Evidence for the Suggestion that the Prion Protein May Function as Metal Binding Protein - Case Study Example

Discussing evidence for the suggestion that the prion protein may function as metal binding protein Discussing evidence for the suggestion that the prion protein may function as metal binding protein Discussing evidence for the suggestion that the prion protein may function as metal binding protein A group of neurodegenerative diseases are linked to the Prion protein or PrP (Hodak et al 2009). The normal function of the prion is not evident yet though we know that it has a copper-binding property. Prion is a harmless neuronal copper-binding protein found in all vertebrates; it causes neuronal death only in its abnormal isoform (Davies, 2008). Normally it is associated with cellular resistance to oxidative stress. It is accepted that prions cause prion diseases (Davies, 2008). The copper-binding property may be linked to this function. The metal-binding property of PrP is being investigated in this paper along with the techniques that are associated with the prion for metal-binding. Studies have suggested two opposing functions for copper bound prions: as anti-oxidants for the neuronal functions and as pro-oxidants increasing the neurodegenerative process (Yokawa et al, 2008). The Human Prion Protein Figure 1 The Amyloid Precursor Protein and Secretase Cleavage Four clear copper binding regions are available on the human prion protein. The seven sites, four in the octarepeat region, the short sequence, neurotoxic region and the helical region are the four regions. Prion Protein is a “GPI anchored glycoprotein having an alpha helical C terminal domain and flexible N-terminal domain” (Stevens, 2009). In neurodegenerative illnesses, a massive accumulation of the scrapie form from the intrinsic cellular form occurs. This scrapie form is a misfolded form of the normal prion protein (Stevens, 2009). However the biochemical process involved is still vague (Yokawa 2009). A report says that the copper bound form of the prion-derived oligopeptides from the four binding sites produce superoxide anions when hydrogen peroxide and neurotransmitters are present, through a process of catalysis. They also catalyze the generation of natural amino-phenols (Yokawa 2009). Micronutrient Copper is essential for neurological function (Stevens 2009). The infection of brain tissues or homogenates with prion is not lost with severe heat treatment or repeated freezing or thawing, hence the severity and fatality of the infection (Yokawa 2009). Hybrid density functional theory is a recent theory which was used to test the copper binding in low concentrations by Hodak et al (2009). At low concentrations 4 histidines residues coordinate the cooper while at higher concentrations of copper, 2 histidine residues are involved. The binding is more when concentrations are higher. Prion diseases are described as fatal neurodegenerative illnesses where neuronal loss together with gliosis occurs after a long duration of incubation period (Thackray, 2002). Prion disease showed a change in the levels of copper and manganese before symptoms appeared. In the early disease, manganese showed a spurt. The analysis of the brains of scrapie –infected mice indicated a reduced copper binding and a decrease in anti-oxidant activity from 30-60 days which are the early changes in the days after the incubation (Thackray, 2002). Thackray and colleagues perceived that changes in the trace element metabolism are the reasons for the pathological features of prion disease. They found that changes in the trace elements could increase the risk of sporadic prion disease. Mice which do not have the prion protein are not infected with the prion inoculum (Thackray, 2002). Mice that belonged to the group of wild type were administered RML mouse scrapie strain. Uninfected mice were also present. Every 30 days, 5 in each group were killed. Freezing the samples of blood, brain, liver and muscle samples on solid carbon dioxide, other samples were collected from mice 2 hours after inoculation; this was considered as zero time point (Thackray, 2002). The terminal stage was 150-160 days after inoculation. Signs of illness began to be noted from the 125th day onwards. The brains of the dead mice were measured or metal levels of copper, manganese, zinc and iron with MS were investigated. The liver showed no changes in the zinc or iron. Blood indicated a minor increase in zinc. Muscle had a minimal increase in iron only in the terminal stages (Thackray, 2002). The brain showed a decrease in copper about 97 days after inoculation. It was elevated in liver from 60 days after. Changes in manganese were more different; they showed increase in blood, brain and