N-terminal truncation of the scrapie-associated form of PrP by lysosomal protease(s): implications regarding the site of conversion of PrP to the protease-resistant state

Abstract

Scrapie and related transmissible spongiform encephalopathies result in the accumulation of a proteaseresistant form of an endogenous brain protein called PrP. As an approach to understanding the scrapieassociated modification of PrP, we have studied the processing and sedimentation properties of proteaseresistant PrP (PrP-res) in scrapie-infected mouse neuroblastoma cells. Like brain-derived PrP-res, the neuroblastoma cell PrP-res aggregated in detergent lysates, providing evidence that the tendency to aggregate is an intrinsic property of PrP-res and not merely a secondary consequence of degenerative brain pathology. The PrP-res species had lower apparent molecular masses than the normal, protease-sensitive PrP species and were not affected by moderate treatments with proteinase K. This suggested that the PrP-res species were partially proteolyzed by the neuroblastoma cells. Immunoblot analysis of PrP-res with a panel of monospecific anti-PrP peptide sera confirmed that the PrP-res species were quantitatively truncated at the N terminus. The metabolic labeling of PrP-res in serum-free medium did not prevent the proteolysis of PrP-res, showing that the protease(s) involved was cellular rather than serum-derived. The PrP-res truncation was inhibited in intact cells by leupeptin and NH4Cl. This provided evidence that a lysosomal protease(s) was involved, and therefore, that PrP-res was translocated to lysosomes. When considered with other studies, these results imply that the conversion of PrP to the protease-resistant state occurs in the plasma membrane or along an endocytic pathway before PrP-res is exposed to endosomal and lysosomal proteases.

Mechanism of PrP‐Amyloid Formation in Mice Without Transmissible Spongiform Encephalopathy

“…Brains from 14 101LL-8a mice at more than 400 days after intracerebral challenge with brain homogenate from mice previously challenged with 8 kDa GSS P102L (101LL-8a mice) were embedded in paraffin wax, cut and labeled with antibodies raised to mouse PrP by methods in routine use in our laboratory (14). The R18 antibody was raised against amino-acid sequence (aa) 142-155; R20 to aa 218-232; R24 to aa 23-37; and R30 to aa 89-103 as previously reported (6). The number and location of plaques in five coronal whole brain or hemibrain slices, two of which contained representation of the corpus callosum, were recorded in each mouse.…”

The scrapie prion protein is present in flotillin‐1‐positive vesicles in central‐ but not peripheral‐derived neuronal cell lines

“…In contrast to PrP c , PrP Sc trafficking was less studied and represents an important challenge. PrP Sc is also localized at the plasma membrane (Vey et al, 1996) and along the internalization pathway of PrP c , in caveolae-like structures (Taraboulos et al, 1995;Kaneko et al, 1997), in late endosome-lysosome vesicles (Caughey et al, 1991;Laszlo et al, 1992;Mayer et al, 1992;Taraboulos et al, 1992;Arnold et al, 1995;Harris, 1999), in the endoplasmic reticulum (Beranger et al, 2002) and nuclei (Mange et al, 2004).…”

The ubiquitin–proteasome system in spongiform degenerative disorders

“…In addition to the UPS, other cellular pathways likely contribute to the vacuolation and degeneration of nervous tissue observed in TSEs. Increases in oxidative stress have been reported (reviewed in [62]) and there is considerable evidence for aberration in the proteolytic processing and trafficking of PrP C [49,[63][64][65][66][67] and lysosomal dysfunction in TSEs [27,49,60,68,69]. These processes are regulated by both proteasome-dependent and -independent ubiquitin signaling (reviewed in [29,70,71]).…”

“…This indicates PrP Sc toxicity is likely dependent upon its localization to the plasma membrane and/or endocytic system. The conformational change from PrP C to PrP Sc probably depends upon a number of factors, but abnormal trafficking to an acidic compartment like the lysosome or MVB may contribute to the propagation of protease-resistant PrP [64]. In fact, subcellular fractionation of scrapie-infected mouse brain [63] reveals that protease-resistant PrP (PrP Sc ) accumulates in fractions that also contain markers for the late endosome and lysosome.…”