Characterization of Aedes aegypti Hsp90 and its interactors SGT and R2TP complex

Author(s)

Natália Galdi Quel

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2019-08-29

Examining board members

Carlos Henrique Inacio Ramos; Juliana Ferreira de Oliveira; Juliana Helena Costa Smetana; Shaker Chuck Farah; Claudio Chrysostomo Werneck

• Advisor: Carlos Henrique Inacio Ramos

Abstract

Proteostasis is maintained by the protein quality control system, constituted by molecular chaperones and the proteasome. Among these chaperones, Hsp90 is essential for cell growth, having a role in protein folding and regulation of signaling proteins. Hsp90’s activity is modulated by its co-chaperones, which influences its ATPase cycle and gives specificity. A group of Hsp90’s co-chaperone involves TPR-containing proteins, that interact with the MEEVD motif localized in the C-terminus of Hsp90. In this work, Hsp90’s interactors were structurally and functionally characterized: SGT (Small glutamine-rich TPR-containing protein), that participates of the translocation of tail-anchored proteins, and R2TP complex (Rvb1-Rvb2-Tah1/RPAP3-Pih1/Pih1D1), which is involved in the assembly and stabilization of complexes associated with several essential processes and interacts with Hsp90 via Tah1/RPAP3 (RNA polymerase II associated protein 3) – both from Aedes aegypti. Proteins were expressed in E. coli and purified with high purity and their secondary structure, thermal stability and hydrodynamic parameters were determined. Hsp90, SGT and Rvb1/Rvb2 were also studied by small angle X-ray scattering. Finally, functional and interaction experiments were conducted. Aedes aegypti Hsp90 showed low ATPase activity, which is in good agreement with that of its orthologues. Furthermore, it maintained an open conformational state during the entire ATPase cycle, similar to human Hsp90 but different from E. coli and yeast Hsp90. Moreover, it was observed an increase in Hsp90 expression upon heat-treatment, which is in good agreement with a protein that is required for this type of stress. Aedes aegypti SGT was a dimer with a non-spherical shape and similar to the human SGT, but different from Leishmania braziliensis and yeast SGTs. Surprisingly, even deleted from its C-terminus, SGT maintained its globular structure, which is different from the suggested for human SGT. Finally, Rvbs were shown as a heterododecamer, RPAP3 was mainly a monomer and Pih1D1 was in a concentration-dependent equilibrium among monomer and dimer. Unexpectedly, Pih1D1 was found to be stable, different from its orthologues. Additionally, we showed evidence of the in vitro assemble of the Aedes aegypti R2TP complex. The characterization of these proteins (Hsp90, SGT and R2TP) is important because it may contribute to the comprehension of the protein quality control system in Aedes aegypti, which is a vector of many diseases (AU)

• FAPESP's process: 14/25967-4 - Characterization of the role of ATPase domains and human co-chaperones Hsp40 and hop in cellular mechanisms of thermal tolerance and suppression of protein aggregation
• Grantee: Natália Galdi Quel
• Support Opportunities: Scholarships in Brazil - Doctorate