muscle from the 60th day. Brain showed a decrease in copper and an increase in manganese. Prion diseases also occur as inherited mutations (Stevens 2009). The octarepeat domain becomes elongated near the N-terminus where micronutrient copper binding occurs. Changes in copper uptake occur depending on the elongation. Elongation beyond a threshold causes a difference in copper uptake and initiates early onset disease (Stevens 2009). Insertional mutations can cause the enhancement of the octarepeat region of the prion, leading to disease (Stevens, 2009). Normal prion protein has 4 octapeptide segments in the N-terminal domain while abnormal forms have shown upto nine octapeptides. The type of disease is dependent on the amount of expansion in the octarepeats. If upto 4 are found, the onset of disease would be around 60. If more are found, age of onset of disease will be 30 or 40 years (Stevens 2009). Biological processes are known to have many divalent metal cations play significant roles in them. PrP is known to bind with many metals: copper, manganese, zinc, nickel and iron. (Davies et al, 2008). Copper is found to bind with the normal isoform of PrP. The copper-binding actually prevents the prion from assuming an abnormal isoform. It also changes the incubation period of disease in animals. Manganese-binding converts the prion into an abnormal isoform (Davies et al, 2008). Zinc and nickel binding occur only in vitro and not in vivo. Different techniques have been used to study the binding of the metals in a physiological state. The metal ions are mediators in the proteins between residues and domains. They also play the role of electron transfer agent and nucleophilic catalyst in active site mechanisms. Interactions and binding features occur between the proteins and metal ions. Prions are distorted forms of the normal host proteins of the body. They are infectious and can produce transmissible brain diseases (Tsenknova, 2004). Creutzfeldt-Jacob disease (vCJD) in human beings and bovine spongiform encephalopathy in cattle are known to be illnesses associated with prions (Wong et al, 2001). Others are the human diseases, sporadic CJD and Fatal Insomnia. Calcium ions bind with recombinant mouse protein producing conformational changes. Increasing concentrations of calcium directly increased the proteins but the fold density decreased to signify the structural change. Copper and zinc are known to produce changes due to the binding property to Prion. Copper binds to the octameric repeat regions which allow the binding of 4 atoms of copper and one more site of binding found later in the N-terminus where two histidine residues present could make this a high affinity site (Davies, 2008). PrP endocytosis occurs, fibril formation is inhibited and intermolecular interactions are increased in the process (Walter et al, 2007). However zinc cannot displace copper already in a bound position. It can only use available binding modes. Walter et al (2007) showed that even large amounts of zinc could not displace copper from the octarepeat region of the prion. When copper levels were low, both copper and zinc were allowed to bind. In high levels of copper, the ratio of histidines to copper was reduced and more zinc was allowed to bind. If the changes did not allow both, it was the zinc that was displaced first (Walter et al, 2007). Ferritin has been known to be involved in the binding with the scrapie prion and being transported across the stomach through the epithelial walls. An extended binding affinity of iron is significant (McDermott, 2005). The binding energy of the iron-prion system was calculated using the density functional theory via the SIESTA code. Manganese causes possible risks to prions in the soil in the environment. Manganese is a risk factor for the survival and transmission of the infectious agent in the prions (Davies and Brown, 2009). In model soils, manganese supports the survival of the prion proteins and thereby increases the infectivity to cells. Brazier and his colleagues (2008) also performed studies for manganese. They found that PrP has an affinity for manganese comparable to other manganese- binding proteins (Brazier, 2008). PrP was found to have two manganese binding sites at concentrations of 63 and 200 mum and at pH 5.5. The binding is at the 5th site where copper also has an affinity for binding (Brazier, 2008). Iatrogenic transmission of the Creutzfeld Jacob prion infection through blood, blood products and instruments in surgery is a major issue in public health policies. Subclinical prion infection in animals is another major problem in public health (Edgeworth et al, 2009). Laboratory animal bioassay is a tedious procedure; the usual sterilization techniques fail in