We have identified eIF5A as the only cellular protein that contains an unusual amino acid, hypusine [N-epsilon-(4-amino-2-hydroxybutyl)lysine], and have established that hypusine biosynthesis occurs by two sequential enzymatic reactions: i) deoxyhypusine synthesis and ii) deoxyhypusine hydroxylation. We have shown that hypusine is essential for the activity of eIF-5A and for eukaryotic cell proliferation. Independent roles of eIF5A and polyamines in cell proliferation The roles of active, hypusine-containming eIF5A and polyamines in cell proliferation were examined in mouse mammary carcinoma FM3A cells treated with an inhibitor of deoxyhypusine synthase, N1-guanyl-1,7-diaminoheptane (GC7), or with an inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine (DFMO), or with DFMO plus an inhibitor of spermine synthase, N1-(3-aminopropyl)cyclohexylamine (APCHA). Growth inhibition was observed by treatment with GC7 or with DFMO, due to reduction of eIF5A or polyamines. The inhibition of growth observed in cells treated with both DFMO and APCHA, before a notable reduction in eIF5A, is probably due to a decrease in the total polyamines. The results indicate that a decrease in either active eIF5A or total polyamine level leads to inhibition of cell growth, and that eIF5A and polyamines are independently involved in the regulation of cell growth. eIF5A as a marker for aberrant cell proliferation Since eIF5A is intimately involved in cell proliferation, we explored eIF5A as a biomarker in human neoplastic tissues using a polyclonal antibody (NIH353) which had been raised against hypusine-containing eIF5A purified from human red blood cells. Whereas commercial antibodies prepared using unmodified eIF5A precursor, react equally with the three forms of eIF5A (the eIF5A precursor, the intermediate and the mature protein), NIH353 is specific for mature eIF5A. The NIH-353 stained cells in the basal and parabasal regenerative layers of normal cervical epithelium in a pattern similar to that obtained for the nuclear protein Ki67, a widely employed marker for proliferating cells. NIH353 also stained vulva intraepithelial neoplasia (VIN3) in a comparable pattern as with Ki67 antibody, suggesting its potential utility in detection of malignancies in this and other human tissues. Structure/Function Studies of eIF5A-1 eIF5A is highly conserved throughout the eukaryotic kingdom. In order to understand the structural requirements of eIF5A for its cellular function, we generated variant forms of human eIF5A-1, with truncations of 6, 11 and 21 amino acids from either the NH2- or COOH-terminus, or with single amino substitutions of conserved amino acid residues, and tested the truncated or mutated human eIF5A-1 genes for their ability to support the growth of yeast. The results indicate that human eIF5A-1 with 6 amino acids deleted from the NH2- or COOH terminus are functional, but those with truncations of eleven or more residues from either end showed substantial or total loss of activity. The amino acid residues thus far identified to be critical for eIF5A functions are Lys47, Gly49, Lys50, Gly52, His51, Lys55A and E143. Differential expression of eIF5A-1 and eIF5A-2 in human cancer cells Of the two human eIF5A genes reported, normally only one isoform (eIF5A-1) is expressed in most human cell lines. Either human eIF5A gene can complement growth of a yeast strain in which the yeast eIF5A genes were disrupted, indicating functional similarity of the two isoforms. eIF5A-1 and eIF5A-2 genes are differentially expressed in mammalian cells. Whereas eIF5A-1 mRNA and protein are constitutively expressed in all human cells, eIF5A-2 mRNAs (0.6-5.5 kb) are expressed in a tissue-specific manner. eIF5A-2 protein was detectable only in the colorectal and ovarian cancer-derived cell lines SW-480 and UACC-1598, which showed high over-expression of eIF5A-2 mRNAs. The multiple forms of eIFFF5A-2 mRNA, with varying lengths of the 3'-UTR were identified as the products of one gene resulting from the use of different polyadenylation signals (AATAAA) in the 3'-UTR. These multiple eIF5A-2 mRNAs appeared to show similar stability. The eIF5A-1 and -2 precursors were modified comparably in UACC-1598 cells and both isoform proteins were similarly stable. These findings suggest that the failure to detect eIF5A-2 protein, even in eIF5A-2 mRNA positive human cells, is due to inefficient translation. The differences in gene-expression patterns, physical characteristics and distinct amino acid sequences in the C-terminal domain imply differentiated, tissue-specific functions of the eIF5A-2 isoform in the mammalian organism and in cancer. Mechanism of Deoxyhypusine synthase reaction/Reversal of deoxyhypusine synthesis Deoxyhypusine synthase normally catalyzes the synthesis of deoxyhypusine residue in the eIF5A precursor protein to form the eIF5A intermediate, eIF5A(Dhp). We recently discovered that this reaction can be efficiently reversed by the same enzyme. When eIF5A([3H]Dhp), radiolabeled in the 4-aminobutyl portion of its deoxyhypusine residue, was incubated with human deoxyhypusine synthase, NAD and 1,3-diaminopropane, [3H]spermidine was formed by a rapid transfer of the radiolabeled 4-aminobutyl side chain of the [3H]deoxyhypusine residue to 1,3-diaminopropane. No reversal was observed with [3H]hypusine protein, suggesting that hydroxylation at the 4-aminobutyl side chain of the deoxyhypusine residue prevents deoxyhypusine synthase-mediated reversal of the modification and that this hydroxylase step is responsible for the irreversibility of hypusine modification. Purified human deoxyhypusine synthase also exhibited homospermidine synthesis activity when incubated with spermidine, NAD and putrescine. A new crystal structure of an active human deoxyhypusine synthase (DHS)and its complex with NAD and the inhibitor GC7 A previous study, performed with DHS crystals formed at high ionic strength and acidic pH, revealed a structure of an inactive enzyme in which each entrance of the four active sites is blocked by a ball-and-chain motif composed of an amino terminal two-turn alpha-helix. A new structure of the DHS:NAD holoenzyme, representing an active enzyme form, was determined for crystals grown at low ionic strength and pH 8.0, near the optimal pH for enzymatic activity. The ball-and-chain motif swings freely and no longer obstructs the active site entrance under these conditions. The open active site of the new crystal form allows for the DHS inhibitor N1-guanyl-1,7-diaminoheptane (GC7) to be bound within the same active site predicted to bind spermidine. This first structure of a DHS:NAD:inhibitor ternary complex provides a structural context in support of the proposed DHS reaction mechanism, and provides a basis for the development of improved inhibitors and anti-proliferative agents.
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