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Decreased accuracy of protein synthesis in extracts from aging human diploid fibroblasts
Abstract:
The accuracy of protein synthesis has been measured in extracts from human diploid fibroblasts of different ages. Extracts were supplied with purified mRNA for the coat protein of the cowpea variant of tobacco mosaic virus (CcTMV), which lacks codons for cysteine and methionine. The presence of 35S-cysteine in CcTMV coat protein synthesized during translation reactions therefore represents translational error. Translation reactions were performed with extracts from young fibroblasts (less than 50% of life span completed) and old fibroblasts (more than 90% of life span completed), and the translation products were purified by immunoprecipitation and analyzed by polyacrylamide gel electrophoresis. The error frequency increased from 4.2 x 10(-5) cysteines/amino acid in young cell extracts to 2.9 x 10(-4) cysteines/amino acid in old cell extracts. Cysteine incorporation was not due to nonspecific binding, and could be increased approximately sixfold by the addition of the misreading antibiotic, paromomycin. It is concluded that translational accuracy is not stable during aging in vitro, and it is proposed that this decrease in the fidelity of information transfer could be responsible for the variety of changes observed in aging cultured human cells.
Insights
Protein synthesis accuracy declines with cellular aging. Older human fibroblasts exhibit higher translational error rates, suggesting a link between reduced fidelity and age-related cellular changes.
Area of Science:
- Molecular Biology
- Cell Biology
- Gerontology
Background:
- Cellular aging is associated with various functional declines.
- The fidelity of protein synthesis is crucial for cellular function.
- Changes in translational accuracy during aging are not well understood.
Purpose of the Study:
- To investigate the impact of cellular aging on protein synthesis accuracy.
- To quantify translational error rates in young versus old human fibroblasts.
- To explore the potential role of decreased translational fidelity in cellular aging.
Main Methods:
- Used human diploid fibroblasts from young and old donors.
- Synthesized cowpea mosaic virus coat protein using cell extracts and specific mRNA.
- Quantified incorporation of 35S-cysteine as a measure of translational error.
- Analyzed translation products using immunoprecipitation and gel electrophoresis.
Main Results:
- Translational error frequency increased significantly with fibroblast age.
- Error rates rose from 4.2 x 10^-5 in young cells to 2.9 x 10^-4 in old cells.
- Paromomycin, an antibiotic, exacerbated misreading, confirming assay validity.
Conclusions:
- Translational accuracy is compromised during in vitro cellular aging.
- Decreased fidelity of protein synthesis may contribute to age-related cellular dysfunction.
- This study provides evidence for impaired information transfer in aging cells.