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Strain back-tracking 101
Three new offline articles in The Lancet
Neuronal storage disease and CJD
Parkinson's Disease: are Lewy bodies congophilic?
Yeast prions form congophilic seed fibers
Both prion allotypes accumulates in sporadic and val 210 ile?
Alpha 1 anti-chymotrypsin polymorphisms in sporadic CJD
Formation of prion fibrils in brain slices

Strain back-tracking 101

18 July 1997 
Commentary by webmaster
TSEs have a property called strain type, which amounts to a molecular memory of their source that can be retained for many passages. This may someday allow the origin of nvCJD, BSE, CWD, and even scrapie to be unambiguously determined, through a process that might be called strain back-tracking.. How does this work and what are the prospects?

Strain back-tracking determines origin based on conformational subtleties of quaternary structure of the the nucleation seed fibril, which are called strain types in TSEs (though sometimes the whole anatomical and behavioral phenotype is thrown in for good measure).

There has been some strain typing done in prions, but, as Collinge has written, there need to be more experimental parameters developed than simple glycosylation ratios for these to be fully resolved. The scatter diagrams seem to spread strains out in two dimensions but unfortunately there is only one actual dimension as they are constrained to the diagonal.

To see the latest example of strains, consider the smooth and wavy forms of the yeast 'prion' seed fibers in the most recent Cell. These are retained over many rounds of recruitment and cytoplasmic inheritance. Transthyretin disorders also have well-studied strain types.

The way strains work, in my view, is that the original rogue prion in the original host animal has a particular stable structure among those available due to its primary sequence and its history of covalent modification, denaturation, and interaction with various chaperones. The final conformer (like most proteins) within the oligomer is at a kinetically available local Gibbs minimum (fairly deep well, one of several) but is not necessarily at a global energy minimum.

In the process of recruitment, the rogue fiber induces a conformational change in recruited normal prion so that it is compatible with existing fiber structure, ie, it joins the 'crystal' by adopting the same conformation as the other monomers. This can happen even though the newly recruited monomer doesn't necessarily have the same covalent structure or history. Once trapped in this conformational potential well, the monomers can stay there for many go-rounds.

You can understand this process quite well by thinking about the military's approach to boot sizes for new soldiers, who later themselves become drill sargeants, or recruitment and generational stability of religous persuasion, or institutional workplace attitude adjustment (learning to be a 'team player').

If the conformer does carry a molecular memory of the initial template, strain back-tracking might work provided there are a large number of experimentally resolvable strain conformers, that these were truly stable ( not just as per some insensitive methodology), and that not too many passages through too many species and genotypes had been involved. Never a proof really, just liklihood.

However it is possible that seed fibers have a dissociative equilibrium and sub-fibers can re-conform themselves once the driving conformer template is gone. Passaging and pooling through a disparate population of varously heterozygous animals of various species then gives a royal heterogeneous mess.

Strain back-tracking also requires the mapping from donor to recipient conformer to be injective (1-1) to be unambiguously invertible. There could be too many marbles and not enough conformer bins: species A and B induce the same conformer in species C.

For scrapie propagating since the seventeenth century, I wouldn't expect much enlightenment from strain back-tracking as to the original source.

I wouldn't be surprised however if strain back-tracking can show that the proximal source (30% ?) of sporadic CJD is scrapie in the UK (perhaps supplanted by proximal PSE in the US and Germany).

If frozen tissue and MBM from the first go-round of BSE were saved and if BSE is essentially a single conformer, the experimental situation is that much easier. I expect strain back-tracking will be able to identify or rule out known scrapie strains as the proximal source of BSE to most people's satisfaction.

I expect strain back-tracking will be able to show convincingly that the proximal source of nvCJD and zoo-TSEs is BSE.

Strain back-tracking is a nightmarish concept in terms of the chain of liability; a parent dies of a certain strain of sporadic CJD and prion in gelatin in the medicine cabinet is later sourced to negligently imported BSE or whatnot.

