Translational fidelity of the ribosome as a critical parameter in human aging

Aging is a time-dependent decline in cellular and organismal functions. A hallmark of aging is the loss of proteostasis(definition), characterized by the accumulation of waste products and a reduced ability to clear them, leading to functional consequences. Protein synthesis is one aspect. The accuracy of translation is important for longevity and aging. Increased translational accuracy extends lifespan, whereas error-prone translation leads to premature aging. In model organisms, age increases translational errors. Human studies are limited. The lack of a sensitive method for detecting translational errors and the limitations of cellular aging models have led to controversial results. In this work, a highly sensitive method for detecting translational errors in cellular systems was developed using the luminescent reporters NanoLuc and Firefly. These methods enabled the detection of ribosomal errors during translation in fibroblasts from young and old donors, as well as in vitro-aged fibroblasts. It was found that translational errors decrease with age, accompanied by a reduction in total protein synthesis. The unfolded protein response, which is upregulated in aging, attenuates translation via phosphorylation of eukaryotic initiation factor 2 (eIF2). Induction of endoplasmic reticulum (ER) stress was found to improve translational fidelity by activating the PKR-like ER kinase (PERK). Downregulation of the binding immunoglobulin protein (BiP), an ER chaperone, can reduce translation errors by reducing PERK inhibition. Nanopore sequencing revealed strong overlap in differentially regulated genes and enriched gene ontologies (GO) between fibroblasts from old donors and fibroblasts from young donors with induced ER stress, compared with fibroblasts from young donors. The addition of proteome analysis by mass spectrometry revealed overlapping GO terms with the transcriptome analysis. The category ribosome is one of them. Ribosomal proteins are reduced with aging, and 80S ribosome assembly is affected in fibroblasts from old donors. Transmission electron microscopy showed a different ER phenotype in fibroblasts from an old donor or after Tunicamycin treatment, compared to fibroblasts from a young donor. Cycloheximide, an inhibitor of translation elongation, was found to improve translational fidelity without affecting protein synthesis at low doses. Fibroblasts from different mouse strains show differences in translation regulation among each other and compared to human fibroblasts. They lack the ability to adjust their protein synthesis and translational fidelity with age, thereby endangering the balance of proteostasis.