Banca de QUALIFICAÇÃO: YANARA ALESSANDRA SANTANA MOURA

MICROALGAE THROMBOLYTIC PROTEINS: CHARACTERIZATION STRUCTURAL, FUNCTIONAL AND CYTOTOXICITY

fibrinolytic enzyme; kinetic; thermodynamic; antitumor; bioinformatic

Fibrinolytic enzymes have been extensively investigated due to their potential as alternative thrombolytic agents. In particular, the fibrinolytic enzyme produced by Arthrospira platensis cyanobacteria possesses effective fibrinolytic activity in vitro and showed stability in human physiological temperature (< 50 ℃) and pH (6,0 - 10,0) ranges. In this sense, this study investigated the biochemical and biological characteristics of the fibrinolytic enzyme from A. platensis (FEAP), as well as analyzed the structures of protein obtained from microalgae and cyanobacteria with thrombolytic potential using bioinformatic methods. For this, mathematical tools were used to estimate the kinetic and thermodynamic parameters of the fibrin hydrolysis by FEAP, as well as its thermal denaturation. Additionally, the enzyme was also investigated for its fibrinogenolytic and plasminogen activation potential. Moreover, the FEAP potential to cause hemolysis in erythrocytes, and its cytotoxicity, genotoxicity, and anti-migration properties were tested against breast cancer cells. Finally, a study in silico was performed to identify the presence of intrinsically disordered regions (IDRs) and short linear motifs (SLiMs) in potentially thrombolytic microalgal and cyanobacterial proteins. The kinetic and thermodynamic properties of the enzyme reaction showed a Michaelis-Menten-type profile of two-plateau with low Km values (0,02 e 6,37 μg･mL−1). Regarding the thermal denaturation parameters, the temperature increase from 40 to 70 ℃ enhanced the denaturation constant from 0,0050 to 0,0134 min−1 and decreased the half-life from 138,62 to 51,72 min. In addition, the evaluation of FEAP’s functional properties showed a thrombolytic degradation of 43% with direct action on fibrin, without effects on fibrinogen or plasminogen. According to cytotoxicity tests, FEAP decreased breast cancer (S-180 e MDA-MB-231) and macrophage (J774A.1) cell viability and did not cause erythrocyte hemolysis. Furthermore, the enzyme also showed an anti-metastatic and non-genotoxic profile. Finally, the evaluation of the potentially thrombolytic protein structures properties revealed that 64.2% of the studied proteins were considered as moderately disordered, while 28,6% are highly disordered. Most of them belong to the plasmin and fibronectin-binding protein A subfamily, which can bind plasminogen and activate it to plasmin. Moreover, themain motifs found in proteins were the MOD_GlcNGglycan and CLV_PCSK_SKI1_1 motifs,which can also carry out different role in thrombolytic activity. These results suggest that FEAP is a thermostable biocatalyzer and is possibly safe for people undergoing cancer treatments, with less possibility of side effects, such as hemorrhages, and possesses antitumor potential preventing metastasis.