Prions: Molecular and Cellular Biology Chapter Abstracts
1. Folding and three-dimensional NMR structure of the recombinant cellular prion protein from the mouse
Rudi Glockshuber, Simone Hornemann, Roland Riek, Martin Billeter, Gerhard Wider, Susanne Liemann, Ralph Zahn and Kurt Wüthrich
The NMR structure of the recombinant mouse prion protein in the cellular form (mPrPC) contains a flexible disordered N-terminal segment of residues 23125 and a novel globular fold with three a-helices and a small ß-sheet formed by the C-terminal residues 126231. The presumed infectious agent of transmissible spongiform encephalopathies (TSEs), PrPSc, differs from PrPC at least with respect to the conformation of the segment 90125, which is resistant to proteolysis in PrPSc but disordered and accessible in PrPC. At acidic pH the fragment mPrP(121231) can adopt an alternative conformation with increased ß-sheet content, which may relate to the presumed conversion of PrPC to PrPSc in acidic endosomes. Biochemical experiments and analysis of the locations in the structure of mPrPC of the point mutation sites linked with familial human TSEs indicate that destabilization of PrPC cannot be a general mechanism favoring formation of PrPSc in inherited TSEs. Amino acid substitutions among the highly conserved mammalian prion proteins are clustered in four distinct regions in the three-dimensional structure of mPrP(121231). Three of these regions represent potential surface recognition sites that may be involved in intermolecular interactions related to the species barrier for infectious transmission of TSEs.
2. Formation of protease-resistant prion protein in cell-free systems
Byron Caughey
In transmissible spongiform encephalopathies (TSE) or prion diseases, the endogenous protease-sensitive prion protein (PrP-sen) of the host is converted to an abnormal pathogenic form that has a characteristic partial protease resistance (PrP-res). Recent studies with cell-free reactions indicate that the PrP-res itself can directly induce this conversion of PrP-sen. This PrP-res induced conversion reaction is highly specific in ways that might account at the molecular level for TSE species barriers, polymorphism barriers, and strains. Not only has this reaction been observed using mostly purified PrP-sen and PrP-res reactants, but also in TSE-infected brain slices. The conversion mechanism appears to involve both the binding of PrP-sen to polymeric PrP-res and a conformational change that results in incorporation into the PrP-res polymer.
3. Expression of Heterogeneous PrP Molecules Blocks Formation of Protease-Resistant Prion Protein In Vitro:Effect of Amino Acid Mismatches at Residue 138
Suzette A. Priola and Bruce Chesebro
In the transmissible spongiform encephalopathies (TSE), the conversion of the normal protease-sensitive host protein PrP-sen to an abnormal protease-resistant form, PrP-res, is a critical step in disease pathogenesis. Amino acid mismatches between PrP-sen and PrP-res can dramatically decrease the amount of PrP-res made and modulate the resistance to crossspecies transmission of TSE infectivity. In mouse scrapie-infected neuroblastoma cells, a hamster-specific methionine at postion 138 in the mouse PrP gene was found to inhibit the species-specific formation of mouse PrP-res. In vivo, PrP-sen and PrP-res molecules which mismatch at this position may have a reduced potential to form PrP-res. This might account for the difficulty in transmitting scrapie and bovine spongiform encephalopathy (BSE) to hamsters, as well as sheep scrapie to certain goats which have a methionine at the homologous residue (position 142 in goat PrP). Conversely, matching of amino acid residues at this position may facilitate cross-species transmission of TSE diseases such as BSE to new species.
4. Cell biological studies of the prion protein
David A. Harris
Studying PrPC and PrPSc in cell culture systems is advantageous because such systems
contain all the organelles, membranes, and molecular cofactors that are likely to play an important role
in the biology of the proteins. Using cultured cells expressing
PrPC, we have discovered that this
isoform constitutively cycles between the cell surface and an endocytic compartment, a
process that is mediated by clathrin-coated pits and a putative
PrPC receptor. We have also
constructed stably transfected lines of CHO cells that express PrP molecules carrying mutations that
are associated with familial prion diseases. The mutant PrP molecules in these cells
are spontaneously converted to the
PrPSc state, a phenomenon which has allowed us to
analyze several key features of prion formation.
