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| Author(s): |
Yuri de Abreu Mendonça
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia |
| Defense date: | 2011-08-29 |
| Examining board members: |
Carlos Henrique Inacio Ramos;
Elizabeth Pacheco Batista Fontes;
Richard John Ward;
Ana Paula Ulian de Araujo;
Renato Vicentini
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| Advisor: | Celso Eduardo Benedetti; Carlos Henrique Inacio Ramos |
| Abstract | |
Gram-negative bacterial pathogens, which have developed sophisticated strategies to infect hosts, use specialized secretion systems to secrete and translocate virulence proteins across the eukaryotic cell membrane into the cytoplasm. The translocation process depends on unfolded or partially folded virulence proteins to be transportated through the secretion system into the target inner cell where they are folded by the host chaperone machinery. Auxiliary proteins are responsible to help folding in cells and are known as molecular chaperones, or heat shock proteins (HSPs). Plants, being sessile organisms, are much more vulnerable to biotic and abiotic stress factors, making the role of HSPs even more important for protein homeostasis and cell viability. The study of the Hsp90 family is widespread due to the key role played in situations of infection and under various types of stress. In this work, an Hsp90 sweet orange (Citrus sinesis) was cloned and purified in order to study the general mechanism of infection of the bacterial pathogen of citrus species, Xanthomonas citri (Xac). First, we investigated the interaction, by combining immunostaining and pull-down assays, between Hsp90 and all four variants of PthA, the major virulence factor of Xac, and the orange Hsp90 cochaperonas cyclophilin (Cyp) and a thioredoxin-like protein (TDX). These proteins have been described to be upregulated in sweet orange during infection with Xac, pointing to a possible role of Hsp90 in the formation of a folding complex able to activate the virulence proteins of Xac inside the infected cells. Furthermore, we took to investigate the structure and function of the Hsp90 from sweet orange, which was folded and soluble as measured by circular dichroism (CD), intrinsic fluorescence spectroscopy and dynamic light scattering (DLS). Hsp90 was a dimer in solution with a Stokes radius of about 62 Å, tolerating up to 90 °C without denaturation, as measured by CD. The protein was functional, as measured by its ability to protect the aggregation of citrate synthase in an light scattering assay. The study of the effect of nucleotides on the conformation and function of Hsp90 by CD, intrinsic fluorescence and small-angle X-ray scattering (SAXS) show evidence of conformation modulation by ATP and ADP (AU) | |