In mink, two different strains evolved out of one. ..The 3D structure is not solely determined by the infecting prion protein, but also at least sometimes a little by the host's prion protein. -- Roland Heynkes


Alpha 1 anti-chymotrypsin signal peptide polymorphism in sporadic CJD

Neurosci Lett 227 (2): 140-142 (May 16 1997)
Salvatore M, Seeber AC, Nacmias B, Petraroli R, Sorbi S, Pocchiari M
In Creutzfeldt-Jakob disease (CJD), a transmissible spongiform encephalopathy, the deposition of the pathological prion protein (PrP-res) in the brain of affected individuals is the key event that triggers the appearance of the disease. Since a polymorphism in the signal peptide of the serine-protease inhibitor alpha1 antichymotrypsin (ACT) is one of the factors that may enhance amyloid formation, we studied this polymorphism in 63 CJD patients and 103 control subjects.

No difference in allele frequencies and genotype distribution was found between CJD cases and controls, nor any difference was found between the ACT genotype and the age at onset and disease duration. Interestingly, there was a significantly different (P = 0.04) ACT distribution between CJD patients and controls in apolipoprotein E (ApoE) E4, and the interaction between ACT and ApoE was almost significant (P = 0.053). Further studies on a larger number of patients will clarify whether this association can identify a possible risk factor for CJD.

Above is an interesting approach to sporadic CJD and to the old question of what other genes, if any (besides the prion gene itself), have any role in the disease. Because amyloid is formed from various proteolytic fragments of prion protein, the authors looked at variants of an important protease inhibitor to see if it was associated with sporadic CJD. They got nothing, but a lot more could be done, since they just looked at signal peptide variants of one particular protease inhibitor. None of the proteases responsible for prion clipping have been identified; this is difficult to infer from end points since several proteases and endopeptidases might act in sequence. --webmaster

Prion allotypes which accumulate in sporadic and familial CJD.

Nat Med 3 (5): 521-525 (May 1997)
Silvestrini MC, Cardone F, Maras B, Pucci P, Barra D, Brunori M, PocchiariM
In the brain of patients heterozygous for isoleucine substituted for valine at codon 210 (Val 21O lle), PrP-res is formed by both the wild-type and mutant PrP-sen. We also found that in a sporadic CJD patient, who was heterozygous (Met/Val) at position 129, PrPres is also formed by both allotypes.

Some comments by webmaster [22 July 1997]:

Although the recent Nature paper that showed a modest species barrier from scrapie or bse to humans did not look at met/val 129 heterozygotes, there has been a fair number of haphazard observations scattered throughout the literature on allotypes that accumulate in CJD. The possibilities range from interference to independence to synergy to not-that-fussy for the heterozygous situation.

I haven't seen the bibliography of this very recent paper yet but hopefully they reviewed this literature carefully for us. They found that in heterozygotes in familial Val 21O lle CJD (which has one normal allele) _both_ peptide species are recruited to amyloid. In other words, Val 21O lle peptide was able to recruit wild type protein, in addition to itself.

They had a similar result in a case of sporadic met 129 val case, though with sporadic CJD, it is hard to say what the original recruiting species would have been or how it varies from case to case.

I would want to see the spectrum of amino- and carboxy termini too, not just whether amyloid had such-and-such a residue somewhere in the middle. And of course the glycosylation ratio, which is our temporary signature for nvCJD. Even the homozygous situation could be quite a mess.

I have been told that published recruitment of wild type by tyr 145 stop isn't being accepted by some researchers. This mutant, as it stands, shoots down a lot of theories as to which residues are important to the species barrier, since it is missing everything downstream from 145.

I would guess that Silvestrini et al did not determine whether "mixed" amyloid was formed, versus two "pure" amyloid seed fibers. Secondary seed fibers, while slow to initialize, might be more efficient than original seed fibers at further recruitment, and 'take over.' Given this, by looking at a multi-year endpoint, one obtains a rather different quantitation of recruitment; the in vitro approach mostly just looks at pump-priming.

By using a disease screen, some recruitment capability is selected, but It is by no means a forgone conclusion that a mutant prion recruits itself over wild type, particularly for mutations in the docking interface, already optimized in wild type. Mutants aren't the equivalent of the fixed polymorphism or inter-species situations because mutants will be less stable generally and worse at self-docking.

