Using Bioinformatics to study prions and other brain eating disorders

Using Bioinformatics to Study Prions and Other Brain Eating Disorders

During the Bioinformatics Summer Program we watched a video entitled "The Brain Eater" by NOVA. This video discussed the topic of Bovine Spongiform Encephalopathy (BSE), otherwise known as Mad Cow Disease. We were all very impressed with the video, yet slightly nervous to go out and eat a hamburger! Our group found the idea of a disease caused by a folding malfunction in a protein very interesting.

After Spontaneous Reaction (our group "name") researched the topic of prions in general, we decided that we were very interested in the fact that this disease "jumped" species. Supposedly there is a species barrier that prevents diseases from spreading between species. Brain eating disorders originated in sheep in a disease called Scrapie. When the disease jumps to a cow it is called BSE, and there is also a human strain called variant Creutzfeld-Jakob's Disease (vCJD). All of these diseases are classified as Transmissible Spongiform Encephalopathies (TSE). Our curiosity sprang from the idea that all of these diseases are related through prion proteins.

How do prions cross a species barrier? Why can't sheep infect humans with Transmissible Spongiform Encephalopathy, if sheep can infect cows, and cows can infect humans?

Background Information

When a new form of an old human disease appeared in Great Britain in 1995, medical specialists immediately suspected that it was Creutzfeldt-Jakob disease (CJD).
Normally, Creutzfeldt-Jakob disease occurs in a person in only three ways. Fifteen percent of all cases were inherited, resulting from gene mutation. Other cases were caused by the use of unsanitary medical tools during surgery. However, most cases occurred sporadically, in people with no family history of the disease. Traditionally, cases of CJD occurred in people over the age of 50. In 1995 people in Great Britain under 30 were contracting what they thought to be CJD. According to the Environmental Research Foundation, "In people younger than the age of 30, CJD is extremely rare, striking an average of 5 people per billion each year." What researchers found was a new variant type of CJD, a totally new disease contracted by eating infected beef. Many people were stunned because they could not believe that humans could have fallen victim to a disease from animals.

This is a microscopic view of brain tissue infected with variant CJD, the human form of this disease.

Picture from University of Iowa's page http://www.uh.org/Providers/TeachingFiles/CNSInfDisR2/Text

When the U.S. government heard that Great Britain’s cows were infected with a disease called Bovine Spongiform Encephalopathy (BSE), they immediately banned British beef products. Even though the U.S. took precautions about this issue, the government did not educate the public about this deadly disease.
In 1986, there was an outbreak in Great Britain of BSE where a total of 178,000 cows died. BSE appears to have originated from a disease called Scrapie that occurs in sheep and has been recognized in Europe since the mid-18^th century. Scrapie is caused by a mother sheep passing the disease to the offspring through the placenta. The British agriculture industry (but not the American agriculture industry) carried out the practice of grinding the carcasses of dead animals to produce protein supplements for farm animals. Scrapie-infected sheep carcasses were rendered into a protein supplement that was fed to cows. Because the genetic makeup of cows and sheep are so closely related, cows contracted BSE, a form of Scrapie, when they ate the infected sheep proteins. When cows fell prey to BSE, they became uncoordinated and experienced uncontrollable shaking. In a period of 2 weeks to 6 months, the brains of the infected cows took on a sponge-like form which eventually led to death.

BSE is the cow version of a larger class of disease; it falls under the category of Transmissible Spongiform Encephalopathies (TSEs). In all species that contract these diseases, the symptoms of TSE are the same: depression, confusion, changes in behavior, and problems with memory, coordination, and sight. There is a progressive destruction of brain cells leading to dementia and eventually death. Another form of TSE is Kuru. Kuru was discovered in a tribe of people that lived in New Guinea in the early 1900s. This tribe practiced cannibalism where they would eat the brains and muscles of their deceased relatives because this was considered to be respectful.

This is a picture of brain infected with the Kuru disease. This disease results in the growth of large, black veins on the brain.

Picture from web site of University of Iowa http://www.uh.org/Providers/TeachingFiles/CNSInfDisR2/Text

TSEs are diseases caused by an infectious protein called prions. The brains of sheep, cows, and humans have a specific gene for a protein called precursor prion protein (PrP, or healthy prion). The normal function of the prion precursor is unknown, but it is thought to be involved in nerve functions. Once the diseased prion comes in contact with the healthy prion, it causes the healthy prion to take on the diseased prion's altered form. The diseased prions multiply at an exponential rate. The prions can link together, creating infectious chains that penetrate the brain matter and kill healthy cells. Due to the fact that prions do not contain nucleic acid and have a very stable form, they cannot be destroyed by conventional cooking or by ultraviolet light. Scientists usually use these procedures to kill other disease causing agents. People resisted the idea that prions can cause disease, because all previously known infectious agents (for example, protozoa, bacteria, and viruses) contained RNA, DNA, or both. So, it was hard for the people to believe that prions are able to cause such deadly diseases.

This shows a microscopic picture of what brain tissue looks like when infected with a TSE. Note the spongy appearance, from which many of these diseases get their names.

Picture from University of Iowa's page http://www.uh.org/Providers/TeachingFiles/CNSInfDisR2/Text

Methods of Research

    We began researching the disease on search engines like Google.com, msn.com, Yahoo.com, and Dogpile.com. We used the keywords "mad cow disease," "Kuru," and "prions." From the links that came up, we selected articles that were published about several different theories. This gave us insight into what causes Mad Cow Disease, and how the disease actually infects the body.
    After discovering the large role prions play in causing the disease, we used Biology Workbench to compare major prion proteins in several different species.   We used these tools: Ndjinn, ClustalW, DrawTree, and BoxShade. We were able to align sequences and notice their differences or similarities.
            We were able to determine that cow prion protein sequences were very similar to those of sheep. We also saw that humans were similar to cows but not so similar to sheep. We then went to the PubMed database to find more articles related to this idea.
           Through research, we found that these diseases are caused by diseased prion proteins, which force the healthy proteins in the brain of the infected animal to change shape. So, we used Protein Explorer to see exactly how the proteins fold. This allowed us to look at the healthy and diseased prion proteins from many different angles to determine what changes occur during the disease's infection.

