Prions

The Infectious Proteins

By

Mehana E.E.
Department of Pathology, College of Veterinary Medicine, Alexandria University, Egypt and

Muhammad Raza (Ph.D.)

Department of Pharmacology, College of Medicine, Qassim University, Qassim, Saudi Arabia

Prions: The Infectious Proteins by Dr. Muhammad Raza and Dr. Mehana EE.

I - Introduction
Is an infectious agent composed of protein in a misfolded form (Prion, 2001)? There is - In contrast to all other known infectious agent (Virus, bacteria, fungus, parasites) which must contain nucleic acids either DNA or RNA or both (Rayan etal., 2004 ). They are the etiological agents of diseases like Transmissible Spongiform Encephalopathies in a variety of mammals, including bovine spongiform encephalopathy (BSE, also known as mad cow disease) in cattle and Creutzfield -Jacob disease (CJD) in humans. All known prion diseases affect the structure of the brain or other neural tissue and are currently untreatable and universally fatal (Prusiner, 1998). Prions and prion diseases have been widely discussed in the media in recent years. The interest in prions and prion disease was stimulated by the outbreak of BSE in Europe in the mid-nineteen-nineties (Megan, 2011). In recent years, researchers have discovered not only non-pathogenic prions that play beneficial roles in biology, but they have also found that prions may even act as essential elements in learning and memory (MIT News, 2012). Proteins are the cells workhorses, and they need to fold into complex and precise shapes to their jobs. Prions are proteins that start out normally, but then at some point misfold -rather like an origami swan that comes out looking and acting instead like a vulture. But prions have another characteristic that enables them to wreak havoc. They recruit other, properly folded proteins into misforming along with them; a process Lindquist calls a conformational cascade. In many organisms, this conformational cascade creates long fibers called amyloids. The brains of animals that have died from prions are literally packed with amyloid clumps (MIT News, 2012). Prions are highly resistant to inactivation by heat, UV light and formaldehyde, but they are inactivated by hypochlorite, sodium hydroxide and autoclaving (WWW.Wikipedia.org/wiki/prion). Also, prions are proteins found on the
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plasma membrane (the membrane that surrounds a cell and defines its physical boundary). In mammals, prions are found in the highest concentration in cells of the CNS. In mammals and yeast, there are several genes coding for different prions. The function of normal prions PrPC is unknown. Aberrant or mutant prions (denoted PrPSC) are thought to be the causative agents of neurological disorders, among which are BSE in cows and CJD in humans. The term prion was coined from proteinaceous infectious particles. The term was coined during early research of the sheep disease called Scrapie. During that time, all that was known about the particles that caused Scrapie was that it was a protein and that it was infectious. Prions have the same system of organization as other proteins. They may have up to four levels of organization. The primamary structure of a protein is the amino acid sequence that makes up the protein. The secondary structure of a protein is the local organization of the primary sequence of the proteinhelical or sheet formations. The tertiary structure of a protein is the overall shape of the protein, caused by interactions between the various local structures. Some proteins have a quaternary level of organization, which is defined by the interactions between the tertiary structures of two or more protein subunits (Megan, 2011). Prions have one characteristic that makes them unique: they can exist in two different confirmations at the level of secondary structure. There is an alpha-helical portion of secondary structure in a normal prion that is refolded into beta-sheet formation in an aberrant prion. The primary structure of the aberrant prion remains the same, but its secondary structure is different. As a result, its tertiary structure will be different as well. Infection of normal cells may occur when an aberrant prion act as a template for the refolding of a normal prion into a new aberrant prion. It is thought there is at least one or more protein involved: the as-yet-unidentified protein X. This protein is believed to mediate the folding from a normal into an abnormal prion. When proteins are synthesized inside of a cell there are other special proteins (Known as chaperones) that help in this process. Chaperones are proteins that
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bind to the newly synthesized protein or protein subunit. In order to ensure that the protein is properly folded into its secondary or tertiary structure, it has been hypothesized that protein X is a type of chaperone (Megan, 2011).

