DNA Science Workshop for VCE Biology Teachers

Prions: proteins that act as if they were alive.

A self-copying molecule is the basis of what we consider a living thing. Of course not every self-copying molecule is alive, crystals aren't, but somewhere in the evolution of life , a fine line is crossed. Plant viroids for instance might be considered alive, and these are just naked little bits of RNA that float around and copy themselves.

Well you might have noticed over the last few months, that the world's headlines have been running hot with stories about Mad Cow disease. What they haven't made clear is that many scientists believe that this transmissible disease is not caused by a virus, bacteria, parasite or any other thing that we consider to be a life form.

Just like the Andromeda team, real world scientists tried to identify the cause of the disease. They started in sheep where the disease is called Scrapie because the animals get a maddening itch and scrape off their wool. What the scientists found was extraordinary. The only thing that seemed to transmit the disease was a protein.

Perhaps this is a good time to remind everybody about Watson and Crick's central dogma -For all living things DNA makes RNA makes protein. DNA is the self-copying blueprint. RNA is the photocopy mostly used for directing the on-site production of proteins, but occasionally employed by retroviruses like AIDs as their informational blueprint. Mind you, to copy itself, the retrovirus has to reverse the central dogma, using its RNA to get a copy of DNA. But proteins, ultra flexible and versatile, are our molecular machines and tissue builders. Not since before the days of Watson and Crick has anyone thought they could do anything else.

So, how could a lone protein be behaving like a lifeform, copying itself and spreading disease between animals? There have been lots of hypotheses. One I remember being told some twelve years ago was that, if RNA could reverse the central dogma, why not protein. Each triplet of amino acids in the protein could theoretically be decoded back to its DNA sequence. After all molecular biologists do this all the time on paper. Well no evidence for that one.

The theory that has the strongest backing these days started off as quirky notion put forth by British physicist J.S. Griffith thirty years ago. Over the last ten years Stanley Prusiner at the University of California, has transformed that quirky notion into the scientific mainstream. Prusiner's version of how a proteinaceous infectious particle or PRION as he calls them could work, goes something like this.

It turns out that the infectious protein, henceforth to be referred to as "the prion", is a dented version of a normal protein. Its most commonly found tethered to the outside of brain cells.

Studies showed that while the normal brain protein had a fairly globular structure and kept to itself, the prion was plate-like and had a tendency to form stacks. For the chemists out there, the normal brain protein has virtually no b-pleated sheet secondary structure, while about 40% of the prion protein is composed of this structural motif.

As I said before proteins are the machines and building blocks of living tissue. For them shape is everything, and its no surprise that a dented protein would change its properties --dramatically.

Prusiner's theory was that the plate-like prion could act like a template, imprinting its structure onto the normal protein. Because each newly converted prion could act this way, what follows is a chain reaction. And the result is that a tiny amount of prion acts as a seed converting just about all the normal brain protein to the ultimately toxic prion aggregates.

While we would not consider a protein a living thing, it nevertheless through this process of templating, acts like a living virus, infecting, copying itself and ultimately killing its host. In fact this process is very reminiscent of theories of self-replicating systems and the evolution of life.

There's a lot of evidence to support this iconoclastic hypothesis.

First as I've told you, scientists have consistently found the abnormal prion protein in infectious material but not much else. Then about 7 years ago, a major link was made. There are a group of hereditary brain diseases in humans that look very much like Scrapie and Mad cow disease. These are Creutzfeld-Jakob Disease , Gerstmann-Str"ussler-Scheinker syndrome and Fatal Familial Insomnia. By studying the genetics of afflicted families, researchers were able to clone the responsible gene. It turned out to be the gene coding for that same brain protein. In each disease, the gene carried a slightly different mutation. But all these mutations occurred in regions believed to be critical to the structure of the protein.

And under the microscope, prions showed up in the brains of afflicted people. Furthermore, though these people had not been infected - their genetic blueprint carried a faulty copy of the gene - their brain tissue was infectious. It was shown to infect primates and unfortunately other humans.

Patients have come down with Creutzfeld-Jakob disease (CJD) after receiving contaminated pituitary extracts, corneal grafts or even after surgery with contaminated instruments.

This genetic evidence adds a hefty weight to the case that the prion diseases are a disease of protein structure. The brain protein can flip between the globular and plate-like forms. The plate is dangerous because it forms stacks that ultimately poison the brain. Normally, the protein stays globular, but transformation to the plate can happen either by introducing seeding prions, by inheriting an unstable form of the protein or through sheer bad luck. Random wobbles of the molecule may shift it into the plate form. This probably happens in about one in a million people. That's the incidence of sporadic CJD, the most common form in the population.

