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theoreticians come out with theories that sometimes take years to prove—such as the existence of quarks or Einstein's theory of relativity// says Taylor. "And quantum chemistry is often used to make predictions in advance of experimental verification. So it really shouldn't be so surprising that we're now reaching the point where biological theory can be ahead of experiment." A group led by Thomas M. Folks, chief of the retrovirus diseases branch of the Centers for Disease Control & Prevention, Atlanta, is currently conducting experiments to determine if HIV-positive individuals harbor antibodies to synthetic peptides based on the proposed fusion proteins. "The preliminary results are encouraging," says Folks. "We have seen some seroreactivity [antibody response to the synthetic peptides] in patient sera. But we don't know yet if it is truly an immune response directed at a gene product made by HIV, or if the antibody is cross-reacting to something else. It could be a staph or strep antigen, or almost anything." Taylor hopes the publication of his paper will spark additional efforts to test the findings experimentally. •
Prion study shows that proteins can 'replicate' A new study shows that a Nucleation-dependent model says naturally occurring protein can convert to a poscrapie agent seeds aggregation tentially pathogenic form Short-lived Prion protein in a cell-free system—promonomer cellular (normal) Conformational viding evidence favoring change the controversial hypothesis that scrapie and related neurological diseases are caused by infective proteins called prions, rather than by viruses or Multiple other traditional infectious StfiDS agents. Prion protein Prions have been conscrapie oligomer (infectious) troversial because they appear to infect animals, replicate, and cause disease like microorganisms do, but ap- demonstrating "reproduction" of priparently lack the specific DNA or RNA on-associated protein. The work was normally believed to be necessary for reported recently in Nature [370, 471 replication. Now, associate professor of (1994)]. chemistry Peter T. Lansbury Jr. of MasThe proposed infectious form of prisachusetts Institute of Technology, By- on protein is insoluble in aqueous soluron Caughey of the National Institutes tion and protease-resistant, but a form of Health's Rocky Mountain Laborato- of the same protein that occurs naturalries (Hamilton, Mont.), and coworkers, ly in animals is soluble and susceptible have developed an in vitro system to proteases. Lansbury, Caughey, and AUGUST 22,1994 C&EN
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SCIENCE/TECHNOLOGY coworkers find that in the presence of the putative infectious protein, the nor mal protein undergoes a conformation al change that causes it to acquire the insolubility and protease resistance characteristic of the infectious form. This is the first time "replication" of protease-resistant prion protein has been demonstrated experimentally. A leading prion researcher, John Collinge of the department of biochemistry and molecular genetics at St. Mary's Hospital Medical School, London, says: "The Nature paper is very exciting. This is obviously an experiment that people have tried before and not succeeded with. It's a very elegant piece of work." Prions have been hypothesized to be infectious proteins that cause transmis sible spongiform encephalopathies (TSEs)—diseases of the nervous system that include scrapie (in sheep), mad cow disease (in cattle), and CreutzfeldtJakob disease, Gerstmann-StrausslerScheinker syndrome, and kuru (in hu mans). TSEs, like Alzheimer's disease, are neurodegenerative diseases that are characterized by deposition in the brain of extracellular protein aggregates. The deposits consist predominantly of an aberrant form of an endogenous (nor mal in-vivo) protein called prion pro tein (PrP). At first sight, TSEs appear to have all the hallmarks of viral diseases: They can be transmitted from one animal to another, the infectious agent can selfreplicate in vivo, there are species bar riers (obstacles to infection of one spe cies by another), and different strains of infectious agent produce distinct forms of disease. But the infectious agents in TSEs sur vive radiation, heat, and extremes of chemical treatment that would normal ly kill viruses. And although viruses are composed largely of nucleic acids, numerous attempts to demonstrate a specific nucleic acid component of pri ons have failed. In the 1960s, some researchers specu lated that the agent responsible for TSEs might be a single protein—even though no protein had ever been found to be the sole cause of a transmissible disease. This hypothesis raised many conceptual questions—for example, how could a single protein replicate? In 1967, mathematics professor J. S. Griffith of Bedford College, London, published a paper in Nature in which he suggested several mechanisms by
