Degradation Of PrP Research Articles

Biochemical evidence for the proteolytic degradation of infectious prion protein PrP sc in hamster brain homogenates by foodborne bacteria

PrP Sc is a general term to describe the infectious agent causing transmissible spongiform encephalopathy (TSE), and the protease-resistant form of cellular PrP C. In this study, we have identified several protease-secreting bacteria able to degrade PrP Sc under more or less native conditions (30 °C, pH 8), focusing on strains isolated mainly from cheese. One hundred and ninty-nine protease-secreting isolates belonging to the Actinomycetales and Bacillales were screened for the expression of PrP Sc degrading activity by a Western blot procedure. Only 6 strains belonging to the following species were found to exhibit such an activity: Arthrobacter nicotianae, Bacillus licheniformis, Brachybacterium conglomeratum, Brachybacterium tyrofermentans and Staphylococcus sciuri and Serratia spp. As revealed by a general protease assay based on dye-labeled Azocoll substrate, the PrP Sc degrading activity was not directly correlated to the total level of secreted proteolytic activity of these organisms. This indicates that specific proteases are required for the degradation of PrP Sc. Our study also suggests the potential use of such starter bacteria or their proteases for application in PrP Sc degradation and decontamination under native conditions.

The Tyrosine Kinase Inhibitor STI571 Induces Cellular Clearance of PrPSc in Prion-infected Cells

The conversion of the cellular prion protein (PrP(c)) into pathologic PrP(Sc) and the accumulation of aggregated PrP(Sc) are hallmarks of prion diseases. A variety of experimental approaches to interfere with prion conversion have been reported. Our interest was whether interference with intracellular signaling events has an impact on this conversion process. We screened approximately 50 prototype inhibitors of specific signaling pathways in prion-infected cells for their capacity to affect prion conversion. The tyrosine kinase inhibitor STI571 was highly effective against PrP(Sc) propagation, with an IC(50) of < or =1 microM. STI571 cleared prion-infected cells in a time- and dose-dependent manner from PrP(Sc) without influencing biogenesis, localization, or biochemical features of PrP(c). Interestingly, this compound did not interfere with the de novo formation of PrP(Sc) but activated the lysosomal degradation of pre-existing PrP(Sc), lowering the half-life of PrP(Sc) from > or =24 h to <9 h. Our data indicate that among the kinases known to be inhibited by STI571, c-Abl is likely responsible for the observed anti-prion effect. Taken together, we demonstrate that treatment with STI571 strongly activates the lysosomal degradation of PrP(Sc) and that substances specifically interfering with cellular signaling pathways might represent a novel class of anti-prion compounds.

Misfolding of the prion protein at the plasma membrane induces endocytosis, intracellular retention and degradation.

Suramin induces misfolding of the cellular prion protein (PrP(C)) and interferes with the propagation of infectious scrapie prions. A mechanistic analysis of this effect revealed that suramin-induced misfolding occurs at the plasma membrane and is dependent on the proximal region of the C-terminal domain (aa 90-158) of PrP(C). The conformational transition induces rapid internalization, mediated by the unstructured N-terminal domain, and subsequent intracellular degradation of PrP(C). As a consequence, PrP Delta N adopts a misfolded conformation at the plasma membrane; however, internalization is significantly delayed. We also found that misfolding and intracellular retention of PrP(C) can be induced by copper and that, moreover, copper interferes with the propagation of the pathogenic prion protein (PrP(Sc)) in scrapie-infected N2a cells. Our study revealed a quality control pathway for aberrant PrP conformers present at the plasma membrane and identified distinct PrP domains involved.

Anti-PrP antibodies block PrPSc replication in prion-infected cell cultures by accelerating PrPC degradation.

The use of anti-PrP antibodies represents one of the most promising strategies for the treatment of prion diseases. In the present study, we screened various anti-PrP antibodies with the aim of identifying those that would block PrP(Sc) replication in prion-infected cell culture. Two antibodies, SAF34 recognizing the flexible octarepeats region on HuPrP protein, and SAF61 directed against PrP amino acid residues (144-152), not only inhibited PrP(Sc) formation in prion-infected neuroblastoma cells but also decreased the PrP(C) levels in non-infected N2a cells. In addition, treatment with both SAF34 and SAF61 antibodies decreased PrP(C) and PrP(Sc) levels in the cells synergistically. In the presence of both antibodies, our results showed that the mode of action which leads to the disappearance of PrP(Sc) in cells is directly coupled to PrP(C) degradation by reducing the half-life of the PrP(C) protein.

