Redox properties of the global regulator fura from anabaena sp. Pcc 7120 and their functional implications
- Autores: Laura Botello Morte
- Directores de la Tesis: María Teresa Bes (dir. tes.), María F. Fillat (dir. tes.)
- Lectura: En la Universidad de Zaragoza ( España ) en 2014
- Idioma: español
- Tribunal Calificador de la Tesis: Francisco Javier Cejudo Fernández (presid.), Maria Pilar Bayona-Bafaluy (secret.), Susana Campoy Sánchez (voc.)
- Materias:
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- Resumen
FUR (ferric uptake regulator) proteins constitute a large family of transcriptional regulators that exhibit a wide diversity of functions. FurA from the filamentous, nitrogen¿fixing cyanobacterium Anabaena sp. PCC 7120 is a global regulator that links iron homeostasis with nitrogen metabolism and the oxidative stress response, among other processes. This DNA-binding protein contains five cysteines whose redox state is critical for the protein to be functionally active. Four cysteines are arranged into two CXXC motifs (C101VKC104 y C141PKC144), whereas the fifth cysteine (Cys133) is specially conserved among cyanobacterial FurA orthologs. Previous studies have demonstrated that a furA¿overexpressing strain of Anabaena sp. PCC 7120 showed decreased levels of reactive oxygen species (ROS) in spite of exhibiting a lower expression of major antioxidant proteins, namely catalase and superoxide dismutase. Moreover, overexpression of FurA in Escherichia coli provides enhanced oxidative stress resistance, through a still unknown mechanism. The work described in this thesis has been addressed to identify the role of cysteine residues in the biological function of FurA as well as their putative implication in the protective effect against oxidative stress. Data obtained in this work has allowed to discard that this effect is produced by FurA oligomerization¿mediated ROS scavenging, by over¿oxidation of FurA cysteines or by transcriptional modulation of the oxidative stress response by FurA in E. coli. Since FurA exhibits two redox CXXC motifs that are essential for the activity of many oxidoreductase enzymes, redox properties of the regulator have been analyzed in detail searching for a novel enzymatic activity responsible for the protective effect of FurA. For the first time for a Fur family member, insulin reduction assays have revealed that FurA exhibits disulfide reductase activity in vitro. Free¿cysteine alkylation assays using Anabaena crude extracts showed that FurA is entirely localized in the cytoplasm in different redox states. Finally, redox potential measurements using gel¿shift assays revealed similar values for the redox CXXC pairs to that of bacterial cytoplasm. For all these reasons, FurA has been proposed to work as a novel CXXC¿based redox sensor. On the other hand, pull down experiments and two hybrid assays were performed to search for potential interacting partners as targets of this novel disulfide reductase activity. In addition of homodimerization, FurA was confirmed to interact with the putative amidase All1140 and the histone¿like regulator HU. FurA was also capable of interacting with some soluble photosynthetic electron carriers, establishing an important connection of the regulator with the photosynthetic electron transport chain. Finally, crosslinking assays, non¿reducing electrophoretic experiments and determination of free¿thiol content of multiple cysteine mutants, together with previous results obtained using simple mutants, allowed to identify the FurA cysteines involved in dimerization of the protein in vitro. These studies have also revealed that the redox state of one of its cysteines is critical for the protein to bind both DNA and the corepressor metal. Furthermore, a thiol/disulfide redox switch is responsible for controlling the redox state of this key residue The mechanism described in this thesis supports a new role for FurA in connecting the intracellular redox state response to iron homeostasis in Anabaena.
