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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Modified Research Career Development Award (K04)
Project #
5K04AG000339-05
Application #
3070524
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1985-09-01
Project End
1987-08-31
Budget Start
1986-09-01
Budget End
1987-08-31
Support Year
5
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Tufts University
Department
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02111
Gurley, R; Dice, J F (1988) Degradation of endocytosed proteins is unaltered in senescent human fibroblasts. Cell Biol Int Rep 12:885-94
Dice, J F; Backer, J M; Chiang, H L et al. (1987) Lysosomal degradation of microinjected proteins. Biochem Soc Trans 15:824-6
Goff, S A; Short-Russell, S R; Dice, J F (1987) Efficient saturation mutagenesis of a pentapeptide coding sequence using mixed oligonucleotides. DNA 6:381-8
Dice, J F; Chiang, H L; Spencer, E P et al. (1986) Regulation of catabolism of microinjected ribonuclease A. Identification of residues 7-11 as the essential pentapeptide. J Biol Chem 261:6853-9
Backer, J M; Dice, J F (1986) Covalent linkage of ribonuclease S-peptide to microinjected proteins causes their intracellular degradation to be enhanced during serum withdrawal. Proc Natl Acad Sci U S A 83:5830-4