(provide by the applicant): There are multiple pathways of intracellular protein degradation that contribute to overall protein turnover within eukaryotic cells. The principal investigator has studied a lysosomal pathway of proteolysis that is activated in cultured fibroblasts by removal of serum growth factors and in rat liver in response to prolonged starvation. This pathway of proteolysis resembles the import of proteins for residence in other organelles such as mitochondria or peroxisomes. Proteins that are targeted to lysosomes for degradation contain within their amino acid sequence a peptide signal, and molecular chaperones both in the cytosol and inside the organelle facilitate the import process. Substrate proteins bind to a receptor protein in the lysosomal membrane before they are imported. This selective pathway of lysosomal proteolysis declines markedly with age, and one of the goals has been to characterize the reason for this decline and to correct the defect to determine its contribution to phenotypes associated with aging.
The specific aims for the upcoming period are: 1) To analyze the role of lamp2a as a receptor and a polypeptide transport channel. The principal investigator will use import into defined liposomes to define regions of lamp2a critical for binding and import. 2) To determine the dynamics between cytosolic hsc73 and lysosomal membrane hsc73. He will determine in the presence and absence of substrate proteins whether lysosomal membrane hsc73 is stable or dynamic. 3) To analyze roles of hsc73 in the lysosomal lumen. He will directly assay the ability of hsc73 to pull protein substrates across the lysosomal membrane and to affect the level of lamp2a in the lysosomal membrane. 4) To correct the defects in chaperone-mediated autophagy in senescent cells. These studies will include correction of the receptor levels in lysosomes of aged cells and analysis of the effects of restoring this protein degradation pathway on levels of aberrant proteins in senescent cells.
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