Identification and characterization of eukaryotic initiation factor 5A‐2

Abstract

The phylogenetically conserved eukaryotic translation initiation factor 5A (eIF5A) is the only known cellular protein to contain the post-translationally derived amino acid hypusine [N e -(4-amino-2-hydroxybutyl)lysine]. Both eIF5A and its hypusine modification are essential for sustained cell proliferation. Normally only one eIF5A protein is expressed in human cells. Recently, we identified a second human EIF5A gene that would encode an isoform (eIF5A-2) of 84% sequence identity. Overexpression of eIF5A-2 mRNA in certain human cancer cells, in contrast to weak normal expression limited to human testis and brain, suggests EIF5A2 as a potential oncogene. However, eIF5A-2 protein has not been described in human or mammalian cells heretofore. Here, we describe the identification of eIF5A-2 protein in human colorectal and ovarian cancer lines, SW-480 and UACC-1598, that overexpress eIF5A-2 mRNAs. Functional characterization of the human isoforms revealed that either human EIF5A gene can complement growth of a yeast strain in which the yeast EIF5A genes were disrupted. This indicates functional similarity of the human isoforms in yeast and suggests that eIF5A-2 has an important role in eukaryotic cell survival similar to that of the ubiquitous eIF5A-1. Detectable structural differences were also noted, including lack of immunological cross-reactivity, formation of different complexes with deoxyhypusine synthase, and K m values (1.5 ± 0.2 vs. 8.3 ± 1.4 lM for eIF5A-1 and -2, respectively) as substrates for deoxyhypusine synthase in vitro. These physical characteristics and distinct amino acid sequences in the C-terminal domain together with differences in gene expression patterns imply differentiated, tissue-specific functions of the eIF5A-2 isoform in the mammalian organism and in cancer.

Crystal structure of human eIF5A1: Insight into functional similarity of human eIF5A1 and eIF5A2

“…2,3,14 Two eIF5A isoforms, eIF5A1 and eIF5A2, occur in humans and have 84% identity in their amino acid sequence. [15][16][17][18] The eif5a1 gene is ubiquitously expressed, whereas eif5a2 is highly expressed in the testis and at moderate levels in the brain. 18 The eIF5A2 protein is also detectable in colorectal and ovarian cancer-derived cell lines.…”

“…[18][19][20] Although the cancer-specific eif5a2 expression implies that eIF5A2 plays a role distinct from eIF5A1 in cancer cells, either human gene can restore the viability of a yeast eif5a mutant, suggesting functional similarity of the two human isoforms in eukaryotic cell survival. 17 The amino acid sequences of the human isoforms are conserved near the hypusination site, 17 suggesting that their function in cell survival might be linked to the hypusine modification.…”

The Role of Acetylation in the Subcellular Localization of an Oncogenic Isoform of Translation Factor eIF5A

“…2B), suggesting that eIF5A2 is hypusinated at lysine-50 as is eIF5A1. 14) Moreover, the acetylation of the K50R mutant was much greater than that of fully hypusinated wild-type eIF5A2 (Fig. 2B), suggesting that hypusination also reduces acetylation in eIF5A2.…”

Posttranslational synthesis of hypusine: evolutionary progression and specificity of the hypusine modification

“…In contrast to S. cerevisiae, suppression of expression of DOHH results in the recessive embryonic lethal phenotypes in C. elegans (Sugimoto, 2004) or Drosophila melanogaster (Spradling et al, 1999) indicating the requirement for fully modified, hypusine-containing eIF5A in higher multicellular eukaryotes. Both human eIF5A isoforms (isoforms 1 and 2) can support the growth of eIF5A null strain of S. cerevisiae (Clement et al, 2003), indicating that the fundamental function of eIF5A has been conserved from yeast to human. The finding that this hydroxylation is vital in the higher eukaryotes suggests either a specialized eIF5A function and/or a critical hydroxylation-dependent regulation mechanism involving eIF5A.…”

“…In yeast, two eIF5A genes, TIF51A (aerobic gene) and TIF51B (anaerobic gene) are reciprocally regulated by oxygen, while the two isoform proteins are functionally indistinguishable. In fish, amphibians and chicken, the two eIF5A genes appear to be co-expressed (Clement et al, 2003). In contrast, most mammalian cells and tissues constitutively express only the predominant isoform, eIF5A-1.…”