Abstract
Prion diseases are rare, neurological disorders caused by the misfolding of the cellular prion protein (PrPC) into cytotoxic fibrils (PrPSc). Intracellular PrPSc aggregates primarily accumulate within late endosomes and lysosomes, organelles that participate in the degradation and turnover of a large subset of the proteome. Thus, intracellular accumulation of PrPSc aggregates has the potential to globally influence protein degradation kinetics within an infected cell. We analyzed the proteome-wide effect of prion infection on protein degradation rates in N2a neuroblastoma cells by dynamic stable isotopic labeling with amino acids in cell culture (dSILAC) and bottom-up proteomics. The analysis quantified the degradation rates of more than 4,700 proteins in prion infected and uninfected cells. As expected, the degradation rate of the prion protein is significantly decreased upon aggregation in infected cells. In contrast, the degradation kinetics of the remainder of the N2a proteome generally increases upon prion infection. This effect occurs concurrently with increases in the cellular activities of autophagy and some lysosomal hydrolases. The resulting enhancement in proteome flux may play a role in the survival of N2a cells upon prion infection.
Highlights
IntroductionNeurological disorders caused by the misfolding of the cellular prion protein (PrPC) into cytotoxic fibrils (PrPSc)
Prion diseases are rare, neurological disorders caused by the misfolding of the cellular prion protein (PrPC) into cytotoxic fibrils (PrPSc)
While increases in the abundances of p62/SQSTM1 and LC3-II can be interpreted as markers of late-stage autophagic inhibition, N2a cells exhibit increased expression of p62/SQSTM1 mRNA, supporting the hypothesis that autophagy is upregulated upon prion infection
Summary
Neurological disorders caused by the misfolding of the cellular prion protein (PrPC) into cytotoxic fibrils (PrPSc). In cultured N2a cells, the activities of lysosomal hydrolases have been shown to be upregulated during prion infection[17] In both mouse and hamster brains infected with prion aggregates, there are increases in the abundances of autophagy-related proteins p62/SQSTM1 and LC3–II18. While increases in the abundances of p62/SQSTM1 and LC3-II can be interpreted as markers of late-stage autophagic inhibition, N2a cells exhibit increased expression of p62/SQSTM1 mRNA, supporting the hypothesis that autophagy is upregulated upon prion infection Together, these studies suggest that host cells upregulate lysosomal degradation as a response to prion infection. Other studies have provided evidence suggesting that accumulation of prion aggregates inhibits lysosomal degradation by interfering with the ability of the cell to endocytose and degrade proteins[19] Considering these opposing effects of prion infection on intracellular protein degradation, the net effect of PrPSc accumulation on global protein turnover kinetics remains unclear. We sought to utilize a proteomic approach to conduct a global analysis of changes in protein degradation kinetics during prion infection
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