Prions : The Infectious Protein Agent

Prions : The Infectious Protein Agent
What causes Mad Cow Disease? Prions. Prions are also behind other neurodegenerative diseases such as the Creutzfeldt-Jakob disease, Kuru, Gerstmann-Straussler-Scheinker disease and some forms of fatal insomnia. These are all prions diseases that have been found to exist in humans. The prion disease for cattle is what we know as the Mad Cow Disease. Prions also exist in other animals such as sheep, mink, mule deer, elk, cats, and some others.
So what?s so special about prions? Unlike other neurodegenerative diseases that are caused by the misfolding of proteins, altered proteins, abnormal gene splicing, improper expression, or ineffective clearing of proteins which slowly leads to disease by accumulation, prions cause disease by acting as an infectious agent. One abnormal prion protein is enough to turn all the normal prion proteins present into itself.
How do prions do that? Scientists are still unsure of how exactly one protein is capable of turning another protein into itself. Many experiments are conducted to help shed light on its mysterious capabilities. In this website, we hope to explain one of these experiments that involved the effect of pH on the structure of prion proteins.

Wait?Prions? Prion Protein? Which is which? Prions is the name assigned to infectious protein agents. Prion protein (PrPC) is the normal cellular protein that can become an infectious agent.

The Official Mad Cow Disease Home Page The prion is a newly discovered pathogen that is vastly different from the known pathogens of today; namely viruses and bacteria. Unlike the bacteria, no antibiotics can cure prions. They are not typical of a prokaryotic organism or a eukaryotic organism, all that is present in this pathogen is the protein PrPSc. This is the mutated form of the protein PrPC, which is encoded by a chromosomal gene. These two proteins differ in their spatial protein structures and their susceptibility to enzyme digestion. PrPC is completely destroyed in enzyme digestion, whereas PrPSc is resistant to any form of digestion.Viruses usually have nucleic acid, protein, and other constituents that aid in the creation of more progeny viruses. As far as prions are concerned, they multiply by infecting the PrPC protein and turning it into a complex such as itself, the PrPSc protein. Prions exist in multiple molecular forms whereas viruses exist in a single form with distinct ultrastructural morphology.Another difference between the virus and the prion is that viruses almost always provoke an immune response in the host that it is infecting. Prions on the other hand, are non immunogens. Prions are not easy to decompose biologically. Not only are they resistant to the digestive enzymes of a typical mammalian host, they are also resistant to high temperatures and disinfectants. Prions can survive even when heated to 100 degrees Celsius, and even the most stringent disinfectants used in hospital operating rooms cannot phase this dangerous mutated protein. Catrogenic Creutzfeldt-Jakob Disease(cjd), a prion disease that affects humans, is most often contracted through surgical tools that were contaminated with the pathogen. Since the disinfectants cannot decompose the PrPSc complex, any materials used on the patient that were contaminated with PrPSc would infect them.

It has been observed in vitro, or in the laboratory, that PrPSc is directly linked to all prionic diseases. It is highly prevalent in any living organism that has a prionic disease, no matter what kind it is, and PrPSc is even thought to be the main cause, not only the indicator, of these pathogenic diseases. Once the PrPSc starts causing the PrPC to be converted into other PrPSc proteins, the process continues exponentially and irreversible brain damage results. The increase in PrPSc is directly related to the increase of infectivity of the host. Although prions do not have as frightening of a reputation as viruses, they have a much higher fatality rate in the diseases that they cause, survive under harsher conditions, and go for longer periods of time undetected. These aforementioned reasons cause them to be much more dangerous to mammals in the rate of infectivity, the treatment of infectivity, and the survival rates of the victims which have been infected.

