Age-related defects in intracellular protein degradation will be analyzed in IMR-90 human diploid fibroblasts through the use of red cell-mediated microinjection. The degradation of ribonuclease A (RNase A) microinjected into senescent cells will be used as a model to probe altered mechanisms of proteolysis in aging. Senescent cells show reduced rates of degradation of microinjected RNase A and an impaired ability to accelerate rates of degradation upon serum withdrawal. Both of these age-related changes in proteolysis result from defects in lysosomal pathways of protein degradation. Degradation of fragments of RNase A will be analyzed to establish the region of the protein required for enhanced degradation in deprivation conditions. Whether this sequence is also present in certain cellular proteins will be determined. The pathways of delivery of RNase A from cytosol to lysosomes will be analyzed for cells maintained in the presence and absence of serum. These studies will involve a combination of biochemical and morphological analyses. Finally, lysosomal uptake and degradation of proteins will be reproduced in cell-free homogenates to better understand the biochemical requirements of the process. Equivalent studies using senescent cells and homogenates will localize the age-related defects responsible for reduced protein catabolism and impaired ability to regulate protein degradation in aging. Protein catabolism is a fundamentally important process in all organisms from bacteria to humans. Age-related reductions in proteolysis may be responsible for several properties of senescent cells including the accumulation of aberrant proteins, the increased cellular protein content, and the slower ability to alter enzyme levels in response to changes in the environment. It may eventually be possible to correct these defects in protein degradation to determine whether cellular lifespans are increased.
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