Cellular prion protein acquires resistance to proteolytic degradation following copper ion binding.

The conversion of cellular prion protein (PrP(C)) into its pathological isoform (PrP(Sc)) conveys an increase in hydrophobicity and induces a partial resistance to proteinase K (PK). Interestingly, co-incubation with high copper ion concentrations also modifies the solubility of PrP(c) and induces a partial PK resistance which was reminiscent of PrP(Sc). However, concerns were raised whether this effect was not due to a copper-induced inhibition of the PK itself. We have therefore analyzed the kinetics of the formation of PK-resistant PrP(C) and excluded possible interference effects by removing unbound copper ions prior to the addition of PK by methanol precipitation or immobilization of PrP(C) followed by washing steps. We found that preincubation of PrPc with copper ions at concentrations as low as 50 microM indeed rendered these proteins completely PK resistant, while control substrates were proteolyzed. No other divalent cations induced a similar effect. However, in addition to this specific stabilizing effect on PrP(C), higher copper ion concentrations in solution (>200 microM) directly blocked the enzymatic activity of PK, possibly by replacing the Ca2+ ions in the active center of the enzyme. Therefore, as a result of this inhibition the proteolytic degradation of PrP(C) as well as PrP(Sc) molecules was suppressed.

Trafficking, turnover and membrane topology of PrP.

Cell biological studies of PrP have contributed enormously to our understanding of prion diseases. Like other membrane proteins, PrP(C) is post-translationally processed in the endoplasmic reticulum and Golgi on its way to the cell surface after synthesis. Cell surface PrP(C) constitutively cycles between the plasma membrane and early endosomes via a clathrin-dependent mechanism, a pathway consistent with a suggested role for PrP(C) in cellular trafficking of copper ions. PrP molecules carrying mutations linked to inherited prion diseases display several abnormalities in their biochemical properties, maturation, and localisation that may explain their pathogenicity. Recent results have clarified the role of the proteasome in degradation of PrP, and the properties of a transmembrane form of PrP which may play a neurotoxic role in prion diseases.

Similar Turnover and Shedding of the Cellular Prion Protein in Primary Lymphoid and Neuronal Cells

The cellular prion protein (PrP(C)) is essential for pathogenesis and transmission of prion diseases. Although prion replication in the brain is accompanied by neurodegeneration, prions multiply efficiently in the lymphoreticular system without any detectable pathology. We have used pulse-chase metabolic radiolabeling experiments to investigate the turnover and processing of PrP(C) in primary cell cultures derived from lymphoid and nervous tissues. Similar kinetics of PrP(C) degradation were observed in these tissues. This indicates that the differences between these two organs with respect to their capacity to replicate prions is not due to differences in the turnover of PrP(C). Substantial amounts of a soluble form of PrP that lacks the glycolipid anchor appeared in the medium of splenocytes and cerebellar granule cells. Soluble PrP was detected in murine and human serum, suggesting that it might be of physiological relevance.

Characterization of BSE and scrapie strains/isolates.

Following the BSE epidemic in cattle and the emergence of a variant form of Creutzfeldt-Jakob disease in humans, the question was raised whether BSE has been transmitted to small ruminants by the inadvertent feeding of infectious meat and bone meal. Such infections could easily be concealed in countries where scrapie is endemic. To address this issue by immuno-chemically analyzing the PrP(Sc) fragments, we have developed two lines of research. Firstly we have focused on the development of criteria for the differential characterization of experimental BSE and scrapie strains/isolates in rodents. To date, three criteria have been identified: quantification of the relative banding intensities of PrP(Sc) glycotypes using a photoimaging technique; the non-uniform kinetic of proteinase K degradation of PrP(Sc); and differences in the molecular masses of their non-glycosylated PrP(Sc) fragments after PK cleavage in immunoblot. The second line of research focused on the implementation of the criteria described above to representative samples from scrapie diseased Irish sheep. Using these three criteria, no evidence was found for the presence of a BSE infection in these animals. However, the final conclusion must take into account the results of mouse incubation time and mouse lesion profile data which are currently being generated.