Global analysis of protein degradation in prion infected cells

Abstract

Prion diseases are rare neurological disorders caused by the misfolding of the cellular prion protein (PrP C ) into cytotoxic fibrils (PrP Sc ). Intracellular PrP Sc 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 PrP Sc aggregates have 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 lysosomal hydrolases. The resulting enhancement in proteome flux may play a role in the survival of N2a cells upon prion infection.

A TCF7L2-responsive suppression of both homeostatic and compensatory remyelination in Huntington disease mice

“…Quantitative mass spectrometry: Callosal tissue Callosal tissue was collected as above (n = 4) and samples were processed as previously described (Hutti et al, 2020). Briefly, callosal cells were homogenized and lysed in 1mL of 5% SDS, 100 mM TEAB, sonicated, then briefly centrifuged to remove debris.…”

“…Quantitative mass spectrometry: Striatal OPCs Since PDGFRA expression was found to diminish with age in these models, while A2B5 expression is maintained and remained selectively expressed by OPCs in the white matter (Gard and Pfeiffer, 1990;Roy et al, 1999), we sorted on the basis of A2B5 antibody binding to extract striatal OPCs for mass spectromtery. Striata (n = 3) were processed as noted above (Hutti et al, 2020), and debris removed via Percoll gradient. Cells were then blocked for 5 min and stained with APC-conjugated A2B5 for 20 min.…”