Prion Doppel Alternative Splicing
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"Alternative splicing" across prion-doppel tandem duplication
Elimination of prions by branched polyamines and implications for therapeutics
Effects of a disrupted disulfide loop
H187R: novel PRNP sequence variant associated with familial encephalopathy.
P101L in mouse alters TSE transmission time
Prominent psychiatric features, early onset in a new insertional mutation
How to improve the clinical diagnosis of CJD
Oral transmission and early lymphoid tropism of CWD
Mutational Analysis of Het-s Prion Analog of Podospora anserina
Interplay of yeast psi prion chaperones

Odd new doppel transcripts

GenBank entries AF192382-85  29 Nov 99 webmaster commentary
Four new GenBank entries describing new doppel mRNAs appeared on 21 Nov 99 from a Japanese group. A paper on ataxic mice is said to be in press. This would be only the second print article on doppel. The authors have no overlap to those of the first paper yet must be using the same mouse strain, C57BL/6/129Sv Ngsk PrP-deficient mice, that have a deletion of the prion coding region that extends 5' to obliterate the splice junction at prion intron2/exon3.

Puzzling out these inadequately annotated sequences with Blastn, it appears that in the deletion strain, 2 unusual mRNAs were found. AF192383 begins with exon 1 and 2 of the prion gene, skips to intergenic exon 2, then continues normally to doppel exon 2a and exon 3. AF192382 is similarly chimeric and also skips intergenic exon 1 but it additionally skips (reads through) the polyA sites of exon 2a and 2b.

Identification of a novel gene encoding a PrP-like protein expressed as chimeric transcripts fused to PrP exon1/2 in ataxic mouse line with a disrupted PrP gene

Cell. Mol. Neurobiol. (2000) In press
Li,A., Sakaguchi,S., Atarashi,R., Roy,B.C., Nakaoke,R., Arima,K., Okimura,N., Kopacek,J. and Shigematu,K
AF192382 3386 bp doppel CDS 256..795 chimeric mRNA
AF192383 2149 bp doppel CDS 256..795 chimeric mRNA
AF192384 3192 bp doppel CDS  62..601 long mRNA
AF192385 1955 bp doppel CDS  62..601 short mRNA
AF192382        U29187 numbering
1 -     48       6205 - 6252 exon 1 6205..6251
46 -   145       8440 - 8539 exon 2 8442..8539
146 -  234      29572 -29660 rpt_family="MLT1B" exon 1 34086..34124 skipped
248 - 3368      36204 -39316 exon 2a 36205..36799 cds 36212..36751
                             exon 2b 36205..37472
                             exon 3  38013..39315
823....2058 missing in others 795 is stop   256 start
In the background strain, chimeric mRNAs are not reported. AF192385 is identical to clone 9 mRNA of the JMB paper; however, AF192385 exhibits read-through unlike any mRNA reported earlier.

In summary, "alternative splicing" across the tandem duplication of prion and doppel creates a rich tapestry of possibilities for mature mRNAs depending on mouse strain, tissue, developmental stage and coordinated regulatory requirements. None of these have the effect of omitting protein-coding exons (as occurs so commonly in other genes) because neither doppel nor prion has internal introns. Simlarly, no fusion protein can be enabled because all polyA sites are external to CDS and stop signals.

The mouse system thus will not serve as a direct model for human (which already differs in not using prion exon 2), though the bottom lines may still be similar.

Looking about for other tandem duplication situations with exon-skipping during splicing of pre-mRNA, an article concerning human CDC42 gene provides an interest counterpart to the mouse prion-doppel situation. [Hum Genet 1999 Jul-Aug;105(1-2):98-103 ] What happened here is that the terminal coding exon #6 and its associated 3' UTR were involved in a tandem duplication, creating the following situation: ....exon5-intron5-exon6utrpolyA-intron6-exon6'utrpolyA...

The splice donor at the start of intron 5 now sees two identical splice acceptors, the original one just before exon 6 and the duplicate before exon 6'. Because these terminal exons each contain polyA signals, whichever splice results in the protein terminating either with the coding sequence of exon 6 or the initially identical one in exon 6' (rather than in a protein with an amino acid repeat 66').

What happens next? Did the two exons "fight" over the splice donor (lest they become truncated pseudogenes)? No, they split along tissue-expression lines: exon 6 became specialized to brain and exon 6' to placenta. Over time, the 3' parts of coded protein (as well as the 3'UTRs) diverged and adapted to slightly different functions, giving 2 genes for the price of 1 (internal partial paralogues).

This is a very special case of alternate splicing because of the tandem repeat nature of the alternative exons and their terminational self-sufficiency.

The history of prion and doppel is similar in many aspects but on a larger scale: the duplication began after 5' UTR exon 2 and encompassed the whole coding region. Again, there were initially 2 identical competing splice acceptors. The specializations may have involved not so much tissue type expression as location (doppel free in matrix or plasma, prion attached to outside of cell).

