Dramatic attenuation of hypusine formation on eukaryotic initiation factor 5A during senescence of IMR-90 human diploid fibroblasts

Abstract

Deoxyhypusine synthase catalyzes the conversion of lysine to deoxyhypusine residue on the eukaryotic initiation factor 5A (elF-5A) precursor using spermidine as the substrate. Subsequent hydroxylation of the deoxyhypusine residue completes hypusine formation on elF-5A. Hypusine formation is one of the most specific polyamine-dependent biochemical events in eukaryotic cells. Although changes in polyamine metabolism have been demonstrated in human diploid fibroblasts during senescence (Chen and Chang, 1986, J. Cell. Physiol., 128:27-32.), it is unclear whether or not polyamine-dependent hypusine formation itself is an age-dependent biochemical event. In the present study, hypusine-forming activity was measured by a radiolabeling assay in cells whose polyamines have been depleted by prior treatment of alpha-difluoromethyl ornithine (DFMO). In addition, an in vitro cross-labeling assay was developed for simultaneous measurement of the deoxyhypusine synthase activity and protein substrate (elF-5A precursor) amount. We showed that the hypusine-forming activity in low-passage presenescent IMR-90 cells [population doubling level (PDL) = 15-23, termed young cells] was prominently induced by serum whereas little or no hypusine-forming activity could be detected in late-passage senescent cells (PDL = 46-54, termed old cells). The striking difference in hypusine-forming activity between young and old cells was due to changes in both deoxyhypusine synthase activity and elF-5A precursor amount in IMR-90 cells during senescence. However, Northern blot analysis showed no significant difference in the elF-5A messenger RNA (mRNA) between young and old cells, suggesting that the age-dependent attenuation of elF-5A precursor protein may be regulated at either translational or post-translational level.

Hypusine Is Required for a Sequence-specific Interaction of Eukaryotic Initiation Factor 5A with Postsystematic Evolution of Ligands by Exponential Enrichment RNA

“…Inhibition of deoxyhypusine synthase in mammalian cells causes growth arrest (7-9), cell death (10), or tumor differentiation (9). In addition, hypusine formation activity exhibits a marked increase in virally transformed cells (11) but a striking attenuation in senescent cells (12).Despite the importance of eIF-5A in cell proliferation and survival, the physiological function of this protein is unclear. The notion that eIF-5A is an initiation factor comes from the earlier observations that it can be isolated from the ribosomebound fraction and that it can stimulate the synthesis of methionyl-puromycin (13-15).…”

“…Inhibition of deoxyhypusine synthase in mammalian cells causes growth arrest (7-9), cell death (10), or tumor differentiation (9). In addition, hypusine formation activity exhibits a marked increase in virally transformed cells (11) but a striking attenuation in senescent cells (12).…”