Age-associated changes in rates of intracellular protein degradation will be determined through the use of red cell-mediated microinjection of radioactive protenis into IMR-90 human diplid fibroblasts. The ability of aging fibroblasts to selectively degrade abnormal polypeptides and the biochemical explanations for altered protein degradation in aging will be studied. Skin fibroblasts from human donors of different ages and from patients with premature aging diseases will be used to confirm results obtained with early passage (""""""""young"""""""") and late passage (""""""""senescent"""""""", """"""""old"""""""") IMR-90 fibroblasts. Protein catabolism is a fundamentally important process involved with the regulation of overall protein content, the control of levels of specific enzymes, and the removal of defective proteins within cells. The few published reports concerning protein degradation in senescent cells and organisms present conflicting results, so the importance of altered protein catabolism in aging remains unresolved. The use of microinjection to study age-associated alterations in protein degradation offers several advantages over studies of catabolism of endogenously labeled proteins: (a) Proteins can be radioactively labeled in vitro by tracers that are not subject to reutilization by the cells' biosynthetic machinery. (b) Proteins can be precisely modified in a variety of ways prior to microinjection to probe the ability of senescent cells to recognize and degrade different types of abnormal polypeptides. (c) Proteins from """"""""young"""""""" cells can be labeled and microinjected into """"""""old"""""""" cells and vice versa to establish whether alterations in the cellular proteins themselves are responsible for age-related changes in their degradative rates. (d) Several studies of the mechanisms and pathways of intracellular protein degradation in aging cells are uniquely possible using microinjection procedures.
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