Navegando por Palavras-chave "Cisteíno Proteases"

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    Expressão heteróloga e avaliação in vitro da atividade de proteínas recombinantes de Haementeria vizottoi sobre os mecanismos de coagulação sanguínea, com foco em aplicação biotecnológica
    (Universidade Federal de São Paulo (UNIFESP), 2020-03-05) Linhares, Debora Do Carmo [UNIFESP]; Tavassi, Ana Marisa Chudzinski [UNIFESP]; Universidade Federal de São Paulo
    Salivary secretions from hematophagous animals have been an important source of proteins with biotechnological potential, since there are a number of protein inhibitors that can be isolated and used to modulate the action of enzymes of interest. Many of these inhibitors have evolved to specifically disable key proteases from their hosts, leading to delayed blood coagulation (anticoagulants) and minor immune response, providing the hematophagous with the conditions for successful feeding. Based on transcriptomics of salivary glands of the leech Haementeria vizottoi, were identified sequences of three genes whose predicted proteins have signatures of potential biological activity as protease inhibitors. The aim of this prospective study was to clone, express and characterize these three H. vizottoi derived proteins, namely: Hviz 1276 (hemerythrin-like), Hviz 78 (antistasin-like) and Hviz 340 (cystatinlike). Due to their structural characteristics, antistin and cystatin-like proteins were expressed in P. pastoris (Komagataella pastoris) and hemerythrin-like protein was expressed in E. coli. The proteins of interest were recovered from the supernatant medium or cell extract, and purified by molecular exclusion chromatography and / or affinity chromatography with different resins. To determine their effects on the coagulation cascade, activated thromboplastin time (aPTT) and prothrombin time (PT) were determined using diagnostic kits. Depending on the result, they were also evaluated for the potential to inhibit specific proteases such as FXa, thrombin, elastase, kallikrein, papain and cathepsin L. Hviz 1276 did not demonstrate the expected biological activity and its study was discontinued. The application of partially purified Hviz 78, in the order of 1 µmol/L, was enough to extend the intrinsic-initiated coagulation time by more than 700%. It is still necessary to work on the refinement of purification of this protein and to perform new function tests, since no specific inhibitory activity against any of the proteases tested was identified. The most promising results were obtained with Hviz 340, purified and identified as inhibitor of cysteine proteases papain and cathepsin L, for the latter with ki=7.9 nmol/L. This activity represents new opportunities to evaluate the potential of Hviz 340 as a modulator of cellular activity related to the immune response, possibly anti-inflammatory, since cathepsins are critical for antigen processing and presentation.