Screen Of EIF5A Interacted Proteins By Yeast Two-hybrid Assasy And Functional Analysis For The Protein Complex

The putative eukaryotic translation initiation factor 5A (eIF5A) is an intriguing protein, since it is the only cellular protein known to contain the unique amino acid hypusine, a modification that appears to be required for cell proliferation. The protein was originally described as a translation initiation factor due to its ability to stimulate the synthesis of methionyl-puromycin in vitro and to transiently attach to ribosomes in the course of initiation of eukaryotic cellular protein synthesis..Actually, depletion of this factor in eukaryotic cell caused only a small reduction in the protein synthesis rate, suggesting that eIF5A did not function for general protein synthesis. Subsequent studies have demonstrated that eIF5A is more than an initiator of protein translation. The finding that eIF5A is a cellular cofactor of HIV-1 Rev and HTLV-1 Rex transactivator proteins in mRNA export suggests that it can have additional activities.Recent data have shown it to be also involved in apoptosis. However, the actual function of eIF5A in apoptosis is still unknown. We recently reported that eEF5A was involved in the apoptosis of tumor cells induced by inhibition of ubiquitin-proteasomes in the studies of proteomics. Especially, the post-translational modification of eIF5A is essential to viability of leukemic cells because blocking the lysine/hypusine transformation inhibits the growth of mammalian cells, and induces apoptosis in tumor cells. In this study, we performed yeast two-hybrid screens to identify eIF5A-interacting proteins and preliminary analysis on the biological function of the interaction, to help us understand its mechanisms.Our results demonstrated, for the first time, that eIF5A and syntenin could engage in a specific interaction both in vitro and in vivo, and have important biological function. In the yeast two-hybrid screens, we used the EFP and eif5a domain of eIF5A as baits to screen an oligo(dT)-primed human mammary cDNA library. Full-length cDNA for syntenin was then obtained from a mammary library. To further confirm the specific interactions between eIF5A and syntenin both in vitro and in vivo, GST pull-down and co-immunoprecipitation experiments was performed, and the results was consistent withthe results of yeast two-hybrid screens. Furthermore, the fluorescence resonance energy transfer (FRET) microscopy was employed to visualize it in vivo and confirmed the physical association of eIF5A with syntenin in living cells. We also constructed different deletions of eIF5A and syntenin, and defined the region, which determines the binding behavior of eIF5A or syntenin with respect to eIF5A.To further investigate the biological significance of the interactions of eIF5A and syntenin, we successfully constructed a Iuciferase reporter system in T47D breast tumor cell lines for the analysis of Sox4 activity. Sox4 plays important roles in the development of breast cancer. In this study, we found eIF5A could inhibit Sox4 transcriptional activity via the interaction with syntenin. Further, our findings revealed a new biological activity for eIF5A as the regulator of p53. Overexpression of eIF5A led to p53 upregulation and activation. However, when eIF5A interacted with its novel partner, syntenin, the eIF5A-induced increase in p53 protein level was significantly inhibited. Therefore, eIF5A seems to be a previously un-recognized regulator of p53 that may define a new pathway for p53-dependent apoptosis, and syntenin might regulate p53 by balancing the regulation of eIF5A signaling to p53 for apoptosis.