Where did the name PrPSc come from?
The Sc suffix of the PrP prion abbreviation, which denotes the mutated form of the normal PrPC protein evident in the human body, comes from the name ?Scrapie?. Scrapie is a prion disease that affects sheep and goats. It is widespread in Europe, the Middle East, and the United States, and interestingly absent in New Zealand and Australia.The earliest records of this disease date back to the 18th century. The name Scrapie comes from the tendency of diseased sheep to scrape off their wool. Other symptoms include difficulty in walking, and the loss of muscle coordination in the infected animal.This is why Scrapie is also known as ?trotting disease? in various other parts of the world, especially in the United Kingdom.

Bovine Spongiform Encephalopathy (bse) was first described in the UK in 1985 as a prion-based disease that affected cattle. In 1996 it was first detected in a human being. The main suspect at the time, which turned out to be correct, was that humans were contracting the disease from eating cows that had been infected with the bse disease. This disease creates mad cow disease in human beings, and always results in fatality. In humans, it is a form of cjd, a human harbored prion disease. After the cattle are infected, it takes three to six years before clinical symptoms start to develop. Some of these symptoms include a decrease in milk production, shaking, and timidness. The cows infected have ranged in age from 20 months to 15 years, so there is no specific age group of cattle that contract this disease. There are three phases of bse in cattle. The first phase is characterized by a low infectivity rate, and the cow does not pose a large threat to humans at this level. In the second phase, symptoms are not evident, but the infectivity level is very high. The prion agent is abundant in both the spinal chord and the brain. A cow in this level is the main risk to public health because it shows no signs of being sick, yet it is highly infected with bse, and if consumed by a human, the human would also contract the sickness. The third phase is characterized by the beginning of clinical symptoms, and then death follows shortly after the appearance of the symptoms. bse is the main cause of cjd in human beings.

Creutzfeldt-Jakob Disease (cjd) is a rare and fatal neurodegenerative condition that occurs in humans world wide. It is classified into two forms, classic cjd and variant cjd. Classic cjd can be transmitted to other species, however other animals cannot carry it. The first and most common case of classic cjd is called sporadic cjd. It accounts for over 85 percent of the classic cjd cases, and is most common in individuals between the ages of 50 and 75. It is characterized by a rapidly increasing dementia in its victims. Studies have shown that approximately one million cases are discovered per year world wide, and the cause of this disease, besides the prion that characterizes it, is unknown. Another kind of classic cjd is catrogenic cjd. This accounts for less than five percent of the documented cases of classic cjd per year. It results from the transmission of the caustic prion via medications and surgical equipment. The medications that have had high levels of cjd related to them are medications for growth hormone treatment. Because of the resistance of PrPSc to sterilization methods, the disease can be transmitted by any surgical equipment or medical tools that have been exposed to the PrPSc protein. Variant cjd, the other classification of cjd, was first identified in 1996.Variant cjd is thought to be caused by the consumption of bse infected meat products. This disease is more commonly known as mad cow disease, and the symptoms include depression, involuntary muscle contractions, and impaired muscle coordination. In contrast to classic cjd, variant cjd seems to affect mostly young patients. The first 10 cases of variant cjd were observed in 1996, ten years after the outbreak of bse in the UK.

Other known prion diseases:

Gerstmann-Straussler-Scheinker Disease
Gerstmann-Straussler-Scheinker Disease, also known as gss, is another prion-derived disease evident in humans. Although this disease is rare, it is one of the only predominately hereditary prion-based diseases. It is characterized by impaired muscle coordination and dementia. It is associated with a mutation in the gene code for the normal human prion protein, PrPC. Death follows the symptoms within 2 to 6 years. Fifty families with gss mutations have been identified to date.

Kuru, one of the most interesting prion diseases ever documented, is transmitted through certain cannibalistic rituals amongst the Fore people in Papua, New Guinea. It is contracted through the consumption of deceased human brains. In this culture, the women and children are left with the brains because the men get the tastier parts of the human being. Therefore, women and children are mostly the victims of this disease. The incubation time can last up to thirty years, but once the symptoms become apparent, the disease progresses very rapidly. Death occurs within three to twelve months of the first symptom. As a result of this disease, cannibalism has been abolished in New Guinea, causing Kuru to virtually disappear.

