In previous studies we have identified eIF5A as the only cellular protein that contains an unusual amino acid, hypusine N-epsilon-(4-amino-2-hydroxybutyl)lysine, and established that hypusine biosynthesis occurs by two sequential enzymatic reactions: i) deoxyhypusine synthesis and ii) deoxyhypusine hydroxylation. We have cloned and characterized the structural and catalytic properties of the two enzymes of the hypusine pathway, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). We and others have demonstrated that hypusine modification is essential for the activity of eIF5A and for mammalian cell proliferation. Previously, we reported biochemical evidence for acetylation of eIF5A at Lys47 and its negative regulation by this acetylation. We have also obtained evidence for selective acetylation of hypusine residue of eIF5A by a polyamine metabolic enzyme, spermidine/spermine acetyltransferase 1 (SSAT1). None of the eIF5A wild type or mutant proteins not containing the hypusine/deoxyhypusine residue worked as a substrate for SSAT1. The amino acid residue of eIF5A acetylated by SSAT1 was identified as acetyl-hypusine or acetyl-deoxyhypusine by ion exchange chromatographic separation. In mammalian cells, cotransfected with eIF5A and SSAT1 vectors, macromolecular interaction between eIF5A and SSAT1 was demonstrated by co-immuno-precipitation, suggesting molecular basis for the specificity of this acetylation. When the activities of bovine testis eIF5A acetylated by SSAT1 was compared with that of non-acetylated eIF5A, the acetylated form was inactive in methionyl-puromycin synthesis assay indicating the importance of the basic side chain of hypusine residue in eIF5A activity. SSAT1 may regulate eIF5A activity by acetylation of its hypusine residue. Hypusine modification is essential for the activity of eIF5A and for eukaryotic cell proliferation. However, the exact role of the basic hypusine residue in eIF5A structure or in its activity is not known. A large quantity of highly pure proteins are needed for structural studies of eIF5A(Hpu), eIF5A(Dhp) and their complexes with modification enzymes and in determination of binding sites of eIF5A on ribosome. However, it is difficult to purify hypusine-containing eIF5A in sufficient quantity from mammalian tissues. We have used a polycistronic vector pST39 to coexpress eIF5A with the modification enzymes, DHS and DOHH for modification of eIF5A(Lys) in E. coli cells. By this method, we have produced eIF5A(Hpu) as well as the intermediate eIF5A(Dhp) in high purity and quantity. The recombinant eIF5A(Hpu) rapidly produced by induction at 37 C for 4 h was inactive in methionyl-puromycin synthesis assay, probably due to improper protein folding. However, recombinant eIF5A(Hpu) induced at 18 C overnight displayed comparable activity as the native eIF5A purified from bovine testis, whereas non-hypusinated eIF5A precursor, eIF5A(Lys) was inactive. Deoxyhypusine-containing eIF5A exhibited a partial activity. The bacterially produced recombinant eIF5A(Dhp) and eIF5A(Hpu) will be useful tools in structure/function studies of eIF5A. To investigate the physiological function of eIF5A isoforms and the hypusine modification enzymes, we performed their gene targeting in mice using the ES cell lines, RRE174 (Eif5a-1 +/-) and RRM039 (Dhps +/-) which have one allele of each gene disrupted by the gene trap method. The gene-targeted heterozygous agouti mice (Eif5a-1 +/-, or Dhps +/-) appeared to be normal and did not show any growth defects or phenotypes. The heterozygous agouti male and female mice were crossed and the pups born from the heterozygous intercrosses were genotyped. No pups were born with the genotype of Eif5a-1-/- or Dhps-/- indicating that homozygous disruption of either gene is embryonic lethal. To clarify the time point of embryonic lethality, we cultured the embryos at developmental stages (E3.5, E6.5, E7.5 and E8.5) and genotyped them by PCR. The Eif5a-1-/- homozygous embryo was identified on the blastocyst stage (E3.5), but not at later stage, indicating that Eif5a-1-/- embryo is viable up to 3.5 days, but not after 6 days. The same was true for Dhps-/- embryos. These findings demonstrate that eIF5A-1 and DHS play an essential role at the early stage of embryonic development.

Project Start
Project End
Budget Start
Budget End
Support Year
18
Fiscal Year
2011
Total Cost
$984,330
Indirect Cost
Name
National Institute of Dental & Craniofacial Research
Department
Type
DUNS #
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Mandal, Ajeet; Mandal, Swati; Park, Myung Hee (2016) Global quantitative proteomics reveal up-regulation of endoplasmic reticulum stress response proteins upon depletion of eIF5A in HeLa cells. Sci Rep 6:25795
Jasniewski, Andrew J; Engstrom, Lisa M; Vu, Van V et al. (2016) X-ray absorption spectroscopic characterization of the diferric-peroxo intermediate of human deoxyhypusine hydroxylase in the presence of its substrate eIF5a. J Biol Inorg Chem 21:605-18
Saxena, Deepti; Spino, Michael; Tricta, Fernando et al. (2016) Drug-Based Lead Discovery: The Novel Ablative Antiretroviral Profile of Deferiprone in HIV-1-Infected Cells and in HIV-Infected Treatment-Naive Subjects of a Double-Blind, Placebo-Controlled, Randomized Exploratory Trial. PLoS One 11:e0154842
Xiao, Su; Chen, Yu Chi; Buehler, Eugen et al. (2016) Genome-scale RNA interference screen identifies antizyme 1 (OAZ1) as a target for improvement of recombinant protein production in mammalian cells. Biotechnol Bioeng 113:2403-15
Mandal, Swati; Mandal, Ajeet; Park, Myung Hee (2015) Depletion of the polyamines spermidine and spermine by overexpression of spermidine/spermine N¹-acetyltransferase 1 (SAT1) leads to mitochondria-mediated apoptosis in mammalian cells. Biochem J 468:435-47
Frey, Avery G; Nandal, Anjali; Park, Jong Hwan et al. (2014) Iron chaperones PCBP1 and PCBP2 mediate the metallation of the dinuclear iron enzyme deoxyhypusine hydroxylase. Proc Natl Acad Sci U S A 111:8031-6
Mittal, Nimisha; Morada, Marie; Tripathi, Pankaj et al. (2014) Cryptosporidium parvum has an active hypusine biosynthesis pathway. Mol Biochem Parasitol 195:14-22
Mandal, Ajeet; Mandal, Swati; Park, Myung Hee (2014) Genome-wide analyses and functional classification of proline repeat-rich proteins: potential role of eIF5A in eukaryotic evolution. PLoS One 9:e111800
Beninati, Simone; Park, Myung Hee; Wolff, Edith et al. (2013) In memoriam: John E. Folk (1925-2010). Amino Acids 44:11-8
Hanauske-Abel, Hartmut M; Saxena, Deepti; Palumbo, Paul E et al. (2013) Drug-induced reactivation of apoptosis abrogates HIV-1 infection. PLoS One 8:e74414

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