The degradation of abnormal proteins will be analyzed in young and senescent IMR-90 human diploid fibroblasts. Three different abnormal proteins that are good substrates for ubiquitin- dependent pathways of proteolysis will be radiolabeled and microinjected into fibroblasts at increasing population doubling levels. Degradative rates will be compared in young and old cells injected with increasing amounts of the abnormal proteins. Young and senescent fibroblasts will be transfected with animal cell vectors carrying beta-galactosidase under control of the early SV40 promoter. The degradation of modified bacterial beta- galactosidase will be followed during transient expression of the foreign DNA. A variety of chimeric genes will be generated that are likely to code for short-lived fusion proteins. These constructions include ubiquitin-beta-galactosidase fusion proteins containing one or multiple ubiquitin molecules attached either to the amino or carboxyl terminus of beta-galactosidase. If the ubiquitin is rapidly cleaved from the beta-galactosidase, site- directed mutagenesis will be used to alter the amino acid at the junction between the two proteins. The fusion proteins that are still cleaved will expose a variety of different amino terminal residues, and the amino terminus is thought to be an important determinant of a protein's susceptibility to ubiquitin-dependent proteolysis. Similar studies with ubiquitin-alkaline phosphatase fusion proteins will determine whether age-related changes in degradation of abnormal forms of beta-galactosidase also apply to an unrelated protein. Abnormal proteins whose degradation is altered in aging will be further analyzed to establish their pathway of degradation and to identify components that are altered in senescent cells. Antibodies to ubiquitin and ubiquitin-protein conjugates will be used to identify ubiquitin-tagged intermediates in the degradation of microinjected proteins, beta-galactosidase derivatives, and alkaline phosphatase derivatives. The amount of free ubiquitin and ubiquitin-protein conjugates will be quantified in young and old cells in order to identify the portions of the ubiquitin- mediated degradation pathway that are altered with aging.
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