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THE SHAPEOF THINGS

TO COMEA number of fatal brain diseases are

linked to misfolded proteins, an effectresearchers are mimicking in the lab.But as they generate new versions of

these malformed molecules, could theybe creating a monster? Roxanne

Khamsi finds out.

NEWS FEATURE NATURE|Vol 439|12 January 2006

In a secure lab in Texas, five machinesare purring away quietly. Workingthrough the night, these boxes churnout billions of malformed proteins.

A seemingly odd thing to mass-produce, these distorted molecules areat the heart of research into a family ofdiseases that destroy the brain.

The equipment was devised by Clau-dio Soto1, a biologist at the University of Texas Medical Branch in Galveston,who works on prion protein, a naturallyoccurring molecule. Misfolded versionsof this protein are thought to cause con-ditions such as mad cow disease and itshuman equivalent, Creutzfeldt–Jakobdisease (CJD). Such abnormal prionsare infectious and, when ingested, maketheir way to the brain, where they slowlydistort the natural prion proteins, caus-ing disease and, ultimately, death.

But this conversion process takes along time — in humans it can be morethan a decade before enough abnormalprion has been made to cause disease,making it hard for scientists to investi-gate the diseases. Soto’s machines havechanged that: they can quickly turn amass of normal proteins into twistedimitations of their former selves.

“We can mimic the process of prionreplication that normally takes a year inthe brain within a matter of hours in thetest tube,” says Soto. And that accelera-tion is allowing researchers to experi-ment with the abnormal prions, gainingfresh insight into how they cause disease. But it begs one important ques-tion: is it safe?

Collectively, the diseases linked toinfectious prions are called transmissi-ble spongiform encephalopathies, orTSEs, because of the spongy appearancethey give the brain. Most TSEs arespecies-specific. Scrapie, for example,occurs in sheep; CJD in humans; andchronic wasting disease (CWD) in deerand elk. But some TSEs have hoppedfrom one species to another — mad cowdisease, for example, is believed to havejumped the ‘species barrier’ to cause disease in both cats and humans.

Given that the abnormal prions arenotoriously hard to destroy — theyresist temperatures as high as 120 �C andare not broken down by the enzymesthat usually degrade proteins — is itwise to be creating new types of them inthe lab?

Like all prion research, Soto’s work iscarried out under strict biosafety condi-tions. He argues that the risk of any pri-ons escaping is very low. “The potential

benefits are much greater than the risk,”he argues, adding that the work his teamis doing could help predict whether aTSE is likely to jump between species.

Other researchers agree. They say thatmaking prions in the test tube will helpthem tackle key problems such as whyone type of abnormal prion behaves dif-ferently from another. “It’s one of themost burning questions in the entirefield,” says Adriano Aguzzi, a prionresearcher at the University Hospital ofZurich in Switzerland.

Scientists have long explored thequestion of whether TSEs can jumpbetween species. Although a particularTSE spreads efficiently within a species,researchers have found it much harderto infect one species with the abnormalprion from another. This led to the ideaof a species barrier that limited suchinfections in the wild.

Broken barriersBut that view was challenged in the late1980s, when Britain’s cow herds werestruck by a new TSE: mad cow disease,or bovine spongiform encephalopathy.Scientists suspect that the species jumpwas caused by the farm animals beinggiven feed created from the renderedcarcasses of scrapie-infected sheep. Andthe disease didn’t stop there — it alsoseemed to jump from cows to humans,causing a condition called variant CJD.

Experts generally agree that abnormalprions from cows can convert normalones in humans, and cite this as anexample of a prion disease crossing thespecies barrier. So just how easily doinfectious prions jump from one animalspecies to another? It’s no idle question.Although governments have bannedcertain animal by-products from feed torestrict the transmission of prion dis-ease among livestock, some experts saythat there may be a need for furtherrestrictions to prevent the possiblespread of TSEs to species such as pigs,which are currently thought to be muchless susceptible to prion disease underexperimental conditions.

But in the 1990s, a new TSE emergedin UK cats, claiming the lives of morethan 80 domestic animals. It even spreadas far as zoo animals, reaching cheetahs,ocelots, pumas, lions and tigers. Again itwas linked to the BSE prion, suggestingthat the species barrier might be easierto breach than was once thought.

To get a clearer picture of infectiousprions and their ability to jump species,researchers are turning to Soto’s mass-production technique. In the brain, theabnormal prions tend to link together inchains, eventually forming visible tan-gles. But this limits the number of prions

Rapid response: Claudio Soto demonstrateshis equipment for mass-producingabnormal prion proteins.

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free to convert normal proteins — onlythose at the end of the chains can causethe switch, making it a very slowprocess. In his protein-misfolding cyclicamplification (PMCA), Soto found aneat way to sidestep this problem.

The technique initially incubates asmall amount of abnormal prion with anexcess of normal protein, so that someconversion takes place. The growingchain of misfolded protein is then blastedwith ultrasound, breaking it down intosmaller chains and so rapidly increasingthe amount of abnormal protein availableto cause conversions. By repeating thecycle, the mass of normal protein israpidly changed into misfolded prion.