the laboratory. The cell culture –based scrapie cell assay is a faster procedure. A bioassay which elaborates the extensive binding affinity of prions to steel surfaces has been indicated by Edgeworth and his team. A higher sensitivity has been achieved with this method than for mouse bioassays (Edgeworth et al, 2009). Prions bind strongly to metal surfaces and this adsorbed material causes a high level of infection to mice and cultured cells. The infectious agent is tightly bound to the steel wires and sensitive cells become infected too. This can be measured using a SCEPA adaptation. A cell-based assay helps to detect the low prion titres on the metal in a mouse model. The metal wire is brought into contact with the homogenate which has the prion infected tissue (Edgeworth et al, 2009). It is then placed in a tissue culture plate and surrounded by susceptible cells. Some of the cells become infected. The adherent cells are harvested and then placed in amulet-well plate. The wells receive one or more of the cells. The infection is allowed to spread covering all the wells. The cells are filtered off onto a 96-well filtration plate. Proteinase K is used to treat the samples. The cells containing the protease-resistant PrPSc are elicited by an antibody-based assay. The Poisson unit is used to calculate the number of tissue culture infectious units. Comparing the steel in the form of wires and disks, it was found that the steel in the wire form was “richer” in infectivity. Different solutions were compared to decide which was most suitable for the assay of prions in low concentrations. Prnp was found better than “uninfected CD-1 mouse brain homogenate, modified reduced serum Eagles’ Minimum Essential Medium and PBS” (Edgeworth et al, 2009). Nuclear magnetic resonance studies include chemical shift mapping, paramagnetic NMR and changes in the backbone dynamics (Basel, 2007). Copper binding at medium concentrations was one technique of copper binding (Hodak, 2009). Thompsett and his colleagues studied high affinity binding between Copper and full length prion protein identified by different techniques: isothermal titration calorimetry (ITC) and competitive metal capture analysis (CMCA) to decide the affinity of Copper for wild mouse and a string of mutants (Thompsett, 2009). In the isothermal titration calorimetry method, the calorimeter was used to make measurements. The recombinant protein is added to the metal-chelator mix. This is kept at 370C and then separated from the chelator solution by filtration using concentration filters. A spectrophotometric assay was used to assess the copper present in the protein and filtrate. Measurements were accurate when compared to coupled plasma mass spectrophotoscopy (Thompsett, 2009). In CMCA, metal binding was performed in the presence of chelators like tris, glycine, methionine and others all listed in increasing order of affinity for copper. Both the methods of ITC and competitive metal capture analysis showed high femtomolar affinity for copper binding sites (Thompsett, 2009). Prion protein bound to copper has many functions which include signaling, sequestration and anti-oxidant functions. It is also involved in oxidative stress in the cell. The behavior of copper binding to Prion protein is similar to the ability to bind copper at the plasma membrane. The binding of extracellular copper is to the ceruloplasmin and serum albumin and the chelators like aminoacids (Thompsett, 2009). High affinity copper binding helps to protect cells from the harmful effect of prions. It blocks “aggregation, stabilizes the structure, disfavors misfolding and subsequent aggregation” (Thompsett, 2009). Prion disease is not allowed to progress. However if the prion protein is diseased, the result is a metal imbalance with breakdown of copper regulation. This leads to oxidative stress and the pathology of prion disease. The copper management in the extracellular region constitutes the normal physiology of the cell (Thompsett, 2009). In Menke’s disease where the copper is deficient, a copper–histidine chelate forms the most effective treatment. The human prion fragment PrP(106-126) has properties similar to the abnormal isoform PrPSc . This allows the use of this fragment for examining the neurotoxic action of PrPSc (Belosi, 2004). Potentiometry, solution calorimetry, VIS spectrophotometry, circular dichroism, EPR and NMR spectroscopy are the methods used for exploring the complex formation equilibria of PrP(106-126) with the copper ion. One more fragment PrP (106-113) also has similarities to PrP(106-126) but is different in that the hydrophobic C terminal is absent. However it contains all the potential donor groups (Belosi, 2004). Summary Prion is a harmless neuronal