Data

Chart 1. BoxShade comparing prion proteins in cow, sheep, and human.

We aligned the sequences of the major prion protein precursor in sheep, cows, and humans. Running a BoxShade on these alignment showed where all three are similar and where only two share an amino acid (see Chart 1). The green highlights show where all three species share the same amino acid. The sections highlighted yellow show where two species share the same amino acid in this area of the sequence. From the amount of green, it became obvious that all three seemed very similar.

Figure 1. Unrooted tree comparing prion protein in cow, sheep, and human.

Then, we made the sequences into an unrooted tree (see Figure 1). This tree shows how similar each animal is by comparing the length of the lines connecting each to another. The longer the line, the less similar their sequence. Cows and sheep appear to be very similar. This gives evidence to our theory that the diseased prions might be able to pass from a sheep to a cow. A new problem arose: how can humans get diseased prions from cows, which seem to have different amino acid sequences for the protein?
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Chart 2. Sequence alignment comparing cow, sheep, and human.

PRP2_BOVIN MVKSHIGSWI LVLFVAMWSD VGLCKKRPKP GGGWNTGGSR YPGQGSPGGN
PRIO_SHEEP MVKSHIGSWI LVLFVAMWSD VGLCKKRPKP GGGWNTGGSR YPGQGSPGGN
PRIO_HUMAN --MANLGCWM LVLFVATWSD LGLCKKRPKP GG-WNTGGSR YPGQGSPGGN

PRP2_BOVIN RYPPQGGGGW GQPHGGGWGQ PHGGGWGQPH GGGWGQPHGG GGWGQGG-SH
PRIO_SHEEP RYPPQGGGGW GQPHGGGWGQ PHGGGWGQPH GGGWGQPHGG GGWGQGG-SH
PRIO_HUMAN RYPPQGGGGW GQPHGGGWGQ PHGGGWGQPH GGGWGQPHGG G-WGQGGGTH

PRP2_BOVIN SQWNKPSKPK TNMKHVAGAA AAGAVVGGLG GYMLGSAMSR PLIHFGNDYE
PRIO_SHEEP SQWNKPSKPK TNMKHVAGAA AAGAVVGGLG GYMLGSAMSR PLIHFGNDYE
PRIO_HUMAN SQWNKPSKPK TNMKHMAGAA AAGAVVGGLG GYMLGSAMSR PIIHFGSDYE

PRP2_BOVIN DRYYRENMHR YPNQVYYRPV DQYSNQNNFV HDCVNITVKE HTVTTTTKGE
PRIO_SHEEP DRYYRENMYR YPNQVYYRPV DRYSNQNNFV HDCVNITVKQ HTVTTTTKGE
PRIO_HUMAN DRYYRENMHR YPNQVYYRPM DEYSNQNNFV HDCVNITIKQ HTVTTTTKGE

PRP2_BOVIN NFTETDIKMM ERVVEQMCIT QYQRESQAYY QRGASVILFS SPPVILLISF
PRIO_SHEEP NFTETDIKIM ERVVEQMCIT QYQRESQAYY QRGASVILFS SPPVILLISF
PRIO_HUMAN NFTETDVKMM ERVVEQMCIT QYERESQAYY QRGSSMVLFS SPPVILLISF

PRP2_BOVIN LIFLIVG
PRIO_SHEEP LIFLIVG
PRIO_HUMAN LIFLIVG
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We had a mystery. How can humans contract diseases caused by a protein found in a cow? We decided to look for amino acids the cow and human share which are different from the sheep. In our research, we read an article that said the key amino acids involved in the disease were contained between codons 96 and 167. We began looking in this area. The two amino acids we found that the cow and human share are highlighted in yellow in the sequence alignment (see Chart 2). These amino acids differ from the sheep while they are the same in the human and the cow. Since humans can contract these prions from a cow and not a sheep, these specific amino acids may be important in the transfer of prions across a species barrier.

Figure 2.

Human Diseased Prion Protein

Figure 3.

Human Prion Protein PrP

The prion protein in Figure 2 is the protein that causes brain eating disorders. The prion protein in Figure 3 is a normal prion that will not cause any diseases, and is normally found in the human brain. There are very few differences between the two. The main difference appears in the main coil that is colored yellow and red in the Figures. In the diseased prion the coil ends abruptly after five coils, where the normal prion continues on for seven coils. Comparison of the sequences shows that they are similar, supporting the theory that these diseases are folding malfunctions.

Conclusion

The BoxShade results show that the cow, sheep, and humans are all fairly similar in their amino acid sequence. However, the unrooted tree shows that the cow and sheep are more closely related to each other than to humans. This close relationship may explain why cows are able to contract prions from sheep.
Based on our data, we also believe that prions are able to cross the species barrier (which separates cattle from humans) because of a comparison utilizing two particular amino acids. The aligned sequences in our data show where the amino acids in the cow and human are the same. Looking at these two amino acids in the sheep, there are no similarities. These are the only two locations where this occurs. These amino acid similarities support the radical idea that humans can contract a fatal disease from a protein in a cow.
Our conclusions came about because of the data we were able to obtain through the resources available to us. This topic is still baffling scientists today and the problem is far from over.