Dramatic attenuation of hypusine formation on eukaryotic initiation factor 5A during senescence of IMR-90 human diploid fibroblasts.
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.
Chen
Z P
ZP
Department of Chemistry, Rutgers, State University of New Jersey, Piscataway 08855-0939, USA.
Chen
K Y
KY
D000970:Antineoplastic Agents / Q000494:pharmacology; D002478:Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D016922:Cellular Senescence / Q000187:drug effects / Q000235:genetics; D004171:Diploidy; D000518:Eflornithine / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D008168:Lung / Q000166:cytology; D008239:Lysine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins; D013379:Substrate Specificity; D000097574:Eukaryotic Translation Initiation Factor 5A
D000970:Antineoplastic Agents; D000518:Eflornithine; D008239:Lysine; D000587:Oxidoreductases Acting on CH-NH Group Donors; D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D000097574:Eukaryotic Translation Initiation Factor 5A; C100028:hypusine; C063507:deoxyhypusine synthase