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 hypusinated eIF5A, but not nonhypusinated eIF5A, by a polyamine metabolic enzyme, spermidine/spermine acetyltransferase 1 (SSAT1). The site of eIF5A acetylation by SSAT1 was identified as terminal amino group of the hypusine side chain by ion exchange chromatographic separation. The bovine testis eIF5A acetylated by SSAT1 was inactive in methionyl-puromycin synthesis assay indicating the importance of the basic side chain of hypusine residue in eIF5A activity. In the course of studying the molecular interaction between eIF5A and SSAT1, we observed suppression of expression of GFP-eIF5A , GFP or any other cotransfected gene by overexpression of SSAT1. SSAT1 activity was required for this inhibition, as overexpression of inactive SSAT1 mutant enzyme did not exert the same effect. The inhibition was not at transcription, since GFP mRNA or GFP-eIF5A mRNA level did not decrease upon cotransfection with SSAT1. This was confirmed by single cell imaging of mRNAs by Stellaris RNA FISH method. Furthermore, single cell fluorescent imaging of synthesis of DNA and RNA revealed that depletion of polyamines by SSAT1 overexpression does not cause inhibition of synthesis of DNA or RNA. Addition of polyamine analogs, such as Bis-ethyl-norspermine (BENSpm) which are not substrates for SSAT1, restored expression of GFP or GFP-eIF5A in the SSAT1 overexpressing cells, indicating that a lack of of polyamines is the underlying cause for inhibition of protein synthesis. In order to characterize biochemical changes in SSAT1 overexpressing cells, we used SSAT1 adenovirus to overexpress SSAT1 in all 293T or HeLa cells. Upon transduction with AdenoSSAT1, a rapid depletion of spermidine and spermine followed by an increase in putrescine and N1-acetylspermidine was observed. Within 24h of AdenoSSAT1 transduction, a total arrest in protein synthesis and cell growth occured, whereas there was no inhibition in cells transduced with AdenoGFP vector. Furthermore, comparison of polysome profiles showed a loss of polysomes in the AdenoSSAT1 transduced cells, but not in the AdenoGFP transduced cells, suggesting a mechanism of a block in translation initiation. Taken together, these findings suggest that the most important function of polyamines in eukaryotic cell growth is to promote translation.
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