In situ formation of protease-resistant prion protein in transmissible spongiform encephalopathy-infected brain slices.

J Biol Chem 272 (24): 15227-15231 (Jun 13 1997)
Bessen RA, Raymond GJ, Caughey B
In this study we demonstrate in situ conversion of protease-sensitive PrPC to PrP-res in TSE-infected brain slices. One step in this process is the binding of soluble PrPC to endogenous PrP-res deposits. The newly formed PrP-res associated with the slices in a pattern that correlated with the pre-existing brain distribution of PrP-res. Punctate in situ PrP conversion was observed in brain regions containing PrP-res amyloid plaques, and a more dispersed conversion product was detected in areas containing diffuse PrP-res deposits.

These studies provide direct evidence that PrP-res formation involves the incorporation of soluble PrPC into both nonfibrillar and fibrillar PrP-res deposits in TSE-infected brain. Our findings suggest that the in situ PrP conversion reaction leads to additional polymerization of endogenous PrP-res aggregates and is analogous to the process of PrP-res fibril and subfibril growth in vivo.

Progress on familial Parkinson Disease

Science, June 27., pg. 1973 and 2045
Parkinson's Disease has just joined CJD, Alzheimer's and 13 others in a common pathogenic mechanism. It seems that Lewy bodies may be congophilic aggregations containing alpha-synuclein.

PD is a very widespread disorder, with 2% of the population ending up with it, but only a tiny proportion familial, with some iatrogenic, but mostly sporadic.

Granted that all these disease are beta-sheet disorders, does that mean that there could be a universal therapy, a single drug that treats all of them? Yes. Congo red itself binds very specifically to all the inter-subunit anti-parallel beta extensions; CR is not that far from being highly chemically reactive already. However, it comes into play too late in the process to serve as a seed fiber nucleation terminator; in fact it might stabilize them. What is needed is an analogue that would both bind and cap an exposed beta strand, ie, a small capped synthetic beta strand plus an activated CR as covalent joiner. I've modeled a Kinemage of a specific prototype agent and juxtaposed it to transthyretin for illustrative purposes, though it would be structurally the same idea for all of them. -- webmaster

The awakening of a-synuclein

Commentary and article in the July 17, 1997 Nature News and Views.            Michel Goedert 
Nature 388, 232ā233 (1997) 
Nature 388, 228ā229 (1997)
Familial Parkinsonľs disease occurs in a rare familial (inherited) form. At long last researchers have identified a gene which, when mutated, is at least in part responsible for the condition „ the mutation changes the amino acid alanine to threonine at a particular position in a protein called a-synuclein. It remains to be seen whether this finding will tell us more about the much more common sporadic form of the disease.

Mutation in the alpha-Synuclein Gene Identified in Families with Parkinson's Disease

Science Volume 276, Number 5321, Issue of 27 June 1997, pp. 2045-2047
Mihael H. Polymeropoulos, *Christian Lavedan, ...Alice M. Lazzarini, Roger C. Duvoisin,  ... Robert L. Nussbaum 
Parkinson's disease (PD) is a common neurodegenerative disorder with a lifetime incidence of approximately 2›percent. A pattern of familial aggregation has been documented for the disorder, and it was recently reported that a PD susceptibility gene in a large Italian kindred is located on the long arm of human chromosome 4.›A mutation was identified in the -synuclein gene, which codes for a presynaptic protein thought to be involved in neuronal plasticity, in the Italian kindred and in three unrelated families of Greek origin with autosomal dominant inheritance for the PD phenotype. This finding of a specific molecular alteration associated with PD will facilitate the detailed understanding of the pathophysiology of the disorder.

Parkinson's disease (PD) was first described by James Parkinson in 1817›(1). The clinical manifestations of this neurodegenerative disorder include resting tremor, muscular rigidity, bradykinesia, and postural instability. A relatively specific pathological feature accompanying the neuronal degeneration is an intracytoplasmic inclusion body, known as the Lewy body, which is found in many regions, including the substantia nigra, locus ceruleus, nucleus basalis, hypothalamus, cerebral cortex, cranial nerve motor nuclei, and the central and peripheral divisions of the autonomic nervous system (1).

