Recent studies have shown that a sizable fraction of PrPSc present in prion-infected tissues is, contrary to previous conceptions, sensitive to digestion by proteinase K (PK). This finding has important implications in the context of diagnosis of prion disease, as PK has been extensively used in attempts to distinguish between PrPSc and PrPC. Even more importantly, PK-sensitive PrPSc (sPrPSc) might be essential to understand the process of conversion and aggregation of PrPC leading to infectivity. We have isolated a fraction of sPrPSc. This material was obtained by differential centrifugation at an intermediate speed of Syrian hamster PrPSc obtained through a conventional procedure based on ultracentrifugation in the presence of detergents. PK-sensitive PrPSc is completely degraded under standard conditions (50 mug/mL of proteinase K at 37 degrees C for 1 h) and can also be digested with trypsin. Centrifugation in a sucrose gradient showed sPrPSc to correspond to the lower molecular weight fractions of the continuous range of oligomers that constitute PrPSc. PK-sensitive PrPSc has the ability to convert PrPC into protease-resistant PrPSc, as assessed by the protein misfolding cyclic amplification assay (PMCA). Limited proteolysis of sPrPSc using trypsin allows for identification of regions that are particularly susceptible to digestion, i.e., are partially exposed and flexible; we have identified as such the regions around residues K110, R136, R151, K220, and R229. PK-sensitive PrPSc isolates should prove useful for structural studies to help understand fundamental issues of the molecular biology of PrPSc and in the quest to design tests to detect preclinical prion disease.
Sensitive quantitation of prions in biological samples is an extremely important and challenging analytical problem. Prions are the cause of several fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). At this time, there are no methods to diagnose TSEs in live animals or to assure a prion-free blood supply for humans. Prions have been shown to be present in blood by transfusion experiments, but based on the amount of infectivity found in these types of experiments, the amount of misfolded prion protein in blood is estimated to be only 30 to 625 amol/mL. More sensitive detection of prions in brain would allow earlier detection of disease and assure a safer food supply. We studied quantitation of the prion protein by use of nanoscale liquid chromatography coupled to a tandem mass spectrometer using the multiple reaction monitoring mode of operation. We developed a method based on the detection of VVEQMCTTQYQK obtained by reduction, alkylation, and digestion with trypsin of the prion protein.
One of the main characteristics of the transmissible isoform of the prion protein (PrPSc) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunodetection of PK-digested PrPSc following Western blot or ELISA. More recently, researchers determined that there is a sizeable fraction of PrPSc that is sensitive to PK hydrolysis (sPrPSc). Our group has previously reported a method to isolate this fraction by centrifugation and showed that it has protein misfolding cyclic amplification (PMCA) converting activity. We compared the infectivity of the sPrPSc versus the PK-resistant (rPrPSc) fractions of PrPSc and analyzed the biochemical characteristics of these fractions under conditions of limited proteolysis. Our results show that sPrPSc and rPrPSc fractions have comparable degrees of infectivity and that although they contain different sized multimers, these multimers share similar structural properties. Furthermore, the PK-sensitive fractions of two hamster strains, 263K and Drowsy (Dy), showed strain-dependent differences in the ratios of the sPrPSc to the rPrPSc forms of PrPSc. Although the sPrPSc and rPrPSc fractions have different resistance to PK-digestion, and have previously been shown to sediment differently, and have a different distribution of multimers, they share a common structure and phenotype.
Prions are infectious proteins that are able to recruit a normal cellular prion protein and convert it into a prion. The mechanism of this conversion is unknown. Detailed analysis of the normal cellular prion protein and a corresponding prion has shown they possess identical post-translational modifications and differ solely in conformation. Recent work has suggested that the oxidized form of the methionine at position 213 (Met213) plays a role in the conversion of the normal cellular prion protein to the prion conformation and is a prion-specific covalent signature. We developed a sensitive method of quantitating the methionine sulfoxide present at position 213 (MetSO213) and used this method to measure the changes in MetSO213 over the time course of an intracranial challenge, using the 263K strain of hamster-adapted scrapie. These results indicate that the proportion of Met213 that is oxidized decreases over the course of the disease. We examined the quantity of MetSO213 in PrP(C) and compared it to the amount found in animals terminally afflicted with the 263K, 139H, and drowsy strains of hamster-adapted scrapie. These strains show only low levels of MetSO213 that is comparable to that of PrP(C). These data suggest that MetSO213 does not appear to be a prion-specific covalent signature.
The cellular prion protein (PrPC) is a highly conserved protein whose exact physiological role remains elusive. In the present study, we investigated age-dependent behavioral abnormalities in PrPC-knockout (Prnp0/0) mice and wild-type (WT) controls. Prnp0/0 mice showed age-dependent behavioral deficits in memory performance, associative learning, basal anxiety, and nest building behavior. Using a hypothesis-free quantitative proteomic investigation, we found that loss of PrPC affected the levels of neurofilament proteins in an age-dependent manner. In order to understand the biochemical basis of these observations, we analyzed the phosphorylation status of neurofilament heavy chain (NF-H). We found a reduction in NF-H phosphorylation in both Prnp0/0 mice and in PrPC-deficient cells. The expression of Fyn and phospho-Fyn, a potential regulator for NF phosphorylation, was associated with PrPC ablation. The number of β-tubulin III-positive neurons in the hippocampus was diminished in Prnp0/0 mice relative to WT mice. These data indicate that PrPC plays an important role in cytoskeletal organization, brain function, and age-related neuroprotection. Our work represents the first direct biochemical link between these proteins and the observed behavioral phenotypes.
We developed a sensitive mass spectrometry-based method of quantitating the prions present in a variety of mammalian species. Calibration curves relating the area ratios of the integrated MRM signals from selected analyte peptides and their oxidized analogues to their homologous stable isotope labeled internal standards were prepared. The limit of detection (LOD) and limit of quantitation (LOQ) for the synthetic peptides from human, sheep, deer, cow, and mouse PrP were determined to be below 100 amol. Nonanalyte peptides that were characteristic of prions were included in the multiple reaction monitoring method, thereby allowing for both the quantitation and confirmation of the presence of prions in the attomole range. This method was used to quantitate the prions present in brains of hamsters or mice 5 weeks after inoculation (ic) with either four hamster-adapted prion strains (139H, drowsy, 22AH, and 22CH) or four mouse-adapted prion strains (Me7, Me7-298, RML, and 79A). The prions from different brain regions of a sheep naturally infected with scrapie were quantitated. All of the rodent-adapted prion strains were detectable in the asymptomatic animals. In sheep, prions were detectable in the obex, anterior portion of the cerebrum, and the nonobex/nonanterior portion of the cerebrum. This mass spectrometry-based approach can be used to quantitate and confirm the presence of prions before detectable pathology.
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