Another very common situation seen in tandem duplicatation is complementary degeneration, eg doppel lost the repeat region and 106-126 through a later deletion (as did the carboxy ester lipase CEL locus: Gene 239 273 1999) and prion may have lost the second disulfide and third glycosylation sites.

It was once thought for the vast majority of tandem duplication cases, that one member was destined to become a pseudogene (relic) but now it seems that a lot of fine-tuning of cellular function can be done by retaining both. The outcome class depends on the boundaries of the duplication relative to initial gene features.

Another gene with very interesting parallels to the prion-doppel doublet is CEL gene doublet mentioned above. The original gene had internal repeats of a proline-rich 11 amino acid region C-terminally. A tandem duplication occurred, mechanistically like that of a retrotransposon, leaving 365 bp flanking direct repeats about the copy.

The original gene then experienced a deletion of exons 2-7, as well as internal stop mutations, leaving it a pseudogene. (It continued to be transcribed at low levels without the former tissue specificity.) This resembles the deletion in doppel, though this did not leave doppel a pseudogene.

The tandom copy took over the former function completely. The duplication took place prior to divergence of old world monkeys and great apes; the deletion came much later in the great ape lineage only. Macaque have the duplication but not the deletion. This suggests doppel lineages could exist lacking the deletion of the repeat and 106-126.

The repeat region in both CEL gene and pseudogene continued to evolve by replication slippage like the prion gene, resulting in different numbers of repeats in various lineages and gene lineages. Sort of an odd behavior for a pseudogene but actually to be expected. The repeat contains a site for O-glycosylation.

The chromosome 22 completion showed further bizarre goings-on, such as active genes fully embedded on the minus strand of introns of larger active genes. A copy of the prion gene, embedded in this manner and degenerated to a pseudogene, might produce the anti-prion mRNA studied some years back. In other words, as the larger gene is transcribed and its mRNA spliced, a plus strand prion mRNA is released. Anti-sense regulation now has a fair number of precedents in other genes.

For that matter, how would we even know if prion-doppel themselves were embedded within an intron of a larger gene? What motivates this is that the vast majority of genes have many, many smallish coding exons whereas prion and doppel have but one. The process of pseudogene formation by retrotransposition purges all intervening introns. That mRNA might have been alternatively spliced, omitting a goodly number of exons from the original gene. This would leave the prion gene as single coding exon with poor homology to the parent gene because of many large gaps (pseudo-orphan), the situation we see today.

H187R: novel PRNP sequence variant associated with familial encephalopathy.

Am J Med Genet 1999 Dec 15;88(6):653-656
Cervenakova L, Buetefisch C, Lee HS, Taller I, Stone G, Gibbs CJ Jr, Brown P, Hallett M, Goldfarb LG
Human transmissible spongiform encephalopathies (TSEs) are a group of chronic progressive neurodegenerative disorders that may be hereditary, infectious, or sporadic. Hereditary TSEs are associated with mutations in the PRNP gene on chromosome 20p12-pter. We report on a family in which seven patients developed limb and truncal ataxia, dysarthria, myoclonic jerks, and cognitive decline. The age of onset in the 30s, 40s, or 50s, prolonged disease duration, cerebellar atrophy on imaging, and the presence of synchronic periodic discharges on electroencephalogram suggested a familial encephalopathy resembling Gerstmann-Straussler-Scheinker disease. A novel H187R mutation has been identified in affected, but not in unaffected, family members or unrelated controls suggesting a pathogenic role for this mutation.

Comment (webmaster): With H187R the issue is with codon 129 haplotype and geneology details (and whether they got back to the founder). For a recent founder and small families, the numbers might just not be there to make an overwelming case for causality though that seems the likliest scenario.

It is important to locate the full kindred because these are the individuals that might benefit from therapeutic developments (because they could receive treatment pre-symptomatically).

A new allelic variant in the bovine prion protein gene (PRNP) coding region.

Anim Genet 1999 Oct;30(5):386-7  [off-line, no abstract]
Schlapfer I,  Saitbekova N, Gaillard C, Dolf G
The 7x repeat bovine sequence appeared on GenBank on 23 Mar 99 and was analyzed on this site earlier.

7x repeats is getting perilously close to pathogenic dimensions so it will be interesting to see whether this was from an affected animal or whether it is a common allele. A rare case of familial BSE remains viable as the initial source of the English epidemic.

7 repeat cow

   G  G  W  N  T  G  G  S  R  Y  P  G  Q  G  S  P  G  G  N  R
 Y  P  P  Q  G  G  G  G  W  G  Q  P  H  G  G  G  W  G  Q  P
 H  G  G  G  W  G  Q  P  H  G  G  G  W  G  Q  P  H  G  G  G
 W  G  Q  P  H  G  G  G  W  G  Q  P  H  G  G  G  G  W  G  Q
 G  V  P  last 2 aa inconsequential PCR frameshift error

Elimination of prions by branched polyamines and implications for therapeutics

Proc. Natl. Acad. Sci. USA 1999 96(25): p. 14529-14534
Surachai Supattapone, Hoang-Oanh B. Nguyen, Fred E. Cohen, Stanley B.Prusiner, and Michael R. Scott
This is an interesting article involving an odd class of compounds. While these branched polyamines did not emerge from a drub-screening program, the methods here do allow drug screening. It is too soon to say whether the compounds here will be medically useful and no data here supports the idea that these compounds will work on amyloid diseases in general. However, it makes sense to search for therapeutics targeted to common features of amyloid rather than do each disease separately -- some are so rare that the effort would not materialize.