In many cases a heritable factor predisposes to the development of the clinical syndrome (2). We have recently shown that genetic markers on human chromosome 4q21-q23 segregate with the PD phenotype in a large family of Italian descent (3). The clinical picture of the PD phenotype in the Italian kindred has been well documented to be typical for PD, including Lewy bodies, with the exception of a relatively earlier age of onset of illness at 46›Ī›13›years. In this family the penetrance of the gene (the proportion of people with the genotype who actually manifest the disease) has been estimated to be 85%, suggesting that a single gene defect will be sufficient to determine the PD phenotype.

The Ala53Thr substitution is localized in a region of the protein whose secondary structure predicts an ›helical formation, bounded by ›sheets. Substitution of the alanine with threonine is predicted to disrupt the ›helix and extend the ›sheet structure. Beta pleated sheets are thought to be involved in the self-aggregation of proteins, which could lead to the formation of amyloid-like structures (9). This was already tested in the case of NAC35, the 35-amino acid peptide derived from -synuclein that was first isolated from plaques found in patients with Alzheimer's disease (4, 9). NAC35 is located in the middle of the -synuclein molecule and extends from amino acid position 61›to 95.›Residue 53,›which is mutated in PD, is outside the NAC35 peptide found in amyloid plaques. However, the true size of the peptide involved in the plaques is not known as the protease used to isolate the peptide cuts at lysine 60›of the -synuclein protein. In cross-linking experiments with ›amyloid, it was demonstrated (9) that residues 1›to 56›and 57›to 97›specifically bind amyloid and that a synthetic peptide consisting of residues 32›to 57›performed similarly.

Three members of the synuclein family have been characterized in the rat, with SYN1 exhibiting 95% similarity to the human -synuclein protein (10). SYN1 of the rat is expressed in many regions of the brain, with high levels found in the olfactory bulb and tract, the hippocampus, dentate gyrus, habenula, amygdala, and piriform cortex, and intermediate levels in the granular layer of the cerebellum, substantia nigra, caudate-putamen, and dorsal raphe (10). This pattern of expression coincides with the distribution of the Lewy bodies found in brains of patients with Parkinson's disease. Decreases in olfaction often accompany the syndromic features of Parkinson's disease, and it is proposed that in many cases hyposmia (decreased sense of smell) is an early sign of the illness (11).

In the zebra finch the homolog to -synuclein, synelfin, is thought to be involved in the process of song learning, suggesting a possible role for synuclein in memory and learning (12). In contrast to humans, rats have a threonine at the same position in their homologs to the human -synuclein gene (Fig. 4). Similarly, the zebra finch synelfin carries a threonine, whereas both Bos taurus and Torpedo californica (13) do not. There are no reports that suggest the presence of Lewy bodies in the brains of the rat or the zebra finch or a phenotype resembling that of PD. Lack of any phenotype could be explained by a combination of factors, such as the relatively short life-span of rodents, the need for interaction with other cellular components not present in the rat, absence of a critical environmental trigger in the rodents, or a requirement for heterozygous status for the production of a phenotype.

Alpha-synuclein, a presynaptic nerve terminal protein, was originally identified as the precursor protein for the non- amyloid component of Alzheimer's disease amyloid plaques NAC (4). The human ›synuclein gene was previously mapped in the 4q21-q22 region (5). Genotype analysis in the Italian PD kindred with additional genetic markers showed recombination events. One recombination was observed for genetic marker D4S2371 at the centromeric end of the PD interval and one recombination was inferred for marker D4S2986 at the telomeric end of the interval. These recombinations redefined the location of the PD gene to an interval of approximately 6›cM between markers D4S2371 and D4S2986 (6)

Thus, alpha-synuclein represented an excellent candidate gene for PD. Sequence analysis of the fourth exon of the -synuclein gene (7) revealed a single base pair change at position 209 from G to A (G209A) relative to the published sequence of the gene (GenBank ID L08850), which results in an Ala to Thr substitution at position 53›(Ala53Thr) and the creation of a novel Tsp45 I restriction site (Fig. 1). Mutation analysis for the G209A change in the Italian kindred showed complete segregation with the PD phenotype with the exception of individual 30,›who is affected but not carrying this mutation (Fig. 2A). This individual apparently inherited a different PD mutation from his father because we have shown that he shares a genetic haplotype with his unaffected maternal uncle, individual 3,›for genetic markers in the PD linkage region.

