In this research paper the author states though the conversion of alpha-helices into beta-sheets underlies the formation of PrPSc, the possibility of an undetected chemical modification of a small fraction of PrPSc initiates this process cannot be eliminated. Prions are composed largely of prion protein (PrPSc in the case of scrapie). Though the formation of PrPSc from the cellular prion protein (PrPC) is a post-translational process, the chemical modification was not identified, suggesting that a conformational change features in PrPSc synthesis. To assess this possibility in this research, both PrPC and PrPSc were purified by using nondenaturing procedures and the secondary structure of each were determined.
Prions are proteinaceous infectious particles that are composed largely of an abnormal isoform of the prion protein (PrP) designated, in the case of scrapie, PrPSc. Prions cause four neurodegenerative diseases of humans and six of animals. PrPSc differs from PrPC only with respect to conformation is likely more possible as post translational chemical modification is not identified. PrPSc differ from PrPC in many properties among which solubility in detergent is one of them. In the brains of some animals and humans that have died of prion diseases, amyloid plaques are found which contain PrP, as determined by immunostaining and Edman protein sequencing studies. To examine the possibility that formation of PrPSc involves the conversion of alpha-helices in prPC into beta-sheets, a PrPC purification protocol was developed utilizing nondenaturing procedures. All chemicals used were of the highest grade commercially available.
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The method included sacrificing animals at 12 weeks age and using their brains. The process included propagation of prions and production of PrPSc in Syrian hamsters, purification of PrPC, PrP 27-30, centrifugation, denaturing of proteins by boiling in buffer etc. Results obtained are were the following. Fourier-transform infrared (FTIR) spectroscopy demonstrated that PrPC has high alpha-helix content (42%) and no beta-sheet (3%), findings that were confirmed by circular dichroism measurements. In contrast, the beta-sheet content of PrPSc was 43% and the alpha-helix 30% as measured by FTIR. N-terminally truncated PrPSc derived by limited proteolysis, designated PrP 27-30, has more higher beta-sheet content (54%) and a lower alpha-helix content (21%).
Also the results obtained are shown in the graphs and table on page 3. The final step in the PrPC purification utilized lectin chromatography (Fig.1a) Fractions 52-57 (pool 2) eluted from the WGA-Sepharose gave only a single band of 33-35 kDa (Fig.1b, lane c) and bound anti-PrP 13A5 mAb (lane f). The amide I' band of the FTIR spectrum of purified PrPC from pool 2 showed a symmetrical peak with a maximum at 1653 cm-' (Fig. 2, solid line). Such spectra are characteristic of proteins with high alpha-helix content. In contrast, the FTIR spectra of PrPSc (Fig. 2, dashed line) and PrP 27-30 (Fig. 2,dotted line) showed patterns which are characteristic of proteins with high beta-sheet content. Class-dependent and naive predictions were performed (Table1). The alpha/alpha class contains proteins which are composed largely of alpha-helices. Similarly, beta/beta class contains proteins that are mostly beta-sheets. The CD spectrum of PrPc shows a minimum at 208 nm and a shoulder at 222 nm, clearly indicating that the protein contains one or more alpha-helices (Fig. 3)
1)From the results shown in the Table 1, it is concluded that the formation of PrPsc involves the conversion of alpha-helices in PrPC into beta-sheets, but it is also seen that an undetected chemical modification of a small fraction of PrPSC initiates this process. This conformational transition seems likely to be the fundamental molecular event that underlies prion propagation, since many lines of evidence argue that PrPsc is an essential component of infectious prion particles.
2) As the accumulation of PrPsc seems to be responsible for the neurodegeneration found in prion diseases, this leads to the conclusion that the conversion of alpha-helices in PrPC into beta-sheets is likely to be the primary lesion in these illnesses. This assertion is supported by the finding that ablation of the PrP gene is apparently not deleterious to mice; thus, the prion diseases appear to be the result of PrPSc accumulation rather than a PrPc deficiency.
3) As all attempts to produce PrPSC in cell-free systems have been unsuccessful, till date, PrPSc synthesis seems to be a complex process. Metabolic radiolabeling studies with scrapie-infected cultured cells demonstrate that inhibition of protein synthesis during the chase period abolishes PrPSc formation. These findings argue that the continuing synthesis of proteins other than PrPC is necessary for PrPsc production.
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4) According to transgenetic studies in prion propagation inoculated prions containing PrPsc trigger the synthesis of homologous prions from the homologous PrPC.The initial studies were performed with transgenic mice expressing Syrian hamster PrP, similar results have been obtained with a chimeric mouse/ Syrian hamster PrP transgene.
5) Some investigators proposed that scrapie agent multiplication proceeds through a crystallization process involving PrP amyloid formation. PrP amyloid plaques are diagnostic of prion diseases when present, but they are often absent from both humans and animals with such diseases. Also, purified infectious preparations isolated from scrapie-infected hamster brains exist as amorphous aggregates (Fig. 4B); only if PrPSC is exposed to detergents and limited proteolysis does the protease-resistant core of PrPSc (PrP 27-30) polymerize into prion rods (Fig. 4C) exhibiting the ultrastructural and tinctorial features of amyloid .
6) Following are some examples of of other proteins with alpha -helices that are converted into beta-sheets and vice versa. In photosystem II reaction center an alpha-helix transforms to 4 beta-sheet when exposed to light. gp120 from human immunodeficiency virus type I which possesses a beta-sheet in aqueous environments is converted into an alpha-helix when the virus binds to the membrane protein CD4. In Alzheimer disease, amyloid plaques contain 13A4 peptide which is derived from amyloid precursor protein (p-APP) that seems to undergo a similar structural transition. BA4 is high in beta-sheet content) but is proteolytically cleaved from a region of beta -APP, a portion of which is thought to form a transmembrane alpha-helix
I agree with the author who states that transition of alpha helices to beta sheets are featured in the formation of PrPSc which seem to be the fundamental events underlying prion ââ‚¬Å“infection,ââ‚¬Â and the existence of scrapie "strains" remains perplexing. Prion "strains" produce distinct patterns of PrPSc deposition in the brain and often have different incubation times. In the continuing absence of any scrapie-specific nucleic acid, the profound conformational changes which PrPC undergoes during its conversion to PrPSc raise the possibility that the information carried in prion "strains" resides within the conformation of PrPsc. The transitions in PrP structure underscore the concepts that (i) prion diseases are disorders of protein conformation and (ii) prions are a class of infectious pathogens which are different from viruses, viroids, bacteria, and fungi.
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FIG. 4. Electron micrographs of negatively stained and immunogold- labeled PrPs. (A) PrPC. (B) PrPSc. (C) Prion rods composed of PrP 27-30, negatively stained. (Bar = 100 nm.)