Aging human fibroblasts exhibit defects in a lysosomal pathway of intracellular protein degradation. This pathway is induced in young cells in response to serum withdrawal. Proteins with specific peptide regions are targeted to this pathway of degradation, and a 73 KDa protein binds to these peptide regions. The 73KDa protein is a member of the heat shock protein 70 KDa family, and its expression is markedly reduced in senescent cells. This serum-regulated pathway of proteolysis will be further defined in young cells, and detailed comparisons with senescent cells will establish the molecular defects associated with aging. The peptide motif required for entry into this pathway of degradation will be experimentally determined using a variety of site-directed mutants in a coding sequence for peptide-beta-galactosidase fusion proteins. These hybrid proteins will be expressed in young and old cells, and degradation of the altered peptide-beta-galactosidase molecules will be measured in the presence and absence of serum. The 73 KDa protein in young cells, but not in senescent cells is induced by serum deprivation. mRNA levels for this protein will be monitored in young and old cells both in the presence and absence of serum. A variety of recombinant DNA and microinjection studies will attempt to increase the level of the 73 KDa protein in senescent cells. The activity of the lysosomal degradation pathway and the proliferative potential of the cell will then be measured. A permeabilized cell system appears to reproduce several aspects of the serum-regulated lysosomal pathway of proteolysis. We will establish the validity of permeabilized cells to study the age-related defect in proteolysis. These cell-free systems will permit the detailed biochemical characterization of the age-related defects in the serum-regulated pathway of proteolysis.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Method to Extend Research in Time (MERIT) Award (R37)
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Pathobiochemistry Study Section (PBC)
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Tufts University
Schools of Medicine
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