Molecular Characteristics

Fate and Transport in the Environment

Methods for Monitoring in the Environment

Exposure Pathways

Methods to Measure Human Exposures

Preventing and Controlling Exposures

Harmful Effects

Organ Site of Toxicity

Absorption and Distribution



Prion Metabolism and Toxicity

Risk Assessment


Prion Metabolism and Toxicity

The prion related protein (PrPc) is a precursor of the prion protein(PrPsc). The PrPc undergoes post-translational events to become the expressed protein(22):

  1. Glycosylation
  2. Formation of a disulfide bond between cysteine residues
  3. Removal of the N-terminal signal peptide
  4. Removal the C-terminal hydrophobic segment
  5. Addition of a phosphatidylinositol glycolipid at the c-terminal

The PrPsc differs from the PrPC protein in a at least a single amino acid residue. Residue 178 contains an asparagine residue in the PrPSC whereas the PrPC has an aspartate residue. Other amino acid substitutions are linked to specific TSE strains. This amino acid substitution is believed to cause a permanent conformational change from alpha helices to beta sheets.

Current evidence also suggests that another protein or external factor may facilitate beta sheet formation.(6) Recent transgenic studies in mice suggest that at least one host factor other than PrPc, tentatively termed Factor X, might be involved in susceptibility to infection.(7) Factor X could be a molecular chaperone that binds to PrPc and assists in altering its conformation.

The most prevalent current prion theory states that the normal protein, PrPc, is converted into its abnormal and infectious form, PrPsc, as a result of a genetic point mutation.(1) Physical measurements have demonstrated dramatic conformational differences in these PrP forms.(2) The PrPc form is approximately 40% alpha helix, with little beta sheet.(3) The PrPsc form contain ~50% beta sheet and only 20% alpha helix.(4) The normal PrP protein may be necessary in preventing neuronal cell death that may occur as a result of excitotoxic mechanisms(5).

There are two proposed models on how the normal protein is converted into the infectious. The fist hypothesis is called the Heterodimer Model. In this model a single PrPsc makes contact with a PrPc and causes the conformational change and then the two infectious units separate. The second hypothesis is called the Seed Polymerization Model. In this model an aggregate of PrPsc molecules come in contact with a PrPc, convert the protein to PrPsc and the aggregate continues to grow. The units do not separate. Evidence supports the Seed Polymerization Model. (22)

Proteinase K is the enzyme which acts on PrPc to digest it into single amino acid units. This enzyme cannot completely digest the PrPsc and thus it is often referred to as PrP-res (res indicating resistant). The Proteinase K can only remove the first approximately 60 amino acids (57-67 in humans) from the N-terminal of the protein (22). Because the enzyme cannot completely digest the PrP-res it has a half-life of greater than 48 hours compared to the 3-6 hours for the normal cellular protein (22). The cellular protein, PrPc, is usually located on the cell surface and anchored to the plasma membrane. The PrP-res, on the other hand, accumulate intracellularly and cause vacoules in the cells, or accumulate extracellularly and cause amyloid plaques (22).

TSE strains appear to be caused by the truncation of PrP-res in conjunction with amino acid substitutions. Different strains appear to have different chain lengths, and these lengths appear to be transmittable from one species to another. A good example of this is nvCJD compared to BSE and CJD.

The Emerging Organophosphate Hypothesis

An alternative hypothesis to the current meat and bone meal/BSE theory, states that the external factor initiating the conversion of PrPc to PrPsc may be phosmet, an organophosphate insecticide.(6) The use of phosmet on cows was compulsory in the U.K. in the 1980's to eradicate the Warble fly. Phosmet was poured along the backbone of cows in high doses (20mg/Kg).(6, 8) The proponents of this organophosphate theory oppose the theory that Bovine Spongiform Encephalopathy (BSE) arose from scrapie and spread through infected meat and bone meal cattle feed. The organophosphate theory is based on a chemical source of disease, as opposed to an infectious source, and that phosmet is at least partially responsible for the BSE epidemic.

Organophosphates act on cells by covalently binding, phosphorylating, and inactivating nerve membrane proteins, particularly acetylcholinesterase.(6) Phosmet has lipophilic properties that allow it to penetrate cell membranes and biological barriers to the central nervous system. Phosmet is the only systemic organophosphate containing phthalimide, the active ingredient of thalidomide. The toxic metabolites of thalidomide are capable of mutating membrane proteins such as PrPc.

Phosmet exposure may be the direct result of chemical treatment, or it may result indirectly through the bioconcentration of phosmet recycled back through feeding contaminated meat and bone meal.(9) This theory proposes that phosmet causes a mutatory mechanism which disrupts normal PrPc synthesis. This leads to PrPc misfolding and the production of a mutant infectious form of PrPc.

Several studies have demonstrated that the same areas of the CNS are targeted by organophosphates as in transmissible spongiform encephalopathies (TSE's).(9) The covalent modification of PrPc may disturb the CNS feedback cycle that regulates the influx of calcium into the nerve cell.(6) The resulting increased influx of calcium may lead to the excessive release of nitric oxide free radicals into the neuron, which could then interact with sites on PrPc and lead to impairment of the folding process.

Misfolding PrPc isoforms attract chaperone stress proteins to bind them, which result in the partial protease resistant PrPsc.(6) Chaperones are vital in stabilizing a protein's structure. Normally the chaperone will be released so that the protein can be denatured. The chaperones that bind to the PrP isoform cannot be released, making PrPsc protease resistant.