5. Inherited prion disease: molecular pathology and cell models
Piero Parchi, Sabina Capellari, Gianluigi Zanusso, Neena Singh, Pierluigi Gambetti and Robert B. Petersen
The prion protein is a normal cellular glycoprotein which, after conversion to a protease resistant pathogenic form appears to be the major, if not the only, component of the infectious agent known as the prion. Numerous strains of prions, differing in their incubation period and neuropathology, have been isolated in the same host genotype, indicating that prions carry information which is independent from the host. Molecular analysis of prion strains has recently focused on human prion diseases and support the view that the prion protein can specify disease phenotypes by differences in its conformation and glycosylation. The observation that approximately 10-15% of all human prion diseases are familiar and result from mutations in the prion protein gene provided a basis for pursuing the factors that favor, or result in, the de novo conversion of the prion protein to a pathogenic form. A cell culture model was established to study the effects of the pathogenic mutations on the metabolism of the mutant prion protein. One of the consistent changes found as a result of the pathogenic mutations clustered in the region of post-translational modification was an alteration of the glycoisoform ratio; an observation that also applies to prion protein derived from prion diseased brain samples.
6. The Use of Genetically Modified Mice in Prion Research
Charles Weissmann, Alex J. Raeber, Doron Shmerling, Antonio Cozzio, Eckhard Flechsig and Adriano Aguzzi
Linkage between the infectious scrapie agent and PrP was first established at the biochemical level and subsequently reinforced by genetic evidence. This led to the prediction that animals devoid of PrP should be resistant to experimental scrapie and fail to propagate infectivity which was then experimentally borne out, adding support to the "protein only" hypothesis. In addition, the availability of PrP knockout mice provided an approach to new lines of investigation.
7. Neurotoxicity and neuroinvasiveness of prions in neuroectodermal transplants
Adriano Aguzzi, Thomas Blättler, Michael A. Klein, Alex J. Räber, Ivan Hegyi, Rico Frigg, Charles Weissmann, and Sebastian Brandner
Although the prime role of PrP in prion diseases is undisputed, the mechanisms of brain damage, and those that control the affinity of the agent for the central nervous system, are unclear. These questions can be addressed by selectively expressing prnp in specific tissues of mice. Towards this goal, we have developed neurografting and bone marrow grafting strategies. Availability of mice overexpressing the prnp gene (which encodes the normal prion protein), along with prnp knockout mice, allows for selective reconstitution experiments aimed at expressing PrP in specific portions of the brain. Here, we summarize how such studies can offer insights into how prions administered to extracerebral sites can gain access to central nervous tissue, and into the molecular requirements for spongiform brain damage.
8. Prion diseases: PrP peptides, pathogenesis and treatment perspectives
F. Tagliavini, M. Salmona, G. Forloni, P. Malesani, G. Giaccone, B. Canciani, R. McArthur, J. Lansen, P. Piccardo, S.R. Dlouhy, B. Ghetti, B. Frangione, F. Prelli, O. Bugiani
The central event in prion diseases is the conformational conversion of the cellular prion
protein (PrPC) into abnormal forms
(PrPres) that have high content of (-sheet secondary structure
and
high tendency to form insoluble aggregates and amyloid fibrils. Amyloid formation occurs to
the highest degree in Gerstmann-Sträussler-Scheinker disease (GSS). Deposition of
PrPres and PrP amyloid in the brain is accompanied by degeneration of neurons and activation of glial
cells. Biochemical studies have shown that the GSS amyloid protein is a fragment of PrP, whose
N-terminus is located within the octapeptide repeat region and C-terminus corresponds to
residue (150. Studies with synthetic peptides homologous to consecutive segments of this
fragment indicate that the region spanning residues 106-126 is able to adopt different conformations
in distinct environments, although has high propensity to form stable (-sheet structures
and assemble into amyloid fibrils which are partially resistant to protease digestion. The
peptide PrP106-126 causes neuronal death by apoptosis, hypertrophy and proliferation of astrocytes,
and activation of microglial cells in vitro. These data suggest that the region corresponding to residues 106-126 may be critical for the structural transition PrPC>PrPres, and that cerebral accumulation of peptides including this sequence may be responsible for the tissue changes that occur in prion diseases. The role of PrP in the pathogenesis of these disorders is
further supported by the observation that compounds able to interact with disease-specific PrP
isoforms delay the accumulation of
PrPres and PrP amyloid and the appearance of nerve cell
degeneration and glial cell reaction in experimental scrapie.
9. The Human Genetic Prion Diseases
Katherine Young, Pedro Piccardo, Stephen Dlouhy, Orso Bugiani, Fabrizio Tagliavini, and Bernardino Ghetti
The genetic prion diseases are autosomal dominant neurodegenerative diseases associated with mutations in the prion protein gene (PRNP). As with the sporadic and transmitted prion diseases, the genetic prion diseases are thought to result from a conformational change in the prion protein (PrP). These diseases are marked at the biochemical level by the accumulation of PrP that is in an abnormal conformation.
