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.
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 rBmTI-A presented a potent inhibitory activity against in vitro vessel formation. In this study, the tertiary structure of BmTI-A was modeled based on the structure of its Sabellastarte magnifica homologue. The structure stabilization was evaluated by molecular dynamics analysis. Circular dichroism data corroborated the secondary structure found by the homology modeling.Thermostability analysis confirmed the thermostability and the relation between the effects of the temperature in the inhibitor activity. The loss of activity observed was gradual, and, after 60 minutes of incubation at 90ºC the inhibitor lost it completely.
28rBmTI-A is a recombinant serine protease inhibitor that belongs to the Kunitz-BPTI 29 family and that was cloned from Rhipicephalus microplus tick. rBmTI-A has inhibitory 30 activities on bovine trypsin, human plasma kallikrein, human neutrophil elastase and 31 plasmin with dissociation constants in nM range. It is characterized by two inhibitory 32 domains and each domain presents six cysteines that form three disulfide bonds, which 33 contribute to the high stability of its structure. Previous studies suggest that serine 34 protease inhibitor rBmTI-A has a protective potential against pulmonary emphysema in 35 mice and anti-inflammatory potential, besides rBmTI-A presented a potent inhibitory 36 activity against in vitro vessel formation. In this study, the tertiary structure of BmTI-A 37 was modeled based on the structure of its Sabellastarte magnifica homologue. The 38 structure stabilization was evaluated by molecular dynamics analysis. Circular 39 dichroism data corroborated the secondary structure found by the homology modeling.
40Thermostability analysis confirmed the thermostability and the relation between the 41 effects of the temperature in the inhibitor activity. The loss of activity observed was 42 gradual, and, after 60 minutes of incubation at 90ºC the inhibitor lost it completely. 43
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