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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA049695-09
Application #
6172186
Study Section
Medical Biochemistry Study Section (MEDB)
Program Officer
Spalholz, Barbara A
Project Start
1990-04-01
Project End
2004-04-30
Budget Start
2000-05-01
Budget End
2004-04-30
Support Year
9
Fiscal Year
2000
Total Cost
$261,288
Indirect Cost
Name
Rutgers University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
038633251
City
New Brunswick
State
NJ
Country
United States
Zip Code
08901
Gosslau, Alexander; Jao, David Li-En; Butler, Renee et al. (2009) Thermal killing of human colon cancer cells is associated with the loss of eukaryotic initiation factor 5A. J Cell Physiol 219:485-93
Chatterjee, Ishita; Gross, Stephane R; Kinzy, Terri Goss et al. (2006) Rapid depletion of mutant eukaryotic initiation factor 5A at restrictive temperature reveals connections to actin cytoskeleton and cell cycle progression. Mol Genet Genomics 275:264-76
Jao, David Li-En; Chen, Kuang Yu (2006) Tandem affinity purification revealed the hypusine-dependent binding of eukaryotic initiation factor 5A to the translating 80S ribosomal complex. J Cell Biochem 97:583-98
Xu, Aiguo; Jao, David Li-En; Chen, Kuang Yu (2004) Identification of mRNA that binds to eukaryotic initiation factor 5A by affinity co-purification and differential display. Biochem J 384:585-90
Li-En Jao, David; Yu Chen, Kuang (2002) Subcellular localization of the hypusine-containing eukaryotic initiation factor 5A by immunofluorescent staining and green fluorescent protein tagging. J Cell Biochem 86:590-600
Xu, A; Chen, K Y (2001) Hypusine is required for a sequence-specific interaction of eukaryotic initiation factor 5A with postsystematic evolution of ligands by exponential enrichment RNA. J Biol Chem 276:2555-61
Lu, J; Chen, Z P; Yan, Y P et al. (2000) Aminohexanoic hydroxamate is a potent inducer of the differentiation of mouse neuroblastoma cells. Cancer Lett 160:59-66
Matuoka, K; Chen, K Y (2000) Possible role of subunit A of nuclear factor Y (NF-YA) in normal human diploid fibroblasts during senescence. Biogerontology 1:261-71
Matuoka, K; Yu Chen, K (1999) Nuclear factor Y (NF-Y) and cellular senescence. Exp Cell Res 253:365-71
Chen, K Y (1997) Transcription factors and the down-regulation of G1/S boundary genes in human diploid fibroblasts during senescence. Front Biosci 2:d417-26

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