There are 5 classical genetic prion diseases: Creutzfeldt-Jakob disease (CJD), Gerstmann- Sträussler-Scheinker disease (GSS), fatal familial insomnia (FFI), prion protein cerebral amyloid angiopathy (PrP-CAA), and atypical dementia. Approximately 15% of CJD cases are familial and 6 different point mutations have been found in CJD. All GSS cases are genetic and 6 GSS mutations have been found. FFI is caused by a mutation at PrP residue 178 and PrP-CAA is caused by a stop codon mutation at residue 145. Heterogeneous phenotypes have been found associated with mutations at PrP residues 171 and 183 and with 5, 6, 7, 8 and 9 insertions in the PrP octapeptide repeat region.
The classic CJD phenotype is one of rapidly progressing dementia often accompanied by myoclonus and periodic synchronized discharges (PSDs) in the electroencephalogram (EEG). In contrast, GSS is a more slowly progressing disease in which ataxia is the predominant sign and dementia usually occurs at a later stage. The pathologic phenotype of CJD consists of spongiform changes, neuronal loss, and gliosis. The hallmark of GSS is the presence of unicentric and multicentric amyloid deposits immunoreactive with antibodies to PrP. Amyloid deposition is accompanied by gliosis, neuronal loss, and occasionally, spongiform degeneration. To these clinical and pathologic criteria we can now add biochemical (immunoblot) and molecular genetic (PRNP mutation screen analysis) assays. As new techniques are applied and more familial cases are identified, more exceptions to the classic CJD and GSS definitions are found, as can be seen in some of the descriptions below.
It is not unusual for a patient with a genetic prion disease to be diagnosed at the onset of symptoms as having olivopontocerebellar atrophy, Huntington disease, or amyotrophic lateral sclerosis (ALS). Several studies have been done to search for pathologic PrP in patients with dementia or other neurodegenerative diseases. Although several PRNP mutations have been found in these patients, in general there is not a significant number of patients with prion diseases who are hidden in other diagnostic groups.
In PRNP there are both pathogenic mutations and benign polymorphisms. Formally speaking, a mutation is defined as a change in a gene. The DNA mutation can be a "silent" mutation which does not change the protein, it can lead to a non-pathogenic amino acid change, or it can lead to a pathogenic amino acid change. A site is defined as polymorphic when there are 2 or more alleles, each at a frequency of at least 1%, in a given population. Since the
genetic prion diseases are rare, and therefore a pathogenic mutation does not exist at
polymorphic frequencies, we will use the term mutation when we refer to a disease-causing change
and
polymorphism when we speak about a benign change. However, it should be kept in mind
that some of the PRNP mutations found in prion disease patients have not been formally linked to the disease and so may turn out to be benign mutations, or "rare polymorphisms".
10. Bovine spongiform encephalopathy and the new variant of Creutzfeldt-Jakob Disease
Dominique Dormont
BSE epidemic has started in the UK in the middle eighties. Today, more than 170,000 cattle have been affected by this disease in Great Britain, and only few cases have been reported outside of British islands. BSE agent has particular biological and physico-chemical properties that are distinct from those of known scrapie agents.; in particular, it can resist to certain procedures that inactivates scrapie agent. Moreover, the oral route is almost as efficient as the parental route for infection, suggesting specific biological properties. The origin of the spread of BSE in the UK is due to the meat and bone meal (MBM) which manufacturing process has been changed in the late seventies and early eighties, the new processes being unable to inactivate the BSE agent. Ban of MBM from the feeding of ruminants has demonstrated its efficacy in reducing the incidence of BSE. A new form of Creutzfeldt-Jakob disease has appeared in the UK and France recently; this new variant has specific characteristics at both clinical and molecular levels. Several experimental data indicate that BSE agent could be the origin of this new form of Creutzfledt-Jakob disease.
11. [URE3] and [PSI] are prions of yeast and evidence for new fungal prions.
Daniel C. Masison, Herman K. Edskes, Marie-Lise Maddelein, Kimberly L. Taylor
and Reed B. Wickner
[URE3] and [PSI] are two non-Mendelian genetic elements discovered over 25 years ago and never assigned to a nucleic acid replicon. Their genetic properties led us to propose that they are prions, altered self-propagating forms of Ure2p and Sup35p, respectively, that cannot properly carry out the normal functions of these proteins. Ure2p is partially protease-resistant in [URE3] strains and Sup35p is aggregated specifically in [PSI] strains supporting this idea. Overexpression of Hsp104 cures [PSI], as does the absence of this protein, suggesting that the prion change of Sup35p in [PSI] strains is aggregation. Strains of [PSI], analogous to those described for scrapie, have now been described as well as an in vitro system for [PSI] propagation. Recently, two new potential prions have been described, one in yeast and the other in the filamentous fungus, Podospora .
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