The Post-translational Synthesis of Hypusine In eIF5A

National Institute Of Dental & Craniofacial Research

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Abstract

In the previous studies, we have identified eIF5A as the only cellular protein that contains an unusual amino acid, hypusine Ne-(4-amino-2-hydroxybutyl)lysine, a modified lysine with 4 aminobutyl moiety derived from the polyamine, spermidine. We have established that hypusine biosynthesis occurs post-translationally by two sequential enzymatic reactions: i) deoxyhypusine synthesis and ii) deoxyhypusine hydroxylation. We demonstrated that hypusine modification is essential for the activity of eIF-5A and for mammalian cell proliferation. Thus, hypusine synthesis defines one specific function of polyamines in cell proliferation. In addition to hypusine synthesis, polyamines bind to nucleic acids and regulate cellular activities at the transcriptional, translational and posttranslational levels. In mammalian cells, this polycationic function by bulk polyamines as well as hypusine synthesis is required for cell proliferation. In this reporting period, (in collaboration with Dr. Herbert Tabors group) we have investigated the role of hypusine and polyamines in the yeast S. cerevisiae. We have also conducted the structure/function studies of eIF5A, and identified the key structural elements of eIF5A critical for its biological activity in supporting growth and protein synthesis. Furthermore, we have obtained strong evidence that eIF5A is indeed a factor essential for eukaryotic protein synthesis. [unreadable] [unreadable] Hypusine modification is the most important function of the polyamine spermidine in the yeast S. cerevisiae: We have investigated the role of eIF5A in yeast polyamine auxotroph strains. Spermidine and its derivative, hypusinated eIF5A, have been shown to be essential for the growth of S. cerevisiae. In wild type cells, 1 % of cellular spermidine is normally utilized for hypusine synthesis. S. cerevisiae polyamine auxotrophs defective in polyamine synthesis grow at a nearly normal rate at low concentrations of spermidine (10-8 M) in the medium. In spe2 cells grown in this medium, cellular spermidine is less than 0.1 % of the normal level (<2 M vs. 2 mM), but as much as 37 % of the spermidine is mobilized for hypusine synthesis. The portion of spermidine used for hypusine synthesis increases dramatically as cellular spermidine becomes limiting. The vital importance of the hypusine modification is further supported by a positive correlation between the ability of spermidine analogs to modify eIF5A to a functional protein and their ability to support growth of the spe2 null mutant. 1-methyl spermidine supported growth whereas caldine, aminopropylcadaverine, 8-methylspermidine did not. Of the R- and S- stereoisomers of 1-methyl spermidine, the S-isomer was consistently better than the R-isomer in supporting growth and as a substrate for deoxyhypusine synthase (unpublished results). The failure of caldine to support growth is likely due to its inability to modify eIF5A. On the other hand, aminopropylcadaverine and 8-methylspermidine, both of which can serve as substrates of DHS to modify eIF5A, failed to support growth, suggesting a stringent structural requirement for the hypusine/deoxyhypusine side chain in eIF5A activity. Taken together, these findings indicate that the hypusine modification of eIF5A is the most critical (and perhaps primary) function of spermidine in supporting the growth of S. cerevisiae.[unreadable] [unreadable] Mutational analyses of human and yeast eIF5A: Identification of amino acid residues critical for the biological activity of eIF5A and the hypusine modification: To investigate the features of eIF5A required for its activity, we generated 49 mutations in human eIF5A-1. Each highly conserved residue was targeted by single site mutation and truncations were made from N- or C-terminus. All mutant proteins were tested for complementing the growth of an S. cerevisiae eIF5A null strain. Only a few mutant eIF5As with substitutions at or near the hypusine modification site (K47D, G49A, K50A, K50D, K50I, K50R, G52A and K55A) or with truncation of 21 amino acids from either the N- or C-terminus failed to support growth. For the Lys50 substitution proteins and G52A and K55A, the inactivity is due to the lack or impairment of deoxyhypusine modification. In contrast, K47D and G49A were effective substrates for deoxyhypusine synthase, yet failed to support growth, suggesting critical roles of Lys47 and Gly49 in eIF5A biological activity, possibly in its interaction with downstream effector(s). Comparison of the three different mutants, K47A, K47D and K47R, offers an interesting clue to the role of Lys47. K47R supports growth as well as the wild type protein, and K47A does so at a reduced rate. In contrast, no growth is observed upon expression of the K47D mutant, indicating the importance of the basic charge at residue 47. In view of evidence for acetylation at this site and loss of activities in K47A and K47D, Lys47 acetylation may serve as a natural mechanism in the regulation of eIF5A activity. [unreadable] A comparative structural and functional characterization was also performed on S. cerevisiae eIF5A (in collaboration with Dr. Sandro Valentinis group). The tertiary structure of yeast eIF5A was modeled based on the structures of its Leishmania mexicana homologues and this model was used to predict the location of site-directed and randomly-generated mutations in the tertiary structure. Most of the 40 new mutants exhibit phenotypes that result from eIF5A protein-folding defects. Our data provide evidence that the C-terminal -helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension is not. Similar to the human eIF5A mutants, yeast eIF5A mutant strains containing substitutions surrounding the hypusine modification site (K51 in the yeast protein) were nonviable (G50A, G50D, H52A, H52D, G53A, G53D, H54D and K56D) or showed temperature-sensitive (K56A) phenotype. These findings demonstrate the stringent requirement for the hypusine loop sequence in the interaction of eIF5A not only with its modification enzymes but also with its downstream effectors.[unreadable] [unreadable] eIF5A is an essential translation factor: Despite the essential nature of eIF5A in eukaryotic cell proliferation, the precise cellular function of eIF5A has remained obscure for decades. eIF5A enhances methionyl-puromycin synthesis in a deoxyhypusine- and hypusine-dependent manner in vitro. eIF5A binds to actively translating ribosomes and conditional mutants of eIF5A are hypersensitive to protein synthesis inhibitors. [unreadable] We have investigated the role of eIF5A in translation using the yeast strain, UBHY-R (derived in the laboratory of Dr. John WB Hershey). In this strain, the two yeast eIF5A genes are inactivated and its growth depends on genetically engineered unstable fusion-eIF5A (UBR5A), expressed under the GAL promoter. Upon shift of this strain from a galactose medium (YPGal) to a glucose medium (YPD), UBR5A was rapidly degraded, protein synthesis declined and cell growth was inhibited. Expression of human wild type eIF5A in the UBHY-R strain restored protein synthesis and growth, whereas expression of inactive mutant eIF5A proteins was without effect. Further evidence for a direct role of eIF5A in translation was obtained from yeast temperature-sensitive eIF5A mutants. Two of the temperature-sensitive yeast strains produced stable mutant proteins, eIF5AK56A and eIF5AQ22H,L93F. Upon shift of these two strains to a restrictive temperature, rapid inhibition of protein synthesis was observed, presumably as a result of an altered conformation of the mutant proteins upon the temperature shift.