TOWARD UNDERSTANDING SMCENTRIN: STUDY OF OLIGOMERIZATION MECHANISMS, FILAMENT FORMATION DYNAMICS AND INHIBITORS ACTIONS

Schistosomiasis is caused by trematode parasites of the Schistosoma genus and represents a global health challenge given that one species of this genus - Schistosoma mansoni - is the main cause of the disease on the American continent. The most widely used drug today is Praziquantel, which acts by destabilizing Ca²⁺ homeostasis in adult worms. Calcium has a dynamic homeostasis that contributes to numerous cellular functions, from gene expression and to cell division and apoptosis. Therefore, calcium-binding proteins are potential targets against this parasite. Centrins are calcium-binding proteins located in centrioles and have a loop that binds Ca²⁺ . S. mansoni centrin has 64% sequential compatibility with HsCen 3 and 51% with HsCent 1 and 2, which form multimers in the presence of calcium. Thus, the aim of this project was to investigate whether S. mansoni centrin (SmCentrin) was capable of oligomerization and, if so, what factors influenced this process, such as salt concentration, the presence of Ca²⁺ and monastrol (inhibitor). The protein was successfully expressed in E. coli Rosetta (DE3) and purified by affinity chromatography and molecular exclusion chromatography. Dynamic light scattering (DLS) experiments showed an increase in particle size when 2 mM and 10 mM calcium were added (in 25 mM Hepes, 20 mM KCl, pH 7), indicating oligomerization and possible polymerization. The oligomerization of SmCentrin (at 100 µM concentration) induced by Ca²⁺ was partially reversible with the addition of EDTA (5 mM). Chemical crosslinking experiments confirmed that concentration is a determining factor for oligomerization, which was observed under different conditions at high concentration (150 µM). Experiments with monastrol in DSL showed a significant increase in particle size (100 μM protein + 100 μM monastrol) when 2 mM and 10 mM CaCl₂ were added, indicating polymerization. When increasing the concentration of monastrol (150 μM), the peak shifted to the left, indicating inhibition of this reaction. The results motivated the continuation of the work and the biophysical characterization of the protein contributed to determining its function and potential use as a molecular target.

This work was supported by Conselho Nac. Des. Cient. Tecnologico CNPq