Fig. (1&2) A normal prion (left), compared to an aberrant, disease-causing prion (right)

Prions: The Infectious Proteins by Dr. Muhammad Raza and Dr. Mehana EE.

II - History
During the 1960s radiation biologist Tikvah Alper and mathematication John Stanley developed the hypothesis that some transmissible spongiform encephalopathies are caused by an infectious agent consisting solely of proteins (Prusiner, 1998). It is believed that the origin of the BSE outbreak in Europe was due to aberrant sheep prions crossing the species barrier to infected cows .During the 1970s there were changes made to the process used to render offal for a livestock feed additives. These changes created conditions that allowed prions to survive the rendering process (Megan, 2011). The sheep disease known as Scrapie is a prion disease. It is believed that prions from Scrapie - infected sheep survived the rendering process and were passed on to cows that ingested infected feed. The practice of using rendered offal as an additive to livestock feed has been discontinued (Megan, 2011). Francis Crick (1970) recognized the potential importance of the Griffith protein - only hypothesis for Scrapie propagation in the second edition of his Central dogma of molecular biology: while asserting that the flow of sequence information from protein to protein, or from protein to RNA and DNA was precluded. In 1982, Stanley B. prusiner of the University of California, San Francisco announced that his team had purified the hypothetical infectious prion, and that the infectious agent consisted mainly of specific protein - though they did not manage to isolate the protein until two years after Prusiners announcement (Taubes, 1986). While the infectious agent was named a prion, the specific protein that the prion was composed of is also known as the prion protein (PrP), though this protein may occur both infectious and non infectious forms. Prusiner was awarded the Nobel Prize in physiology or Medicine in 1997 for his research into prions (The Nobel prize in physiology, 1997).

Prions: The Infectious Proteins by Dr. Muhammad Raza and Dr. Mehana EE.

III - Mechanism of prion-inducing diseases

Prions propagated by transmitting a misfolded protein state. When prion enters a healthy organism, it induces existing, properly folded proteins to convert into the disease associated, prion form ; the prion acts as a template to guide the misfolding of more proteins into prion form (Aguzzi, 2008). These newly formed prions can then go on to convert more proteins themselves; this triggers a chain reaction that produces large amount of the prion form. All known prions induce the formation of an amyloid fold, in which the protein polymerises into an aggregate consisting of tightly packed beta sheets. Amyloid aggregates are fibrils, growing at their ends, and replicating when breakage causes two growing ends to become four growing ends (Aguzzi, 2008). The incubation period of prion diseases is determined by the experimental growth rate associated with prion replication, which is a balance between the linear growth and the breakage of aggregates. All known mammalian prion diseases are caused by the so-called prion protein, PrP. The endogenous, properly folded, form is denoted PrPC while the diseaselinked misfolded form is denoted PrPSc (for Scarpie disease, after one of the disease first linked to prions and neurodegenerative (Krull etal., 2004 and Lauren etal., 2009). Proteins showing prion-type behavior are also found in some fungi, which have been useful in helping to understand mammalian prions (Lindquist et al., 2001).

IV - Prion Diseases
All known prion diseases, collectively called transmissible spongiform encephalopathies (TSEs), are untreatable and fatal (Gilch etal., 2001). Prions cause neurodegenerative disease by aggregating extracellular within the CNS to form plaques known as amyloid, which disrupt the normal tissue structure. This disruption is characterized by holes in the tissues with resultant spongy architecture due to the vacuole formation in the neuron (Robbins etal., 1999). Other
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histological changes include astrogliosis and absence of an inflammatory reaction (Belay, 1999). While the incubation period for prion diseases is generally quite long; once symptoms appear the disease progress rapidly, leading to brain damage and death (Prion Diseases, 2006). Also, the neurodegenerative symptoms can include convulsion, dementia, ataxia (balance and coordination dysfunction), and behavioral or personality changes. Many different mammalian species can be affected by prion diseases, as the prion protein (PrP) is very similar in all mammals (Collinge, 2001). Due to small differences in PrP between different species it is unusual for a prion disease to be transmitted from one species to another. The human prion disease variant (Creutzfeld-JaKob disease), however, is believed to be caused by a prion which typically infects cattle, causing bovine spongiform encephalopathies and is transmitted via infected meat (Ironside, 2006).