Studies of early onset AD have previously documented that missense mutations can cause an adult onset neurodegenerative disorder. Of the 31›mutations described so far in the loci for presenilin 1›and 2,›30›were missense and 1›was a splice variant (14). Missense mutations in the prion protein have also been implicated in the amyloid production seen in GSS and Creutzfeld-Jakob diseases, both forms of spongiform encephalopathy (15). Studies in these neurodegenerative disorders have pointed to the importance of the physical chemical properties of mutant cellular proteins in initiating and propagating neuronal lesions leading to disease. Similar studies in the synuclein protein family may provide valuable insights into the etiology and pathogenesis of PD.

Although the mutation identified in the alpha-synuclein gene is unlikely to account for the majority of sporadic and familial cases of PD, it may account for a significant proportion of those early-onset families with PD characterized by a highly penetrant, autosomal dominant inheritance. Even if the mutation we have described is directly related to only a small fraction of the total number of PD patients, it provides a clue that should lead to the understanding of the underlying pathways resulting in the symptoms of PD.

Commentary by Gretchen Vogel: The link to -synuclein does not provide any easy answers to what causes Parkinson's, but it does offer some tantalizing clues. Proteins must fold up into a three-dimensional structure to function, and the researchers suggest that the mutation causes -synuclein to misfold. It might then produce abnormal deposits in the brain, much as the accumulation of a protein called amyloid in neurotoxic deposits may contribute to nerve degeneration in Alzheimer's disease. Just as the brains of Alzheimer's patients are riddled with these plaques, Parkinson's brains are studded with inclusions called Lewy bodies.

Polymeropoulos notes that the -synuclein mutation, which replaces the amino acid alanine with a threonine, has the potential to cause the protein to misfold. Although the exact conformation of -synuclein is not known, alanine is commonly found in a coiled structure, called an helix, while threonine is common in a more rigid and insoluble structure called a sheet--the same kind of formation that is suspect in the formation of Alzheimer's plaques. The next step, Polymeropoulos says, is to find out if the Lewy bodies actually contain -synuclein.

The possibility that -synuclein misfolding leads to Parkinson's is very much an unproven hypothesis, however, and it does not jibe with a current leading theory of what causes the disease. Evidence has been building that oxidative stress--the buildup of cell-damaging compounds called free radicals--may be behind the neuron loss. "It's not apparent how to link the two theories," says John Trojanowski, a pathologist at the University of Pennsylvania Medical Center in Philadelphia who has studied both Alzheimer's disease and Lewy bodies.

Deepening the mystery of how the mutation causes disease are the versions of -synuclein found in the rat and mouse. These proteins already have a threonine where the normal human protein has an alanine, yet the threonine causes no apparent problems in the animals. The rodents' short life-span may be one explanation for the paradox, the authors suggest: The animals may simply die before they can develop the disease. The researchers also raise the possibility that the mutation in humans may disrupt--or encourage--the interaction of -synuclein with another protein not present in rodents. Finally, Polymeropoulos notes that the mutation in the Italian family is dominant--only one of the two gene copies is mutated--and the normal and mutant forms of the protein may have to interact to cause problems.

To try to figure out just what the -synuclein mutation does, researchers would like to transfer the mutant gene into mice to see whether they can re-create Parkinson's in the animals. The natural threonine in the mouse suggests that the transgenic animals might not get sick, Polymeropoulos concedes. The researchers hope they might succeed by mimicking the genetic endowment of the Italian and Greek patients: knocking out both copies of the mouse gene and then substituting one normal human gene and one with the threonine substitution.