At an incidence of one in a million, the prion diseases should not be much to worry about. But a few misadventures by the human population have made this rare accident of protein folding, a much more significant phenomenon. Prions, unlike viruses , have no deliberate mechanism of spreading. But because of their stacked structure, they tend to resist degradation. This means that if accidentally eaten or injected, they survive to circulate in the body. While we don't know exactly how they break through the blood-brain barrier, they may get their ticket to the central nervous system by tracking up sensory nerve endings. Once in the brain, enough chance meetings with the normal brain protein and the chain reaction begins.

It's astonishing but vanishing amounts of the prion can do it. One Melbourne neurosurgeon told me, they just have to throw out contaminated instruments, no sterilization process is guaranteed to get rid of them.

But as long as humans do not inject themselves with brain extracts or eat the brains of that rare person carrying CJD, there should not be much risk. In the highlands of New Guinea, the Fore tribe were once devastated by the transmissible prion disease, Kuru. They had a ritual of eating the brains of deceased relatives. As nobel laureate Carleton Gajdusek and others have hypothesized, an ancestor probably had a rare spontaneous case of CJD that was tragically perpetuated by this ritual. Since the practice stopped in 1959, the disease has virtually disappeared.

And if it had not been for British farmers feeding rendered fatty sheep protein to cows, there were be no Mad Cow disease.

So is there a risk to humans from eating contaminated British beef? Well I think there's no denying the risk is there. Prions find it difficult but not impossible to cross the species barrier. The vulnerable brain protein varies slightly between species and for prions to convert most efficiently they seem to require a perfect match. So mouse prions will convert mouse brains more efficiently that hamster brains, but given long enough the hamsters will be converted too. And of course sheep prions have been shown to do the trick in cows, and also in British cats and zoo animals. Experiments are now underway to see if cow prions will convert human brains. Of course they can't use volunteers, so they use transgenic mice whose brains carry the human protein. So far these mice show no conversion, but short-lived mice may die before this slow-progressing disease has a chance to show up.

Most worrisome, ten patients were identified this year in the UK. with a new form of CJD. It was most untypical. CJD affects elderly people. These cases were all in people under 40 with a number being teenagers. And while CJD usually presents with a variable pathology, these cases were all remarkably uniform. They all showed big splotches or plaques in the brain filled with prions. It was reminiscent of Kuru and pathologists John Collinge and Martin Rossor writing in Lancet wondered whether an oral route of infection might be the common factor.

It's scary I admit, but there's no proof these cases were caused by eating diseased beef. And there's the argument that no-one has ever been documented as coming down with CJD from eating sheep, where the prion disease Scrapie is much more common.

And, as to whether British beef is safe now? Well , the belief is that the only way cows got it was by being fed diseased sheep protein. There's no evidence for transmission from cow to cow, or cow to calf, or through milk. Since that practice ended in the late eighties and herds have been culled, I guess its pretty safe now.

Now I wouldn't want you to go away thinking that the prion story is all wrapped up. Like just about any field of Science there is disagreement and some intriguing mysteries.

I said that there is no way that the prion spreads naturally, hence once the Fore stopped cannibalizing their diseased relatives, Kuru disappeared and likewise for cows eating diseased sheep protein. But, there is an exception. The disease does appear to spread in sheep and no-one knows why. There is the mystery of Icelandic sheep. Affected herds were destroyed and after several years a disease-free stock was re-introduced. These herds subsequently came down with Scrapie. Perhaps, prions remained deep-frozen in the soil?

The other thing is, that while I have been romanced away by the notion of prions as a glimpse of how self-copying systems can accidentally evolve, there is a respectable body of scientists who do not buy the prion story or at least remain open-minded about the possibility that a virus-like molecule is involved.

Retroviruses especially can be extremely elusive. They can hang around as mere shreds of RNA, but these little shreds while appearing lifeless to the biochemist can stealthily repair themselves at some opportunity and go on to multiply. And certainly shreds of DNA or RNA have been found associated with prions. Finally consider the case of Mad Horse Disease. For years no virus could be found. Then armed with new techniques Ian Lipkin at the University of California at Irvine isolated Bornavirus, the causative agent. It has also recently been implicated in human psychiatric disease.

Still I think I place my bets on the prion model. It's yet to be conclusively proven. That proof will require somebody to take pure normal brain protein with its plump globular structure and find a way to massage it into the plate form. Then they have to show that the plates alone act like prions. Recently a group at the Rocky Mountain Laboratories in Colorado, have been able to carry out the first step, converting pure brain protein to the plate-form in a test tube. We'll have to wait and see if they can demonstrate these altered proteins are infectious.