Chronic Wasting Disease
Chronic Wasting Disease, or cwd, occurs in elk and deer in North America. It was first observed in the late 1960?s, and has become more prominent with time. Common symptoms of this disease include emaciation, excessive salivation, and behavioral changes in the infected animals. In some parts of the United States, up to five percent of the hunter-harvested animals are infected with this disease.

This experiment, testing for the effects of pH on the structure of the normal prion protein, was done using
Molecular Dynamics Simulations

A little introduction?
- mds calculates the time dependent behavior of a molecular system
- mds provides detailed information of the fluctuations and conformational changes of proteins and nucleic acids
- routinely used to investigate the structure, dynamics, and thermodynamics of biological molecules and their complexes
- permits the complex study of molecular movement that occurs in a biological system. The aspects depicted and looked at are the following:
?protein stability
?conformational change (extremely relevant in our topic)
?protein folding
?molecular recognition (proteins, dna, membranes, complexes)
?ion transport in biological systems
?drug design
?structure determination
?geometry ? bond angles/lengths
?energy ? heat of formation, and activation energy
?electronic properties ? charges, ionization, electron affinity
?bulk properties ? volume, surface areas, diffusion, viscosity

In this experiment, Syrian hamster prion structures in a water box were subjected to mds at low and neutral/high pH concentrations.

Some historical background?
-first introduced by Alder and Wainwright in the 1950?s to study the interactions of hard spheres
-next advance was in 1964, Rahman carried out the first simulation using a realistic potential for liquid argon
-first protein simulations in 1977, simulation of the bovine pancreatic trypsin inhibitor
-in the present day, they are of solvated proteins, protein dna complexes, and lipid systems

The statistical mechanics?
Molecular dynamics combines energy calculations from force field methodology with the laws of Newtonian mechanics. The simulation is performed by numerically combining Newton?s equations of motion of small fractions of time. The simulation begins when the properties and force vectors are assigned to each atom. From this point the calculations dive into deep mathematics and is beyond my understanding. However, the basic interpretations and uses of the statistical mechanics are listed below:
-generates information at the microscopic level of atomic positions and velocities
-allows for conversions between microscopic information to something that is macroscopic observable (i.e. pressure, energy, and heat capacities)
-the connection between microscopic simulations and macroscopic properties is made via statistical mechanics
-provides mathematical expressions that relate macroscopic properties to the distribution and motion of the molecules
-enables us to study thermodynamic properties and/or time dependent (kinetic) phenomenon
-allows the studies of macroscopic systems from a molecular point of view
-the goal is to understand and predict macroscopic phenomena from the properties of individual molecules making up the system
-an experiment is usually made on a macroscopic sample that contains an extremely large number of atoms or molecules sampling an enormous number of confirmations
-an ensemble average is an average taken over a large number of replicas of the system considered simultaneously

Some miscellaneous information?
-MD simulations in their simplest form involve solving Newton?s equations of motion for a system of atoms by numerical integration
-structures can be presented in 3D on a screen

The results of the Molecular Dynamics Simulations on the Syrian hamster prion structure in low and neutral pH concentrations are as follows:

The two graphs below show that as time passes, measured in nanoseconds, the movement of the alpha-carbons remain relatively low for neutral pH whereas during low pH, there is a larger degree of movement or deviation from the orginal structure.
Graph a shows the average deviations of all the molecules over time(ns)

Graph b shows the deviations for each alpha-carbon of each amino acid or residue

The deviation results above can be better understood when looking at a model of the PrPC at neutral pH and its unraveling at low pH
(The labels above correspond to the labels of the residues in graph b)

