The candidate is a junior researcher with considerable experience in intracellular protein degradation and some training in aging research. In order to become a fully independent researcher studying changes in protein metabolism with age, an additional supervised training period will be very important. Dr. J. Fred Dice, due to his extensive research experience in protein degradation in aging, would be the ideal mentor for this training. The research project will be carried out at Tufts University which offers a stimulating scientific environment and a well- established research program in cellular metabolism and physiology. The proposed four year career development plan is oriented to: i) confer the candidate strong background in aging, ii)improve her research experience, including learning new experimental approaches, iii)facilitate productive collaborations with well established researchers and iv)develop her teaching, training and group leader skills. The main goal of the research project is to identify the cause(s) of the decrease in the specific degradation of cytosolic proteins in lysosmers with age. The reduction of this degradative system may contribute to the cytosolic accumulation of abnormal proteins in senescent cells and the subsequent generalized failure of many cellular functions. The selective lysosomal pathway is induced in response to nutrient deprivation, and it is mediated by a cytosolic heat shock protein of 73KDa (hsc73), that stimulates binding of substrates to the lysosomal membrane, and by a lysosomal hsc73, essential for the import of substrate proteins. The characterization of the different components involved in this lysosomal uptake, and the further comparison of their levels in young and old rats, will be decisive in identifying the origin of the lysosomal failure with age. The steps followed by the substrates of this pathway will be analyzed in an in vitro system with isolated rat liver lysosomes. Immunological procedures and protein interaction assays will be use to determine changes in levels or activity of the components of the system with age. Overexpression of some of those components in cultured cells will be used to determine their participation in the selective degradation of proteins in lysosomes and to analyze the contribution of their age-dependent changes to some of the characteristics of senescent cells. These studies will also evaluate the possible reversibility of the lysosomal defect with age.
