Dramatic attenuation of hypusine formation on eukaryotic initiation factor 5A during senescence of IMR-90 human diploid fibroblasts
✍ Scribed by Zong Ping Chen; Kuang Yu Chen
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 211 KB
- Volume
- 170
- Category
- Article
- ISSN
- 0021-9541
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✦ Synopsis
Deoxyhypusine synthase catalyzes the conversion of lysine to deoxyhypusine residue on the eukaryotic initiation factor 5A (eIF-5A) precursor using spermidine as the substrate. Subsequent hydroxylation of the deoxyhypusine residue completes hypusine formation on eIF-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 a-difluoromethyl ornithine (DFMO). In addition, an in vitro cross-labeling assay was developed for simultaneous measurement of the deoxyhypusine synthase activity and protein substrate (eIF-5A precursor) amount. We showed that the hypusine-forming activity in low-passage presenescent IMR-90 cells [population doubling level (PDL) Å 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 Å 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 eIF-5A precursor amount in IMR-90 cells during senescence. However, Northern blot analysis showed no significant difference in the eIF-5A messenger RNA (mRNA) between young and old cells, suggesting that the age-dependent attenuation of eIF-5A precursor protein may be regulated at either translational or posttranslational level.