But even if researchers can pin down how the -synuclein gene mutation leads to Parkinson's in the Italian family, they won't completely solve the riddle of the disease. "Parkinson's disease is going to be a 100-piece puzzle," Polymeropoulos says. "-Synuclein may be a central piece of the puzzle and will hopefully give us a picture of what it will look like when it is done. But we should be prepared for another 99 pieces."

Dominantly inherited dementia and parkinsonism, with non-Alzheimer amyloid plaques: a new neurogenetic disorder.

Ann Neurol 25 (2): 152-158 (Feb 1989) 
Rosenberg RN, Green JB, White CL 3d, Sparkman DR, DeArmond SJ, Kepes JJ
A family is described in which a dominant form of inheritance, probably autosomal dominant, expresses severe dementia and parkinsonism as the major clinical features. Neuropathological correlates in two autopsied members of this family consisted of extracellular hyaline eosinophilic, congophilic amyloid plaques in decreasing order of frequency in the cerebral cortex, basal ganglia, thalamus, and substantia nigra, and atrophy and gliosis of the basal ganglia and substantia nigra. The extracellular plaques did not stain with antibody raised against the prion protein nor with two separate anti-amyloid A4 antibodies. The combination of dominantly inherited dementia with parkinsonism and extracellular plaques in this distribution that are amyloid and prion protein antibody negative has not been previously reported and thus may represent a new neurological genetic disorder.

Prion-inducing domain 2-114 of yeast sup35 protein transforms in vitro into amyloid-like filaments.

Proc Natl Acad Sci U S A 94 (13): 6618-6622 (Jun 24 1997) 
(contains a  good color image of the congophilic fibrils)
 King CY, Tittmann P, Gross H, Gebert R, Aebi M, Wuthrich K
see also recent article in Science
The yeast non-Mendelian genetic factor PS], which enhances the efficiency of tRNA-mediated nonsense suppression in Saccharomyces cerevisiae, is thought to be an abnormal cellular isoform of the Sup35 protein. Genetic studies have established that the N-terminal part of the Sup35 protein is sufficient for the genesis as well as the maintenance of PSI. Here we demonstrate that the N-terminal polypeptide fragment consisting of residues 2-114 of Sup35p, Sup35pN, spontaneously aggregates to form thin filaments in vitro. The filaments show a beta-sheet-type circular dichroism spectrum, exhibit increased protease resistance, and show amyloid-like optical properties.

It is further shown that filament growth in freshly prepared Sup35pN solutions can be induced by seeding with a dilute suspension of preformed filaments. These results suggest that the abnormal cellular isoform of Sup35p is an amyloid-like aggregate and further indicate that seeding might be responsible for the maintenance of the PSI element in vivo.

Self-seeded fibers formed by Sup35, the protein determinant of [PSI+], a heritable prion-like factor of S. cerevisiae.

Cell 89 (5): 811-819 (May 30 1997) 
Glover JR, Kowal AS, Schirmer EC, Patino MM, Liu JJ, Lindquist S
The PSI+ factor of S. cerevisiae represents a new form of inheritance: cytosolic transmission of an altered phenotype is apparently based upon inheritance of an altered protein structure rather than an altered nucleic acid. The molecular basis of its propagation is unknown. We report that purified Sup35 and subdomains that induce [PSI+] elements in vivo form highly ordered fibers in vitro. Fibers bind Congo red and are rich in beta sheet, characteristics of amyloids found in certain human diseases, including the prion diseases. Some fibers have distinct structures and these, once initiated, are self-perpetuating. Preformed fibers greatly accelerate fiber formation by unpolymerized protein. These data support a "protein-only" seeded polymerization model for the inheritance of PSI+.

Quotes from the text:

By electron microscopy, these fibers have at least two distinct structures. Once a particular structure is initiated, it is apparently self-perpetuating, continuing along the entire length of the fiber. The time course of fiber formation indicates that this is a self-seeded pro

We conclude that Hsp104 is not absolutely required for the coalescence of Sup35 into [PSI+]-like foci when this protein is expressed at a high level.

