Hypusine formation on elF-5A precursor is by tar the most specific polyamine-dependent biological event in living cells. Deoxyhypusine synthase catalyzes the oxidative transfer of the aminobutyl moiety from spermidine to a unique lysine residue on elF-SA precursor to form deoxyhypusine residue which becomes hypusine after further hydroxylation. Deletion of either deoxyhypusine synthase or elF-5A gene in yeast gives a lethal phenotype. Although in vivo studies suggest that hypusine formation is tightly coupled to cell proliferation, the precise function of elF-5A and the physiological significance of hypusine formation are not clear. It has been suggested that elF-5A is the cellular target of HIV-1 viral protein Rev. We have obtained preliminary data indicating that modified eIF-5A, but not the unmodified precursor, caused gel mobility supershift of the Rev-RRE RNA complex, suggesting that deoxyhypusine/hypusine modification is required for the direct interaction between elF-5A and Rev. Rev is an RNA binding protein involved in the export of selected mRNAs. If the elF-5A interacting protein is Rev-like, it would imply that elF-5A may have a role in pre-mRNA processing and selective gene expression. We will use Rev and other Rev-like proteins (Rex, NS1 etc) as a model to gain insight of the specificity and structural requirement of these interactions, including the role of RNA species. We will use the yeast two-hybrid system, a powerful technique for studying protein- protein interaction, in conjunction with other more conventional methods, including co-immunoprecipitation, protein affinity chromatography photo-cross-inking and expression library probing, to search for the elF-5A interacting proteins. We have generated all necessary molecular tools (antibodies, cDNA clones, unmodified and modified recombinant elF-5As) for this purpose. The putative elF-5A interacting proteins identified by any of the above screening methods will be further characterized to confirm the interaction both in vitro and in vivo. Functional domains on elF- 5A, particularly the protein binding sites for Rev and for elF-5A interacting proteins will be defined by mutational analysis and site-specific cross-linking. Finally, the regulation and physiological function of elF-5A and its interacting proteins will be investigated in mouse neuroblastoma cells using pharmacological, histochemical and biochemical means. Preliminary studies have shown that specific inhibition of deoxyhypusine synthase could promote neuroblastoma differentiation. Possible effect of hypusine formation on the expression of cell cycle-dependent growth associated genes will be examined in normal human diploid fibroblasts. It is likely that elF-5A, mediated through its interacting proteins, may be involved in the regulation of a small class of genes essential for proliferation and for differentiation.
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