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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.

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The human cytomegalovirus (HCMV) UL111A gene encodes several homologs of the cellular interleukin 10 (cIL-10). Alternative splicing in the UL111A region produces two relatively well-characterized transcripts designated cmvIL-10 (isoform A) and LAcmvIL-10 (isoform B). The cmvIL-10 protein is the best characterized, both structurally and functionally, and has many immunosuppressive activities similar to cIL-10, while LAcmvIL-10 has more restricted biological activities. Alternative splicing also results in five less studied UL111A transcripts encoding additional proteins homologous to cIL-10 (isoforms C to G). These transcripts were identified during productive HCMV infection of MRC-5 cells with the high passage laboratory adapted AD169 strain, and the structure and properties of the corresponding proteins are largely unknown. Moreover, it is unclear whether these protein isoforms are able to bind the cellular IL-10 receptor and induce signalling. In the present study, we investigated the expression spectrum of UL111A transcripts in fully permissive MRC-5 cells and semi permissive U251 cells infected with the low passage HCMV strain TB40E. We identified a new spliced transcript (H) expressed during productive infection. Using computational methods, we carried out molecular modelling studies on the three-dimensional structures of the HCMV IL-10 proteins encoded by the transcripts detected in our work (cmvIL-10 (A), LAcmvIL-10 (B), E, F and H) and on their interaction with the human IL-10 receptor (IL-10R1). The modelling predicts clear differences between the isoform structures. Furthermore, the in silico simulations (molecular dynamics simulation and normal-mode analyses) allowed us to evaluate regions that contain potential receptor binding sites in each isoform. The analyses demonstrate that the complexes between the isoforms and IL-10R1 present different types of molecular interactions and consequently different affinities and stabilities. The knowledge about structure and expression of specific viral IL-10 isoforms has implications for understanding of their properties and role in HCMV immune evasion and pathogenesis.

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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