Estudos comparativos da interação de Hsp90 de diferentes organismos com nucleotídeos adenosina e caracterização da Aha4 de Plasmodium falciparum
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Hsp90 (heat shock protein of 90 kDa) are among the most abundant proteins in non-stressed cells. They interact with proteins that are not in the native state, correcting their incorrect folding, they participate in signal transduction pathways, apoptosis and they are also involved in cell growth and development, protein maturation and activation, among others. In protozoa such as Plasmodium falciparum and Leishmania braziliensis, Hsp90 participate in the growth, development, differentiation and survival of the parasites. In this way, they become attractive targets for treatment of diseases such as malaria, leishmaniasis and cancer. Hsp90 do not act alone because they are involved in a complex network with other proteins, especially the co-chaperones. Aha protein (Activator of the Hsp90-ATPase Activity) is a co-chaperone that activate the ATPase activity of Hsp90, which is composed by two canonical domains localized at the N- and C-terminus, respectively, binding at the chaperone dimer interfaces. This interaction is also targeted for the treatment of diseases. The objectives of the first chapter of this work were to perform a comparative study to characterize the interaction of yeast, L. braziliensis, P. falciparum, sugarcane and human α and β isoforms with nucleotides adenosine ATP and ADP by isothermal titration calorimetry, nuclear magnetic resonance and molecular dynamics. The estimation of the ATPase activity of the Hsp90 proteins of P. falciparum and L. braziliensis, as well as the evaluation of the inhibition of Hsp90 by classical inhibitors of Hsp90 (geldanamycin and radicicol) were also performed. In parallel, for PfAha4 protein (Aha type 4 of P. falciparum), structural studies such as circular dichroism spectrophotometry, fluorescence spectroscopy, analytical ultracentrifugation and small angle X-ray scattering (SAXS) were performed (shown in chapter 2 of this thesis). For the interaction characterization, ITC experiments were performed in order to test the PfAha4 interaction with P. falciparum Hsp90 protein (PfHsp90). The experiments showed that the mode of interaction of Hsp90 with ATP and ADP are different, probably due to the microstates that the binders experience at the interaction site. Thus, despite the high sequence similarity of these proteins, selective inhibition can be achieved. For PfAha4, which is composed by the canonical C-terminal Aha domain, the results showed that it has similar properties with other Ahas. It is a globular monomer in solution and showed high affinity for PfHsp90 in a stoichiometry of 2:1.