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w o u l d cause the w h o l e theoretical structure of molecular biology to come tumbling down," Griffith wrote. Later, Stanley B. Prusiner of the de partments of neurology and of bio Coauthors of Nature paper: Peter T. Lansbury chemistry and biophysics at the Uni versity of California, San Francisco, (left in photo at left) and Jon H. Come; and formulated the concept of a protein in (bottom photo,front,from left) David A. fectious agent in more specific terms— Kocisko, Byron Caughey, Suzette A. Priola, coining the term "prion" to describe Gregory J. Raymond, and Bruce Chesebro. the agent. f-JHHBBHX The infectious agent in TSEs has been Mil proposed to be a form of PrP called pri on protein scrapie (PrP50). PrP 50 is an in soluble, protease-resistant form of prion protein cellular (PrP c ), the endogenous protein. According to the "protein-only" hypothesis of TSE transmission, PrP 50 in fects a cell and somehow converts P r P c into more PrP 50 . Griffith first proposed that the differ ence between P r P c and PrP 50 is a confor mational one, and this was later demon strated experimentally. But there is on going debate on the mechanism by which the proposed infectious material in one conformation converts noninfec tious material in another conformation into more infectious material. One model of prion formation origi nally proposed by Griffith and later
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SCIENCE/TECHNOLOGY championed by Prusiner and others is the heterodimer mechanism. This model proposes that PrPSc monomer is the infectious agent, and that a slow conformational interconversion from PrP 0 monomer to PrP^ monomer occurs via formation of PrP c -PrP Sc heterodimer, which then converts to PrP^-PrP50 homodimer. Dissociation of the homodimer releases new PrP^ monomer. Another model is the nucleationdependent mechanism of aggregate formation. This model also was initially proposed by Griffith and then was expanded on by pediatrician and virologist D. Carleton Gajdusek of the National Institutes of Health, who earned the 1976 Nobel Prize in Physiology or Medicine for his work on kuru. The model proposes that PrP Sc is not a monomer but an oligomer, a small amount of which can act like a crystallization "seed" to nucleate aggregation of PrP c -derived protein. Lansbury's group at MIT proposed the first detailed, testable chemical mechanism of nucleation-dependent aggregation. Prusiner, UCSF professor of pharmaceutical chemistry Fred E. Cohen, and coworkers recently proposed a "template" concept of prion formation that emphasizes common features of the heterodimer and nucleation-dependent mechanisms. At some point in the prion replication process, says Cohen, "there is an interface where PrPSc contacts PrP c or a slightly modified version of PrP c . Many molecules of PrPSc could be lined up, but one of them could present an interface to PrP c . We would call that heterodimer formation, but it's also exactly what's implied by the seed model." Cohen believes the mechanism of prion replication will be found to draw upon features of both the heterodimer and nucleation-dependent mechanisms. Lansbury strongly disagrees, contending that the two mechanisms are distinctly different and mutually exclusive. In their Nature paper, Lansbury, Caughey, and coworkers now report the in vitro conversion of radiolabeled PrP c to an insoluble, protease-resistant, PrPSc-like form of the protein. The researchers mixed labeled P r P c from scrapie-free cells with unlabeled PrPSc. After two days, the mixture yielded labeled protein fragments that were protease-resistant and were similar to fragments found when PrP Sc is taken from scrapie-infected animals and treated with protease.
SOFTWARE/DATABASE UPDATE "PrPSc appears to replicate itself even though it does not have its own genetic material," says David A. Kocisko, Lansbury's graduate student and lead author on the study. "The conversion of PrP c to the irregular form related to scrapie . . . required the presence of preexisting PrP , providing direct evidence that PrPSc derives from specific PrPc-PrPSc interactions." Caughey says the findings "are consistent with . . . protein-only models of PrPSc formation. However, our results do not rule out the involvement of other non-PrP constituents such as sugars or nucleic acids . . . that might be cofactors in the conversion.,/ The work indicates that PrP c obtains some type of template assistance from PrPSc in its conversion into more PrPSc. However, the results do not preferentially favor either the heterodimer mechanism or the nucleation-dependent model. So the debate over the mechanism of prion formation remains undecided for now. A key limitation of the study is that about a hundredfold excess of PrPSc (over PrP c ) was needed to effect the conversion. Hence, the high level of preexisting infectivity from PrPSc overwhelmed any capability to detect new scrapie infectivity associated with the conversion of radiolabeled PrP c to a protease-resistant form. "We have not yet addressed the question of whether PrPSc formation is equivalent to scrapie agent replication," says Caughey. 'Obviously what the study doesn't do is show an increase in infective titer, which is the ultimate experiment really," comments Collinge. Lansbury, Caughey, and coworkers are "clearly not able to do that with the current system/' he says, "in that they start with a vast molar excess of the Sc form. . . . The key question is whether they can find the right experimental situation in which to be able to detect infectivity convincingly." Indeed, in studies conducted since the Nature paper was submitted, Lansbury and Caughey say they have greatly improved the yield of the conversion reaction by decreasing the ratio of PrPSc to PrP . In future work, says Lansbury, "it is our hope that we can use our in vitro conditions to generate infectious material from purified PrP c " and thus confirm the protein-only hypothesis. Stu Borman
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