Fig. ( 3 ) : Abnormal protein

Fig.( 4 ) : Status spongiosus

Prions: The Infectious Proteins by Dr. Muhammad Raza and Dr. Mehana EE.

Fig. ( 5 ) : Bovine spongy-form encephalopathy brain

Example of TSE: Scrapie Disease

Disease of sheep, domestic goats called Scrapie due to the characteristic clinical signs, Purities which often results in loss of wool in sheep.

Clinical signs
Purities and loss wool In coordination Recumbency Eventually death

Incubation period: Several months to 4-5 year. Characters of the disease

Not induce gross lesions in CNS or generally nervous system Long incubation period Lack of typical immune responses
8 Prions: The Infectious Proteins by Dr. Muhammad Raza and Dr. Mehana EE.

Grossly
Loss of body condition Skin purities No gross in CNS

Microscopic
Limited to CNS (diencephalons, brain stem and cerebellum) Neuronal degeneration and chromatolysis, Astrogliosis and gray matter spongiosus including shrinkage with increase basophilia Amyloid plaque (cerebrovascular amyloidosis) Astrocytosis Gray matter spongiform ---Vacuolation of neurons and dilatation of N. process Suffolk sheep naturally infected with disease

V - Transmission
It has been recognized that prion diseases can be arises in three different ways: acquired, familial, or sporadic (Groschup and Kretzschmar, 2001). It often assumed that the diseases form directly interacts with normal form to make it rearrange its structure. The primary method of infection in animals is through ingestion. It is thought that prions may be deposited in the environment through the remains of dead animals and via urine, saliva, and other body fluids. They may then linger in the soil by binding to clay and other minerals (Johnson et al,. 2007).

VI - Sterilization
Sterilizing prions therefore involves the denaturationof the protein to a state where the molecule is no longer able to induce the abnormal folding of normal proteins. Prions are generally quite resistant to proteases, heat, radiation, and formalin treatment (Qin etal., 2006), although their infectivity can be reduced by such treatments. Effective prion decontamination
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relies upon protein hydrolysis or reduction or destruction of protein tertiary structure. Examples include bleach, caustic soda, and strongly acidic detergents such as LPH. Temperature as high as 134C (274 F) for 18 minutes in a pressurized steam autoclave may not be enough to deactivate the agent of disease (Collins etal., 2004). Ozone sterilization is currently being studied as a potential method for prion denaturatioin and deactivation (Ozone Sterilization, 2008). The World Health Organization recommends any of the following three procedures for the sterilization of all heat-resistant surgical instruments to ensure that they are not contaminated with prions. Immerse in a pan containing 1N NaOH and heat in a gravity displacement autoclaved at 121C for 30 minutes; clean; rinse in water; and then perform routine sterilization processes. Immerse in 1N NaOH or sodium hypochlorite (20000 ppm available chlorine) for 1 hour; transfer instruments to water; heat in a gravity-displacement autoclaved at 121C for 1 hour; clean; then perform routine sterilization process. Immerse in 1N NaOH or sodium hypochlorite (20000 ppm available chlorine ) for 1 hour; remove and rinse in water , then transfer to an open pan and heat in a gravity displacement (121C ) or in a porous load (134C ) autoclave for 1 hour; clean; and then perform routine sterilization processes (Sutton etal., 2006 ) .

VII - Potential treatments

There are some compounds which can serve as a treatment for prion-caused diseases, such bas one compound found to bind a cavity in the PrPC and stabilize the conformation, reducing the amount of harmful PrPsc (Kuwata etal., 2007). Recently, antiprion antibodies capable of crossing the blood -brain barrier and targeting cytosolic prion protein have been described. In the last decade, some progress has been reported dealing with ultra-high

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Prions: The Infectious Proteins by Dr. Muhammad Raza and Dr. Mehana EE.

pressure inactivation of prion infectivity in processed meat. In 2011 it was discovered that prions could be degraded by Lichens (Yam, 2011).