copper-binding protein found in all vertebrates; only in its abnormal isoform, it causes neuronal death. Prion diseases are described as fatal neurodegenerative illnesses where neuronal loss together with gliosis occurs after a long duration of incubation period (Thackray, 2002). The metal binding property of PrP is being investigated in this paper along with the techniques that are associated with the prion for metal binding. Copper binds to the octameric repeat regions which allow the binding of 4 atoms of copper and one more site of binding was found later in the N-terminus where two histidine residues present could make this a high affinity site (Davies, 2008). Ferritin has been known to be involved in the binding with the scrapie prion and being transported across the stomach through the epithelial walls. Zinc cannot displace copper already in a bound position. The isothermal titration calorimetry (ITC) and competitive metal capture analysis (CMCA) were used to decide the affinity of Copper for wild mouse and a string of mutants (Thompsett, 2009). Prion protein bound to copper has many functions which include signaling, sequestration and anti-oxidant functions. It is also involved in oxidative stress in the cell (Thompset , 2009). The behavior of copper binding to Prion protein is similar to the ability to bind copper at the plasma membrane. If the prion protein is diseased, the result is a metal imbalance with breakdown of copper regulation. This leads to oxidative stress and the pathology of prion disease. References: Belosi, B., Gaggeli, E., Guerrini, R. Kozlowski, H., Luczkowski, M. and Mancini, F. et al. (2004). Copper binding to the neurotoxic peptide PrP(106-126): thermodynamic and structural studies. Chembiochem, Vol. 5, p. 349-359 Brazier, M.W., Davies, P., Player, E., Marken, F.Viles, J.H. and Brwon, D.R. (2009). Manganese biding to the prion protein. Journal of Biological Chemistry. Vol. 283, p. 12831-12839 Davies, P. and Brown, D.R. (2009). Manganese enhances the prion protein survival in model soils andincreases prion infectivity to cells. PLoS One, Vol. 4, No. 10. Davies, P., Fontaine, S.N., Moulla, D., Wang, X., Wright, J.A. and Brown, D.R. (2008). Amyloidogenic metal binding proteins: new investigative pathways. Biochemical Society Transactions, Vol. 36, Part 6. Biochemical Society. Edgeworth, J.A., Jackson, G.S., Clarke, A.R., Weissman, C. and Collinge, J. (2009). Highly sensitive quantitative cell-based assay for prions adsorbed to solid surfaces. Proc. Natl.Acad. Science, Vol. 106, No. 9. P. 3479-3483. Hodak, M., Chisnell, R., Lu, W. Bernholc, J. (2009). Functional implications of multi-stge copper binding to the prion protein. Proc Natl Acad, Sc., Vol. 106, No. 28, p. 11576-11581 McDermott, D.M. and Davis, U.C. (2005). A density functional approach to iron binding in Prions. The REU Project The SIESTA Manual. Stevens, D.J., Walter, E.D., Rodriguez, A., Draper, D., Davies, P.,Brown, D.R. et al (2009). Early onset prion disease fromm octarepeat expansion correlates with copper binding properties. PLoS Pathogenesis, Vol. 5, No. 4 Thackray, A.M., Knight, R., Bujdoso, R. and Brown, D.R. (2002). Metal imbalance and compromised anti-oxidant function are early changes in prion disease. Biochemistry Journal, Vol. 362, p. 253-258 Thompsett, A.R., Abdelraheim, S.R., Daniel, M. and Brown, D.R. (2009). High affinity binding between copper and full-length prion protein identified by two different techniques. The American Society for Biochemistry and Molecular Biology, Inc. Tsenkova, R.N., Iordanova. I.K., Toyoda, K. and Brown, D.R. (2004). 9th International Symposium on Biochromatography: From nanoseparations to macropurifications. Biochemical and Biophysical Research Communications, Vol. 325, No. 3, p. 1005-1012 Water, E.D., Stevens, D.J., Visconte, M.P. and Milhauser, G.L. (2007). The Prion protein is a combined zinc and copper binding protein: Zinc alters the distribution of Copper coordination modes. Journal of American Chemistry Society, Vol. 129, No. 50, p. 15440-15441. Pubmed Wong, B.S., Chen, S.G., Colucci, M. Xie, Z., Pan, T.and Liu, T. et al, (2001). Aberrant metal binding by prion protein in human prion disease. Journal of Neurochemistry, Vol, 78, No. 6, p. 1400-1408 Yokawa, K., Kagenishi, T. and Kawano, T. (2009). Thermostable nature of aromatic monoamine dependent superoxide-generating activity of human prion-derived Cu-binding peptides, Bioscience Biotechnology Biochemistry, Vol. 73, No. 5, p. 1218-1220 Yokawa,K., Kagenishi, T., Goto, K. and Kawano, T. (2008). Free tyrosine and tyrosine-rich peptide-dependent superoxide generation catalyzed by a copper-binding, threonine-rich neurotoxic peptide derived from prion protein Int J Biol Sci. 2009; 5(1): 53–63. Read More