Eukaryotic Initiation factor 6 (eIF6) is a peculiar translation initiation factor that binds to the large 60S ribosomal subunits, controlling translation initiation and participating in ribosome biogenesis. In the past, knowledge about the mechanisms adopted by the cells for controlling protein synthesis by extracellular stimuli has focused on two translation initiation factors (eIF4E and eIF2), however, recent data suggest eIF6 as a newcomer in the control of downstream of signal transduction pathways. eIF6 is over-expressed in tumors and its decreased expression renders cells less prone to tumor growth. A previous work from our laboratory has disclosed that over-expression of eIF6 in transformed cell lines markedly increased cell migration and invasion. Here, we performed a quantitative proteomic analysis of membrane-associated proteins in A2780 ovarian cancer cells over-expressing eIF6. Differentially expressed proteins upon eIF6 overproduction were further investigated in silico by Ingenuity Pathway Analysis (IPA). RT-qPCR and Western blot were performed in order to validate the proteomic data. Furthermore, the effects of a potent and selective inhibitor ML-141 in A2780 cells were evaluated using transwell migration assay. Finally, we explored the effects of eIF6 over-expression on WM793 primary melanoma cell lines. We demonstrated that: (i) the genes up-regulated upon eIF6 overproduction mapped to a functional network corresponding to cellular movements in a highly significant way; (ii) cdc42 plays a pivotal role as an effector of enhanced migratory phenotype induced upon eIF6 over-expression; (iii) the variations in abundance observed for cdc42 protein occur at a post-transcriptional level; (iv) the increased cell migration/invasion upon eIF6 over-expression was generalizable to other cell line models. Collectively, our data confirm and further extend the role of eIF6 in enhancing cell migration/invasion. We show that a number of membrane-associated proteins indeed vary in abundance upon eIF6 over-expression, and that the up-regulated proteins can be located within a functional network controlling cell motility and tumor metastasis. Full understanding of the role eIF6 plays in the metastatic process is important, also in view of the fact that this factor is a potentially druggable target to be exploited for new anti-cancer therapies.

The eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for the viability of the cells whose proposed function is to prevent the stalling of the ribosomes during translation elongation. eIF5A activity requires a unique and functionally essential post-translational modification, the change of a lysine to hypusine. eIF5A is recognized as a promoter of cell proliferation, but it has also been suggested to induce apoptosis. To date, the precise molecular mechanism through which eIF5A affects these processes remains elusive. In the present study, we explored whether eIF5A is involved in controlling the stress-induced expression of the key cellular regulator p53. Our results show that treatment of HCT-116 colon cancer cells with the deoxyhypusine (DHS) inhibitor N1-guanyl-1,7-diamineheptane (GC7) caused both inhibition of eIF5A hypusination and a significant reduction of p53 expression in UV-treated cells, and that eIF5A controls p53 expression at the level of protein synthesis. Furthermore, we show that treatment with GC7 followed by UV-induced stress counteracts the pro-apoptotic process triggered by p53 up-regulation. More in general, the importance of eIF5A in the cellular stress response is illustrated by the finding that exposure to UV light promotes the binding of eIF5A to the ribosomes, whereas UV treatment complemented by the presence of GC7 inhibits such binding, allowing a decrease of de novo synthesis of p53 protein.

Translation factor a/eIF5A is highly conserved in Eukarya and Archaea. The eukaryal eIF5A protein is required for transit of ribosomes across consecutive proline codons, whereas the function of the archaeal orthologue remains unknown. Here, we provide a first hint for an involvement of Sulfolobus solfataricus (Sso) aIF5A in translation. CRISPR-mediated knock down of the aif5A gene resulted in strong growth retardation, underlining a pivotal function. Moreover, in vitro studies revealed that Sso aIF5A is endowed with endoribonucleolytic activity. Thus, aIF5A appears to be a moonlighting protein that might be involved in protein synthesis as well as in RNA metabolism.

Fish are an interesting taxon comprising species adapted to a wide range of environments. In this work, we analyzed the transcriptional contribution of transposable elements (TEs) in the gill transcriptomes of three fish species exposed to different salinity conditions. We considered the giant marbled eel Anguilla marmorata and the chum salmon Oncorhynchus keta, both diadromous, and the marine medaka Oryzias melastigma, an euryhaline organism sensu stricto. Our analyses revealed an interesting activity of TEs in the case of juvenile eels, commonly adapted to salty water, when exposed to brackish and freshwater conditions. Moreover, the expression assessment of genes involved in TE silencing mechanisms (six in heterochromatin formation, fourteen known to be part of the nucleosome remodeling deacetylase (NuRD) complex, and four of the Argonaute subfamily) unveiled that they are active. Finally, our results evidenced for the first time a krüppel-associated box (KRAB)-like domain specific to actinopterygians that, together with TRIM33, might allow the functioning of NuRD complex also in fish species. The possible interaction between these two proteins was supported by structural prediction analyses.

Translation factor 5A (eIF5A) is one of the most conserved proteins involved in protein synthesis. It plays a key role during the elongation of polypeptide chains, and its activity is critically dependent on hypusination, a post-translational modification of a specific lysine residue through two consecutive enzymatic steps carried out by deoxyhypusine synthase (DHS), with spermidine as substrate, and deoxyhypusine hydroxylase (DOHH). It is well-established that eIF5A is overexpressed in several cancer types, and it is involved in various diseases such as HIV-1 infection, malaria, and diabetes; therefore, the development of inhibitors targeting both steps of the hypusination process is considered a promising and challenging therapeutic strategy. One of the most efficient inhibitors of the hypusination process is the spermidine analog N1-guanyl-1,7-diaminoheptane (GC7). GC7 interacts in a specific binding pocket of the DHS completely blocking its activity; however, its therapeutic use is limited by poor selectivity and restricted bioavailability. Here we have performed a comparative study between human DHS (hDHS) and archaeal DHS from crenarchaeonSulfolobus solfataricus(aDHS) to understand the structural and dynamical features of the GC7 inhibition. The advanced metadynamics (MetaD) classical molecular dynamics simulations show that the GC7 interaction is less stable in the thermophilic enzyme compared to hDHS that could underlie a lower inhibitory capacity of the hypusination process inSulfolobus solfataricus. To confirm this hypothesis, we have tested GC7 activity onS. solfataricusby measuring cellular growth, and results have shown the lack of inhibition of aIF5A hypusination in contrast to the established effect on eukaryotic cellular growth. These results provide, for the first time, detailed molecular insights into the binding mechanism of GC7 to aDHS generating the basis for the design of new and more specific DHS inhibitors.