Tangled webSoto and his team have used the tech-nique to probe the limits of the speciesbarrier. In unpublished work, they incu-bated normal protein from lab animalssuch as mice with a range of infectiousprions, including those that cause madcow disease, scrapie and CWD. Theresulting prions caused disease wheninjected into the brains of rodents. “Thedata from our experiments suggest thatthere is no absolute species barrier. Youcan always break it,” says Soto. “It’s just aquestion of how far you push the system.”

Soto’s lab is now extending theseexperiments to look at other possiblespecies jumps, including that frominfected deer to humans. Although eat-ing meat from scrapie-infected sheepdoes not seem to cause prion disease inhumans, the effects of eating venisoncontaminated with CWD remainunclear. It has not yet been proved thatCWD can jump to humans, but studieshave shown that injecting CWD prionsinto monkeys can cause disease2, saysPaul Brown, former medical director of

the Laboratory of Central Nervous Sys-tem Studies in Bethesda, Maryland. Hestresses that CWD does not pose a pub-lic health risk as deer remains do nottypically get cycled into the food chain.

“The concept of an absolute speciesbarrier is probably a myth,” says Brown,adding that there are simply “varyingdegrees of ease” in terms of how readilythe prions can be transmitted. Underexperimental conditions, skunks, pigsand even gerbils seem susceptible toTSEs from other species3. As yet,researchers have not succeeded ininfecting dogs or rabbits with a TSE.

Taking the strainAnother big question is what makes aparticular prion ‘strain’ act in the way itdoes. Strains are defined by the charac-teristics of the illness they cause in agiven animal, including incubation timeand the type of damage done to thebrain. Often, different strains will havedistinctive biochemical properties —although two strains within the samespecies can have the same amino-acidsequence and behave differently.

Byron Caughey and his team at theRocky Mountain Laboratories in Hamil-ton, Montana, have been working onmaking prions in the test tube for morethan a decade. In 1995, they showed that

infectious prions made in the absence ofcells retain strain-specific qualities, suchas tending to convert only prions fromthe same species4,5, which suggests thatno other cellular components are neededfor the prion’s activity.

Some studies suggest that the speciesbarrier is dictated largely by the three-dimensional structure of the abnormalprion species6. In other words, the givenprion’s structure has to be able to workwith the native normal protein. Bychanging just two key amino acids in theproteins, Witold Surewicz of Case West-ern Reserve University in Cleveland,Ohio, and his team have created newprion fragments that defy the speciesbarrier7,8. The result was new structuresthat could adopt the shape of prion frag-ments from a different species.

Using Soto’s technique, such investi-gations into the species barrier will onlyaccelerate. But as biologists mix normaland infectious prions from differentspecies in the lab, they will create novelprions with unpredictable abilities. Therisks of creating a ‘super-strain’ are notnew — scientists recently debated themerits of recreating and tinkering withthe virus strain that caused the 1918 flupandemic.

Proponents of the prion work say thatthe threat of creating an accidentalmonster is minimal. They stress thatunlike the genetic manipulation of fluviruses, which could produce an inhaledsuperbug, similar work in prions isunlikely to produce infectious materialthat can be transmitted through the air.

Others point out that scientists havebeen creating new prion strains formany years — albeit in animals, ratherthan in the test tube. “I do not see thegeneration of new strains in cell-freesystems as a fundamentally new type ofbiohazard,” says Caughey.

And understanding how easily partic-ular infectious prions can pass from onespecies to another will help officialsdesign adequate safety measures to pro-tect us and our food chain, says Soto.“It’s very important to evaluate the danger and be prepared.” ■

Roxanne Khamsi reports for Naturefrom New York.

1. Saborio, G. P., Permanne, B. & Soto, C. Nature 411,810–813 (2001).

2. Marsh, R. F., Kincaid, A. E., Bessen, R. A. & Bartz, J. C. J. Virol. 79, 13794–13796 (2005).

3. Brown, P. Transmissible Spongiform Encephalopathyas a Zoonotic Disease (International Life SciencesInstitute Europe, Brussels, 2003).

4. Kocisko, D. A. et al. Proc. Natl Acad. Sci. USA 92,3923–3927 (1995).

5. Bessen, R. A. et al. Nature 375, 698–700 (1995).6. Peretz, D. et al. Neuron 34, 921–932 (2002). 7. Jones, E. M. & Surewicz, W. K. Cell 121,63–72 (2005).8. Vanik, D. L., Surewicz, K. A. & Surewicz, W. K.

Mol. Cell 14, 139–145 (2004).

“Theconcept ofan absolutespeciesbarrier isprobably amyth.” —Paul Brown

Twisted into shape: just minor changes to a normal human prion protein (left) can change its three-dimensional structure (right) to resemble the infectious, disease causing prion found in hamsters (centre).

Pumas areamong a few

big cats incaptivity to

have contractedan illness

similar to madcow disease.

ARD

EA

REF.

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