Differential expression of eIF5A‐1 and eIF5A‐2 in human cancer cells

Abstract

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the unusual amino acid hypusine [Nε‐(4‐amino‐2‐hydroxybutyl)lysine]. Vertebrates carry two genes that encode two eIF5A isoforms, eIF5A‐1 and eIF5A‐2, which, in humans, are 84% identical. eIF5A‐1 mRNA (1.3 kb) and protein (18 kDa) are constitutively expressed in human cells. In contrast, expression of eIF5A‐2 mRNA (0.7–5.6 kb) and eIF5A‐2 protein (20 kDa) varies widely. Whereas eIF5A‐2 mRNA was demonstrable in most cells, eIF5A‐2 protein was detectable only in the colorectal and ovarian cancer‐derived cell lines SW‐480 and UACC‐1598, which showed high overexpression of eIF5A‐2 mRNA. Multiple forms of eIF5A‐2 mRNA (5.6, 3.8, 1.6 and 0.7 kb) were identified as the products of one gene with various lengths of 3′‐UTR, resulting from the use of different polyadenylation (AAUAAA) signals. The eIF5A‐1 and eIF5A‐2 precursor proteins were modified comparably in UACC‐1598 cells and both were similarly stable. When eIF5A‐1 and eIF5A‐2 coding sequences were expressed from mammalian vectors in 293T cells, eIF5A‐2 precursor was synthesized at a level comparable to that of eIF5A‐1 precursor, indicating that the elements causing inefficient translation of eIF5A‐2 mRNA reside outside of the open reading frame. On sucrose gradient separation of cytoplasmic RNA, only a small portion of total eIF5A‐2 mRNA was associated with the polysomal fraction, compared with a much larger portion of eIF5A‐1 mRNA in the polysomes. These findings suggest that the failure to detect eIF5A‐2 protein even in eIF5A‐2 mRNA positive cells is, at least in part, due to inefficient translation.

The unique hypusine modification of eIF5A promotes islet β cell inflammation and dysfunction in mice

“…Hypusine is formed posttranslationally in a reaction catalyzed by DHS and DOHH and involving transfer of a 4-aminobutyl moiety from spermidine to Lys50 of eIF5A (15). The novelty of this modification in mammalian cells is reflected in the observation that only a single protein (eIF5A1/2) is detectable upon incubation of cells with 3 H-spermidine (25). eIF5A and its hypusinated form exhibit prolonged half-lives (≥24 hours) in many mammalian cells (40,41); strikingly, however, pulsechase studies revealed that eIF5A Hyp exhibits only approximately 6 hours of half-life in primary islets and islet-derived cell lines (present study).…”

“…Mammalian eIF5A exists as 2 isoforms, eIF5A1 and eIF5A2, which exhibit differing tissue distributions (25,26). As shown in Supplemental Figure 1 (supplemental material available online with this article; doi:10.1172/ JCI38924DS1), islets and islet-derived cell lines contain only the eIF5A1 isoform, which will henceforth be referred to simply as "eIF5A."…”

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

“…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 spermidine analogue GC7 (N1-guanyl-1,7-diamineoheptane) induces autophagy through a mechanism not involving the hypusination of eIF5A

“…K50A eIF5A mutant p3XFLAG-CMV-10.1 encoding for human eIF5A-1 was kindly provided by Myung Hee Park National Institute of Dental and Craniofacial Research "NIDCR" Bethesda, MD (Clement et al 2006). The mutant K50A was obtained in our lab using Quick Change Site-Directed Mutagenesis Kit (Stratagene).…”