A proteomic analysis of the apoplastic fluid (APF) of coffee leaves was conducted to investigate the cellular processes associated with incompatible (resistant) and compatible (susceptible) Coffea arabica-Hemileia vastatrix interactions, during the 24–96 hai period. The APF proteins were extracted by leaf vacuum infiltration and protein profiles were obtained by 2-DE. The comparative analysis of the gels revealed 210 polypeptide spots whose volume changed in abundance between samples (control, resistant and susceptible) during the 24–96 hai period. The proteins identified were involved mainly in protein degradation, cell wall metabolism and stress/defense responses, most of them being hydrolases (around 70%), particularly sugar hydrolases and peptidases/proteases. The changes in the APF proteome along the infection process revealed two distinct phases of defense responses, an initial/basal one (24–48 hai) and a late/specific one (72–96 hai). Compared to susceptibility, resistance was associated with a higher number of proteins, which was more evident in the late/specific phase. Proteins involved in the resistance response were mainly, glycohydrolases of the cell wall, serine proteases and pathogen related-like proteins (PR-proteins), suggesting that some of these proteins could be putative candidates for resistant markers of coffee to H. vastatrix. Antibodies were produced against chitinase, pectin methylesterase, serine carboxypeptidase, reticuline oxidase and subtilase and by an immunodetection assay it was observed an increase of these proteins in the resistant sample. With this methodology we have identified proteins that are candidate markers of resistance and that will be useful in coffee breeding programs to assist in the selection of cultivars with resistance to H. vastatrix.

To become metastatic, a tumor cell must acquire new adhesion properties that allow migration into the surrounding connective tissue, transmigration across endothelial cells to reach the blood stream and, at the site of metastasis, adhesion to endothelial cells and transmigration to colonize a new tissue. Hydrogen peroxide (H2O2) is a redox signaling molecule produced in tumor cell microenvironment with high relevance for tumor development. However, the molecular mechanisms regulated by H2O2 in tumor cells are still poorly known. The identification of H2O2-target proteins in tumor cells and the understanding of their role in tumor cell adhesion are essential for the development of novel redox-based therapies for cancer. In this paper, we identified Ribosomal Protein SA (RPSA) as a target of H2O2 and showed that RPSA in the oxidized state accumulates in clusters that contain specific adhesion molecules. Furthermore, we showed that RPSA oxidation improves cell adhesion efficiency to laminin in vitro and promotes cell extravasation in vivo. Our results unravel a new mechanism for H2O2-dependent modulation of cell adhesion properties and identify RPSA as the H2O2 sensor in this process. This work indicates that high levels of RPSA expression might confer a selective advantage to tumor cells in an oxidative environment.

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Ribosomal Protein SA (RPSA), a component of the 40S ribosomal subunit, was identified as a H2O2 target in HeLa cells [1]. In order to analyze the intracellular localization of RPSA in different redox states we overexpressed wild-type RPSA (RPSAwt) or RPSA containing two cysteine to serine residue substitutions at positions 148 and 163 (RPSAmut) in HeLa cells. The transfected cells were exposed to H2O2 or N-acetylcysteine (NAC) and RPSA subcellular localization was assessed by immunofluorescence in permeabilized cells. In addition, co-immunofluorescence for RPSA and Ribosomal Protein S6 (RPS6) was performed in cells overexpressing RPSAwt or RPSAmut. Finally, the ribosomal expression of endogenous RPSA in the presence or absence of H2O2 was analyzed by Western blot.The data presented in this work is related to the research article entitled “Hydrogen peroxide regulates cell adhesion through the redox sensor RPSA” [1].