References
Aguzzi A (2008): Unraveling prion strains with cell biology and organic chemistry. Proceedings of the National Academy of Sciences of the United States of America 105(1): 11-13. Belay ED (1999): Transmissible spongiform encephalopathies in humans. Annual Review of Microbiology 53: 283-314. Collinge J (2001): Prion diseases of humans and animals: their causes and molecular basis. Annual Review of Neuroscience 24:519-50. Collins SJ; Lawson VA and Masters CL (2004): Transmissible spongiform encephalopathies. Lancet 363(9402): 51-61. Crick F (1970): Central dogma of molecular biology .Nature 227(5258): 561-3. Ironside JW (2006): Variant Creutzfeld Jakob disease: risk of transmission by blood transfusion and blood therapies. Haemophilia: The Official Journal of the World Federation of Hemophilia 12 Suppl 1:8-15. Gilch S ; Winklhofer KF and Groschup MH (2001) : Intracellular re-routing of prion protein prevents propagation of PrP(SC) and delays onset of prion disease .The EMBO journal 20 (15): 3957-66. Groschup MH and Kretzschmar HA (2001): Prion diseases diagnosis and pathogenesis. Archives of Virology. Suppl. 16. Johnson CJ; Pedersen JA Chappel etal., (2007): Oral transmissibility of prion disease is enhanced by binding to soil particles. PLoS Pathogens 3(7): e93. Krull IS; Brian K and Nunnally S (2004): Prions and mad cow disease .New York, N.Y.: Marcel Dekker .PP.6. Kuwata K; Nishida N and Matsumoto T (2007): Hot spots in prion protein for pathogenic conversion. Proceeding of the National Academy of Science of the United States of America 104(29):11921-6.
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Lauren J; Gimbel DA and Strittmatter SM (2009): Cellular prion protein mediates impairment of synaptic plasticity by amyloid-beta oligomers. Nature 457(7233): 1128-32 Lindquist S; Krobitsch S and Sondheimer LI (2001): Investing protein conformation based inheritance and disease in yeast .Biological Sciences 356(1406): 169-76. Megan S (2011): Prions: Infectious proteins Responsible for Mad Cow Disease. The Science Creative Quarterly .6: 1-6. MIT News (2012): Prions misfold and recruit others to follow suit .David Cameron, Whitehead Institute todays news .1-5. Ozone Sterilization (2008): UK Health Protection Agency .Archived from the original. 5-22. Prion (2001): Oxford English Dictionary (3rd ed) Oxford University Press. Prion Diseases (2006): US centers for Disease Control .26-1-2006. Prusiner SB (1998): Prions Proceeding of the National Academy of Science of the United States of America 95(23): 13363-83. Qin K; Donnell MZ and Zhao RY (2006): Doppel more rival than double to prion. Neuroscience 141(1): 1-8. Rayan KJ; Ray CG etal., (2004): Sherris Medical Microbiology (4th ed.) .McGraw Hill .PP.624-632. ISBN 0-8385-8529-9. Robbins SL; Cortan RS and Kumar VC (1999): Robbins pathologic basis of disease .Philadelphia: Saunders. ISBN 0-7216-7335-X. Sutton JM; Dickinson J and Raven JT (2006): Methods to minimize the risks of CFJD transmission by surgical procedures: where to set the standard .Clinical infectious Diseases 43(6): 75764. Taubes G (1986): The game of name is fame. But is it science? Discover 7(12): 28-41. The Nobel prize in physiology (1997): Nobel prize.org WWW.Wikipedia.org/wiki/prion Yam P (2011): Natural born prion killers: Lichens Degrade Mad Cow Related brain pathogens. 2011-05-19.

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