The following diagram depicts the snapshots of the secondary structures of the PrPC every 2 ns. It reveals that as time passes in low pH, the strands of the beta sheet, the red arrows, increase from only two strands to five strands. The alpha helix, highlighted in blue, also decreases in size. Increase in beta strands, thus the extension of the beta sheet, and the decrease of alpha helices is consistent with the structure of the abnormal PrPSc.
Not only through diagrams of the molecule is this evident, but also through a statistical count of the number of amino acids in alpha helixes and beta sheets. Through the graphs below, the same phenomenon can be seen. In neutral pH, there are high amounts of alpha helices and a good number of extended beta sheets. At low pH however, the amount of alpha helices decrease, and the number of extended beta sheets increases.
These results suggest that in low pH, or a more acidic environment, normal PrPC is capable of changing into and taking on the structural characteristics of abnormal PrPSc.

The main distinguisher between the two proteins is the ratio of their alpha helices and beta strands. The normal protein, (PrPC), has three alpha helices and two short beta strands. The PrPSc protein, on the other hand, has two alpha helices and more beta strands. Many things could cause the pH to drop down between pH 4 and 6. However, the connection between pH and prion diseases is still unclear. It was made clear that there is a connection between more acidic pH?s and the transformation of normal proteins into neurodegenerative proteins, however it was still not clear what the role of acidic pH is in real prion diseases. It was also not known if this transformation occurred before the first PrPC to PrPSc mutation. Does the pH level cause the prion disease, or does the prion disease cause the pH conditions? If the prion disease causes the change in pH, is this later in the degenerative disease in order to continue the conversion process between PrPC and PrPSc, or is it right from the beginning, and a direct result right when the first conversions are made??? Prion diseases can either be sporadic, inherited, or infectious. The infectious prion diseases could not be caused by pH levels, they would have to be caused by the prion they were introduced to via the host. The other types, sporadic and genetic, would be more likely candidates to have pH play a direct role in their development. The pH could cause the mutation in the sporadic case. In the inherited case, elevated pH levels could be one of the characteristics that the victims inherit. pH does not affect the kind of prion disease a person has. The prions are specialized, and this specialization occurs in the tertiary and quaternary structures. pH seems to affect more the rapidity of the mutations than be a catalyst for the original mutations. Nonetheless, pH has been shown to directly affect the transformation from a normal PrPC protein with three alpha helices and two beta strands to the PrPSc protein with two alpha helices and more beta strands.

Prion diseases are becoming better understood, but the question that still plagues everyone?s thoughts is: ?What causes them?? In this experiment one possible cause was localized for observation, and this cause was pH. Through new, state of the art technology it was concluded that very acidic pH is directly correlated to the transformation of healthy PrPC proteins to neurodegenerative PrPSc proteins. The pH changes the structure of the healthy protein, specifically the number of alpha helices and beta strands. A healthy verson of this protein has three alpha helices and two beta strands. The neurodegenerative form of this protein has only two alpha helices and many more beta strands.The PrPSc proteins that result from acidic pH levels cause prion diseases.Acidic pH causes this transformation to occur, therefore it can be concluded that this pH condition is associated with prionic diseases.One cannot be sure if the pH levels are the sole catalysts for this transformation or if there are other contributing factors. It has, however, been unanimously concluded by members of our group after reading the experiment that acidic pH levels are associated with prionic diseases. The applications are really endless when dealing with a science frontier such as prions. Once the questions of how and why these molecules just randomly conform to the altered (infectious) form are answered, there can be rapid drug research to find a solution to this pathway. Hopefully in the future, there will be enough advancement in knowledge and technology for these mutations to be reversed, so people with prion diseases or other neurodegenerative diseases will be able to returned to their normal mental state or at least stop at their current state. In the drug design process, Molecular Dynamic Simulations can be a useful tool to create new drugs to cure neurodegenerative diseases that are the by-product of the mutated prion protein. With this new knowledge of how the pathway occurs that causes the change to the infectious PrPSc form, we hope pharmaceutical companies will be able to cure or suppress diseases like Alzheimer?s, Parkinson?s, and Creutzfeldt-Jakob disease

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