ON THE ASSEMBLY OF Aedes aegypti R2TP COMPLEX

R2TP plays an important role in the assembly of several macromolecular complexes involved in essential pathways in the cell. It comprises RuvBL1 and RuvBL2 - AAA+ proteins that have several roles within the cell and are essential for cell growth and proliferation - along with RPAP3 and Pih1D1, which mediate interaction with client proteins and chaperones. The present study aimed to unveil the assembly of Aedes aegypti R2TP. Both RuvBLs, RPAP3 and Pih1D1 were individually characterized on their secondary structure content, thermo-induced unfolding profile, and hydrodynamic parameters, using circular dichroism spectropolarimetry and SEC-MALS-QELS (SEC coupled to Multi-angle and Quasi-Elastic Light Scattering). To assess the assembly of the R2TP complex, we conducted interaction experiments such as pull-down and analytical SEC followed by SDS-PAGE. All R2TP components were produced pure (>95%) and folded, with an alpha-helix characteristic CD spectrum (except for Pih1D1, whose CD spectrum indicated a mixture of alpha-helix and beta-sheet). RPAP3 and Pih1D1 were found to be elongated monomers in solution, whereas the RuvBLs1/2 had a spherical shape pattern, corroborating the classical donut shape from the literature. Their molecular mass, hydrodynamic radius, and diffusion coefficient were also obtained and will be shown. Afterward, we managed to assess the interaction between RuvBLs1/2, RPAP3, and Pih1D1, demonstrating the assembly of a novel R2TP complex. By this means, we can contribute to the knowledge regarding protein assembly in this epidemiologically important mosquito, which can further be useful for the development of new strategies in disease control.