Structural modelling and thermostability of a serine protease inhibitor belonging to the Kunitz-BPTI family from the Rhipicephalus microplus tick.

rBmTI-A is a recombinant serine protease inhibitor that belongs to the Kunitz-BPTI family and that was cloned from Rhipicephalus microplus tick. rBmTI-A has inhibitory activities on bovine trypsin, human plasma kallikrein, human neutrophil elastase and plasmin with dissociation constants in nM range. It is characterized by two inhibitory domains and each domain presents six cysteines that form three disulfide bonds, which contribute to the high stability of its structure. Previous studies suggest that serine protease inhibitor rBmTI-A has a protective potential against pulmonary emphysema in mice and anti-inflammatory potential. Besides that, rBmTI-A presented a potent inhibitory activity against in vitro vessel formation. In this study, the tertiary structure of rBmTI-A was modeled. The structure stabilization was evaluated by molecular dynamics analysis. Circular dichroism spectroscopy data corroborated the secondary structure found by the homology modelling. Also, in circular dichroism data it was shown a thermostability of rBmTI-A until approximately 70 °C, corroborated by inhibitory assays toward trypsin.

Neutrophil elastase inhibitor purification strategy from cowpea seeds.

Serine proteases and its inhibitors are involved in physiological process and its deregulation lead to various diseases like Chronic Obstructive Pulmonary Disease (COPD), pulmonary emphysema, skin diseases, atherosclerosis, coagulation diseases, cancer, inflammatory diseases, neuronal disorders and other diseases. Serine protease inhibitors have been described in many species, as well as in plants, including cowpea beans (Vigna unguiculata (L.) Walp). Here, we purified and characterized a protease inhibitor, named VuEI (Vigna unguiculata elastase inhibitor), from Vigna unguiculata, with inhibitory activity against HNE (human neutrophil elastase) and chymotrypsin but has no inhibitory activity against trypsin and thrombin. VuEI was obtained by alkaline protein extraction followed by three different chromatographic steps in sequence. First, an ion exchange chromatography using Hitrap Q column was employed, followed by two reversed-phase chromatography using Source15RPC and ACE18 columns. The molecular mass of VuEI was estimated in 10.99 kDa by MALDI-TOF mass spectrometry. The dissociation constant (Ki) to HNE was 9 pM. These data indicate that VuEI is a potent inhibitor of human neutrophil elastase, besides to inhibit chymotrypsin.

rBmTI-6 attenuates pathophysiological and inflammatory parameters of induced emphysema in mice.

Protease/anti-protease imbalance is the main pathogenic mechanism of emphysema and protease inhibitors have been recognized as potential molecules to treat the disease conditions. In this work the rBmTI-6 first domain (rBmTI-6-D1), a recombinant Kunitz-type serine proteinase inhibitor, was used to verify its effect in prevention or minimization of PPE-induced emphysema in mice. C57BL/6 mice were submitted to a PPE-induced emphysema model and treated with rBmTI-6-D1 before the emphysema development. We showed that the rBmTI-6-D1 treatment was sufficient to avoid the loss of elastic recoil, an effective decrease in alveolar enlargement and in the number of macrophages and lymphocytes in bronchoalveolar lavage fluid. Proteolytic analysis showed a significant increase in elastase activity in PPE-VE (induced emphysema) group that is controlled by rBmTI-6-D1. Kallikrein activity was decreased in the PPE-rBmTI6 (induced emphysema and inhibitor treated) group when compared to PPE-VE group. Although rBmTI-6-D1, did not present a neutrophil elastase (NE) inhibitory activity, the results show that the inhibitor interfered in the pathway of NE secretion in PPE-emphysema mice model. The role of rBmTI-6-D1 in the prevention of emphysema development in the mice model, apparently, is related with a control of inflammatory response due the trypsin/kallikrein inhibitory activity of rBmTI-6-D1.