN type I. Therefore, the basical functional structure of the rHuIFN- and rHuIFN- were kept, allowing the study of the roles of the -helixes and grips in the diferent biological function of these IFN, particularly in their specie specifity. The constructions presented diferences in the production and purification of the protein. The modifications done in the structure of the HuIFN- and HuIFN-, showed alterations in the antiviral and specie specifity in Vero, Wish, MDBK and L929 cells when compared to those of the rHuIFN-2b. Two hybrids that were constructed (rHuIFN-97 and rHuIFN44106) presented lower antiviral activity in Vero cells, when compared the controls rHuIFN- and rHuIFN-97; however, the antiviral activity observed in MBDK cells were equal to those observed with the controls. Furthermore, neither of the constructed IFN presented antiviral activity in L-929 cells. All the constructed IFN were eficient in the induction of genes already reported as induced by IFN. The acumulation of mRNA of all the genes (6-16, 2´5´OAS e MxA) assayed in Wish cells were observed, with some acumulation level variations of these mRNA. Although, in L-929 cells, the mRNA acumulation of the GBP gene, induced by IFN, was not observed for neither of the tested IFN. One explanation for this absence of mRNA acumulation can be the fact that these IFN did not show antiviral activity in these cells. This study brought contributions for a better understanding of the structures and function of the humans IFN type I (HuIFN-2 and HuIFN-), as also rised other questions related to these molecules, what leads to perspectives for future studies with these cytokines.