Caracterização bioquímica e farmacológica de proteases do látex de carica candamarcensis com atividade mitogênica

Abstract

Latex from Caricaceae contains proteolytic enzymes localized in the fruit, which are widely used industrially as well as in pharmaceutical preparations. Ethnopharmacologically, Carica candamarcensis, a member of Caricaceae family common to many areas of South America, is used to treat digestive disorders. Cysteine proteinases from Caricaceae latex are believed to be part of a plant defense mechanism. Upon injury, these enzymes promote the formation of a protein clot around the wounded area, as it occurs with mammalian systems. Some of these proteins display proliferative properties when probed with mammalian cells suggesting a role in reconstruction of wounded tissue. There are evidences that some of these enzymes are able to protect and enhance healing of chemically induced gastric ulcers in rats. In addition, it was demonstrated that these proteinases enhance the healing rate of induced dermal abrasions. In this study, we first aimed the isolation and characterization of proteinases displaying mitogenic activity. Subsequently, our goals were to determine the structure of the mitogenic protein and analyze a possible mechanism of action of this molecule. After three chromatography steps we isolated two mitogenic cysteine proteinases (CMS2MS2 and CMS2MS3) displaying a relative mass of 23 kDa. CMS2MS2 at approximately 1 nM stimulates proliferation above 50% of the control levels on insulinoma, fibroblastic, and osteoblastic cells. The mitogenic activity of CMS2MS2 in fibroblast was not affected by its inhibition with E-64, a specific inhibitor of cysteine protease. Furthermore, a specific MEK inhibitor (PD98059), that blocks the MAP-kinases pathway, abolishes the mitogenic activity of CMS2MS2. This inhibitory effect was also observed when CMS2MS2 was inhibited with E-64. In addition, the mitogenic effect was slightly suppressed (10%) when fibroblasts were pre-incubated with AG1478, a specific inhibitor of the EGF-receptor kinase. This phenomenon was correlated with growth factors released from cells acting in an autocrine way. CMS2MS2 contains 214 residues including the catalytic triad composed by Cys25, His159, Asn175. A phylogenetic tree analysis demonstrated that CMS2MS2 ranks closer to chymopapain than to papain, both isolated from Carica papaya latex. The overall predicted three-dimensional structure is similar to proteases from the papain family. These results suggest that minor structural differences within CMS2MS2 must account for its proliferative action that is independent of its proteolytic activity; but it is mediated by activation of MAP-kinases. This mitogenic property attributed to a purified cysteine proteinase may explain some of the therapeutical actions attributed to these enzymes.