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Altered sensitivity of protein synthesis to paromomycin in extracts from aging human diploid fibroblasts
Abstract:
Age-related differences in the effects of paromomycin (Pm) on protein synthesis have been investigated in translation reactions with extracts derived from young and old human diploid fibroblasts. Translation products from reactions directed by endogenous or exogenous mRNA were analyzed by polyacrylamide gel electrophoresis and fluorography. The exogenous mRNA lacked codons for cysteine, and therefore cysteine incorporation into translation products represented translational error. This laboratory has previously used this assay to show that the basal translational error level in the absence of Pm increases in extracts from old fibroblasts. In this report, Pm stimulated the misincorporation of cysteine by 6-7 fold over cysteine misincorporation levels in the absence of Pm. This degree of Pm stimulation was similar in extracts from young and old fibroblasts. However, other results showed quantitative differences in the responses to Pm between young and old cell extracts. Old cell extracts were less sensitive to the stimulation of the rate of protein synthesis, and more sensitive to the inhibition of protein synthesis, by Pm. It is proposed that aging human diploid fibroblasts contain altered ribosomes which react differently with Pm.
Insights
Paromomycin (Pm) affects protein synthesis differently in aging cells. While Pm equally increased errors in young and old human fibroblasts, older cells showed reduced protein synthesis stimulation and increased inhibition by Pm.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Aging is associated with increased translational error in human fibroblasts.
- Paromomycin (Pm) is an aminoglycoside antibiotic known to affect protein synthesis.
Purpose of the Study:
- To investigate age-related differences in the effects of paromomycin on protein synthesis in human diploid fibroblasts.
- To determine if paromomycin affects translational error rates differently in young versus old cells.
Main Methods:
- Protein synthesis was studied using cell-free extracts from young and old human diploid fibroblasts.
- Translational error was quantified by measuring cysteine misincorporation into proteins synthesized from cysteine-free mRNA in the presence of paromomycin.
Main Results:
- Paromomycin stimulated cysteine misincorporation (translational error) 6-7 fold in both young and old cell extracts.
- Old cell extracts exhibited reduced sensitivity to paromomycin-induced stimulation of protein synthesis rate.
- Old cell extracts showed increased sensitivity to paromomycin-induced inhibition of protein synthesis.
Conclusions:
- Aging human fibroblasts possess altered ribosomes that exhibit differential responses to paromomycin.
- These findings suggest age-dependent changes in ribosomal function and drug sensitivity.