Fibers formed by whole Sup35, and by NM, also bound Congo red, although we did not observe apple green birefringence by polarized light microscopy. Bound dye exhibited a spectral shift with a maximum difference at 540 nm (Figure 3A), the same shift reported for Congo red bound to amyloid proteins (Klunk et al., 1989 ). Scatchard analysis of Congo red binding to preformed NM fibers indicated an average of 4.4 binding sites per NM monomer, with a Kd of 250 nM (Figure 3B).

We also examined fiber formation with a mutant protein lacking amino acids 22-69 (including two of the four nonapeptide repeats that characterize this domain) that is defective in [PSI+] induction in vivo

the backbone of Sup35 can assume distinct structural forms, "straight" or "wavy." These data indicate that (1) self-association of N dictates the formation of the fiber axis, (2) M packs against the exterior of this core, and (3) C is located on the periphery and orderly packing of this domain is not critical for the stability of the fiber. ...Most remarkably, transitions between wavy and straight fibers were never observed. Thus, once a specific type of packing is established in the fiber, it is self-perpetuating along the length of the fiber.

Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy

J. Virol. 68, 7858-7868.
Bessen, R.A., and Marsh, R.F. (1994)

Genesis and variability of [PSI] prion factors in Saccharomyces cerevisiae.

Genetics 1996 Dec;144(4):1375-1386
Derkatch IL, Chernoff YO, Kushnirov VV, Inge-Vechtomov SG, Liebman SW
Finally, we find that [PSI] factors of different efficiencies and different mitotic stabilities are induced in the same yeast strain by overproduction of the identical Sup35 protein. We suggest that the different [PSI]-containing derivatives are analogous to the mysterious mammalian prion strains and result from different conformational variants of Sup35p.

In Vitro Propagation of the Prion-Like State of Yeast Sup35 Protein.

 Science 1997 Jul 18;277(5324):381-383
Paushkin SV, Kushnirov VV, Smirnov VN, Ter-Avanesyan MD
The yeast cytoplasmically inherited genetic determinant [PSI+] is presumed to be a manifestation of the prion-like properties of the Sup35 protein (Sup35p). Here, cell-free conversion of Sup35p from [psi-] cells (Sup35ppsi-) to the prion-like [PSI+]-specific form (Sup35pPSI+) was observed. The conversion reaction could be repeated for several consecutive cycles, thus modeling in vitro continuous [PSI+] propagation. Size fractionation of lysates of [PSI+] cells demonstrated that the converting activity was associated solely with Sup35pPSI+ aggregates, which agrees with the nucleation model for [PSI+] propagation. Sup35pPSI+ was purified and showed high conversion activity, thus confirming the prion hypothesis for Sup35p.

The Lancet July 18, 1997

(None of the 3 articles are online.)

Is the neuropathology of new variant CJD and kuru similiar?

P L Lantos, and others

Type of prion protein in UK farmers with CJD

A F Hill and others

High sequence homology of the PrP gene in mule deer and Rocky Mountain elk

L Cerven∑kov∑ and others

Heritable disorder resembling neuronal storage disease in mice expressing prion protein with deletion of an alpha-helix.

Nat Med 1997 Jul;3(7):750-755 
Muramoto T, DeArmond SJ, Scott M, Telling GC, Cohen FE, Prusiner SB
Mice were constructed carrying prion protein (PrP) transgenes with individual regions of putative secondary structure deleted. Transgenic mice with amino-terminal regions deleted remained healthy at >400 days of age, whereas those with either of carboxy-terminal alpha-helices deleted spontaneously developed fatal CNS illnesses similar to neuronal storage diseases.

Deletion of either C-terminal helix resulted in PrP accumulation within cytoplasmic inclusions in enlarged neurons. Deletion of the penultimate C-terminal helix resulted in proliferation of rough endoplasmic reticulum. Mice with the C-terminal helix deleted were affected with nerve cell loss in the hippocampus and proliferation of smooth endoplasmic reticulum. Whether children with the human counterpart of this malady will be found remains to be determined.

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