As nitric oxide free radicals impair the normal folding process, PrPc is no longer able to mediate the uptake of calcium. This then leads to greater production of nitric oxide free radicals and further impairment of the normal folding process. The excessive uptake of calcium into the cell has been shown to result in a neurotoxic response with the formation of spongiform vacuoles, which are characteristic of TSE pathology.(6, 9, 10)

A related theory attempts to explain the prion diseases that result in a longer time to onset. It is proposed that in-utero exposure to phosmet may alter the conformational shape of PrPc, resulting in the disturbance of the cell adhesion system.(6, 11) This change is more subtle and prion disease would develop years later, rather than immediately after acute exposure.

The most recent theory suggests that it is the copper chelating action of phosmet that indirectly causes the misfolding of the prion protein.(6, 12) Copper 2+ is a co factor for PrPc and helps anchor the protein to the membrance.(22) In in-vitro experiments copper ions can also help the PrPc refold if it has been denatured or partially unfolded.(22) Copper chelation may occur during phosmet metabolism, leading to a depleted supply of copper ions in the CNS. The copper domain on the PrPc is deprived and is then open to manganese invasion. The invasion of manganese ions may be capable of transforming PrPc to PrPsc. The problem may be made worse by the phosmet induced up-regulation of misfolded PrP at cell surfaces.(13) During the period when cattle were being treated with phosmet, they were fed a manganese-rich chicken manure-based feed supplement.(9, 12, 14, 15) The resulting copper deficiency and rich manganese diet is believed by some to be the key to the origin of BSE in British cattle. The subsequent feeding of meat and bone meal from cattle contaminated with organophosphate and/or manganese-modified prion protein may have caused the spread of the disease to other cattle.

A copper deficiency may predispose cattle and humans to PrPc transformation and misfolding. This theory states that the BSE variant in humans (vCJD) is caused by a low copper and high manganese content in soil and food consumed by humans.(14) When PrPc binds manganese instead of copper it becomes more protease resistant over time and loses superoxide dismutase activity.(22) It is believed that excess manganese entered the soil by the use of sewage sludge as fertilizer and manganese-based pesticides. A group of researchers have concluded that PrPc plays a role in copper metabolism or transport and that disturbance of this function may be involved in prion-related neurotoxicity.(16)

In conclusion, phosmet may have caused an initial mutation of PrPc and a reduction in copper ions in the CNS. Excessive amounts of calcium may be taken into the cell as a result of the impaired ability of PrPc to regulate calcium levels. The resulting production of free radicals may lead to even greater misfolding and altered PrPc. Copper is also necessary for the normal folding process. The presence of phosmet, free radicals, and the lack of copper may lead to misfolding and PrPc conformation changes. The uptake of manganese in place of copper may produce symptoms characteristic of manganese toxicity (slow movement, tremor and/or impaired postural reflexes, and spasm).(14, 17)

References and Additional Information:

1. Prusiner SB. Molecular Biology of Prion Diseases. Science. 1991;252:1515-1522.
2. Horwich AL, Weissman HS. Deadly conformation - protein misfolding in prion disease. Cell, 1997;89:499-510.
3. Pan KM, Baldwin M, Nguyen J, et al. Conversion of alpha-helices into b-sheets features in the formation of the scrapie prion proteins. Proceedings of the National Academy of Science, USA, 1993;90:10962-10966.
4. Safar J, Roller PP, Gajdusek DC, Gibbs CJ. Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity. Protein Science, 1993;2:2206-2216.
5. Sakaguchi S, Katamine S, Nishida N, et al. Loss of cerebellar Purkinje cells in aged mice homozygous for a disrupted PrP gene. Nature, 1996;380:528-531.
6. from 2002.
7. Telling GC, Scott M, Mastrianni J, et al. Prion propagation in mice expressing human and chimeric PrP transgenes implicates the interaction of cellular PrP with another protein. Cell, 1995;83:79-90.
8. Andrews AH. Abnormal reactions and their frequency in cattle following the use of OP warble fly dressings. Vet Record 1981;109:171-175.
9. Purdey M. High-dose exposure to systemic phosmet insecticide modifies the phosphatidylinositol anchor on the prion protein: the origins of new variant transmissible spongiform encephalopathies? Medical Hypotheses 1998;50:91-111.
10. Purdey M. The UK epidemic of BSE:Slow virus or chronic pesticide initiated modification of the prion protein. (Pts 1 and 2). Medical Hypotheses 1996; 46:429-454.
11. Hope J. Update BSE. Journal of Biological Education 1990; 24:225-228.
12. Purdey M. Ecosystems supporting clusters of sporadic TSEs demonstrate excesses of the radical-generating divalent cation manganese and deficiencies of antioxidant co-factors Cu, Se, Fe, Zn. Medical Hypotheses, 2000;54(2):278-306.
13. Prusiner S.B. Prions: Proceedings of the National Academy of Science, 1998;95:13370.
14. Dresher WH, Greetham G, Harrison BJ. A case for the role of copper deficiency in Mad-Cow disease and human Creutzfeldt-Jakob disease. Accessed November, 2001.
15. Purdey M. Are organophosphate pesticides involved in the causation of bovine spongiform encephalopathy (BSE)? Hypothesis based upon a literature review and limited trials on BSE cattle. Journal of Nutritional Medicine, 1994;4:43-82.
16. Jackson GS, Murray LL, Hosszu N, Gibbs JP, et al. Location and properties of metal-binding sites on the human prion protein. Proceedings of the National Academy of Science USA, 2001;98(15):8531-8535.
17. Finley JW, Davis CD. Manganese deficiency and toxicity: are high or low dietary amounts of manganese cause for concern? Biofactors, 1999;10:15-24.
22. Caughey, Byron. Advances in Protein Chemistry. Volume 57 Prion Proteins. 2001.

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