Proteomic and Interactome Approaches Reveal PAK4, PHB-2, and 14-3-3η as Targets of Overactivated Cdc42 in Cellular Responses to Genomic Instability.

Cdc42, a member of the Rho GTPase family, is an intracellular signaling protein known for its roles in cytoskeleton rearrangements and, more recently, in apoptosis/senescence triggered by genotoxic stress. In some tumor cells, the overactivation of Cdc42 through the expression of constitutively active mutants (G12V or Q61L), GEF activation, or GAP downregulation functions as an antiproliferative or pro-aging mechanism. In this study, human cell lines with different P53 protein profiles were exposed to UV radiation, and the interactions between Cdc42 and proteins that are putatively involved in the DNA damage response and repair mechanisms were screened. The affinity-purified proteins obtained through pull-down experiments of the cell lysates using the recombinant protein baits GST, GST-Cdc42-WT, or GST-Cdc42-G12V were identified by mass spectrometry. The resulting data were filtered and used for the construction of protein-protein interaction networks. Among several promising proteins, three targets, namely, PAK4, PHB-2, and 14-3-3η, which are involved in the cell cycle, apoptosis, DNA repair, and chromatin remodeling processes, were identified. Biochemical validation experiments showed physical and proximal interactions between Cdc42 and the three targets in the cells, particularly after exposure to UV. The results suggest that the molecular mechanisms coordinated by overactivated Cdc42 (with the G12V mutation) to increase the cellular sensitivity to UV radiation and the susceptibility to cell death are collectively mediated by these three proteins. Therefore, the Cdc42 GTPase can potentially be considered another player involved in maintenance of the genomic stability of human cells during exposure to genotoxic stress.

Participation of 14-3-3ε and 14-3-3ζ proteins in the phagocytosis, component of cellular immune response, in Aedes mosquito cell lines.

BACKGROUND: Better knowledge of the innate immune system of insects will improve our understanding of mosquitoes as potential vectors of diverse pathogens. The ubiquitously expressed 14-3-3 protein family is evolutionarily conserved from yeast to mammals, and at least two isoforms of 14-3-3, the ε and ζ, have been identified in insects. These proteins have been shown to participate in both humoral and cellular immune responses in Drosophila. As mosquitoes of the genus Aedes are the primary vectors for arboviruses, causing several diseases such as dengue fever, yellow fever, Zika and chikungunya fevers, cell lines derived from these mosquitoes, Aag-2 from Aedes aegypti and C6/36 HT from Aedes albopictus, are currently used to study the insect immune system. Here, we investigated the role of 14-3-3 proteins (ε and ζ isoform) in phagocytosis, the main cellular immune responses executed by the insects, using Aedes spp. cell lines. RESULTS: We evaluated the mRNA and protein expression of 14-3-3ε and 14-3-3ζ in C6/36 HT and Aag-2 cells, and demonstrated that both proteins were localised in the cytoplasm. Further, in C6/36 HT cells treated with a 14-3-3 specific inhibitor we observed a notable modification of cell morphology with filopodia-like structure caused through cytoskeleton reorganisation (co-localization of 14-3-3 proteins with F-actin), more importantly the decrease in Salmonella typhimurium, Staphylococcus aureus and E. coli phagocytosis and reduction in phagolysosome formation. Additionally, silencing of 14-3-3ε and 14-3-3ζ expression by mean of specific DsiRNA confirmed the decreased phagocytosis and phagolysosome formation of pHrodo labelled E. coli and S. aureus bacteria by Aag-2 cells. CONCLUSION: The 14-3-3ε and 14-3-3ζ proteins modulate cytoskeletal remodelling, and are essential for phagocytosis of Gram-positive and Gram-negative bacteria in Aedes spp. cell lines.