The authors note that except for familial CJD, there is not adequate lead time even if a miracle compound were in hand. There is no way to screen 270 million people regularly even if a blood test were established, ie, no way to anticipate sporadic CJD. Even for high risk populations, such as in the UK, the logistics of determining met/met and the incidence of side effects make matters problematical.


We report that branched polyamines, including polyamidoamide dendimers, polypropyleneimine, and polyethyleneimine, are able to purge PrPSc, the protease-resistant isoform of the prion protein, from scrapie-infected neuroblastoma (ScN2a) cells in culture. The removal of PrPSc by these compounds depends on both the concentration of branched polymer and the duration of exposure. Chronic exposure of ScN2a cells to low noncytotoxic concentrations of branched polyamines for 1 wk reduced PrPSc to an undetectable level, a condition that persisted at least 3 wk after removal of the compound. Structure-activity analysis revealed that a high surface density of primary amino groups is required for polyamines to eliminate PrPSc effectively from cells. The removal of PrPSc by branched polyamines is attenuated by chloroquine in living cells, and exposure of scrapie-infected brain extracts with branched polyamines at acidic pH rendered the PrPSc susceptible to protease in vitro, suggesting that endosomes or lysozomes may be the site of action. Our studies suggest that branched polyamines might be useful therapeutic agents for treatment of prion diseases and perhaps a variety of other degenerative disorders.

Here we report that noncytotoxic concentrations of branched polyamines can rapidly eliminate PrPSc from chronically infected ScN2a cells. These compounds appear to act by stimulating normal cellular mechanisms to destroy PrPSc. Once purged of prions, the treated cells remain free from evidence of scrapie infection during repeated serial passage in polyamine-free media. It remains to be determined whether branched polyamines will find use as therapeutic agents in prion diseases and other degenerative central nervous system disorders characterized by deposits of abnormal proteins such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and frontotemporal dementia

SuperFect transfection reagent was purchased from QIAGEN. SuperFect is a mixture of branched polyamines derived from heat-induced degradation of a polyamidoamide (PAMAM) dendrimer (30). Therefore, we investigated the ability of several other branched and unbranched polymers to eliminate PrPSc from ScN2a cells.

The preceding results demonstrate the potent ability of branched polyamines to clear PrPSc from ScN2a cells within a few hours of treatment. To explore whether these compounds could be used as a potential therapeutic for treatment of prion disease, we tested whether they were cytotoxic for ScN2a cells, using as criteria cell growth, morphology, and viability as measured by trypan blue staining. None of the compounds was cytotoxic to ScN2a cells after exposure for 1 wk at concentrations up to 7.5 µg/ml (data not shown)... Cells may be maintained indefinitely in culture in the presence of therapeutic levels of branched polyamines.

The ability of chloroquine to attenuate the ability of branched polyamines to remove PrPSc is consistent with the notion that these agents act in either endosomes or lysosomes. More work is required to determine the precise mechanism by which chloroquine attenuates the clearance of PrPSc by branched polyamines.

Clarification of the exact mechanism of PrPSc elimination by branched polyamines is an important objective. Although a number of possible scenarios exist, several possibilities may be excluded already. One possibility that we considered, that polyamines act by induction of chaperones such as heat-shock proteins that mediate PrP refolding, can be eliminated because of our apparent success in reproducing the phenomenon in vitro.

Sulfated polyanions may inhibit PrPSc accumulation in ScN2a cells by directly binding to PrPC, but because branched polyamines are able to clear preexisting PrPSc, their mechanism of action cannot simply involve binding to PrPC and inhibiting de novo synthesis....Observations rule out endosome rupture as the mechanism by which branched polyamines remove PrPSc.

Several scenarios remain possible. (i) Branched polyamines may bind directly to PrPSc arranged as an amyloid with exposed negatively charged moieties and induce a conformational change under acidic conditions. (ii) Treatment of PrP 27-30 with acid decreases turbidity and increases -helical content, suggesting that such conditions might dissociate PrPSc into monomers (43). It is therefore possible that polyamines bind to an equilibrium unfolding intermediate of PrPSc present under acidic conditions. (iii) Alternatively, polyamines might sequester a cryptic negatively charged component bound to PrPSc that is essential for protease resistance, but which is released only when PrPSc undergoes an acid-induced conformational change. Such a component might act as a chaperone for PrPSc inside endosomes or lysosomes. (iv) Finally, another possibility is that polyamines activate an endosomal or lysosomal factor that can induce a conformational change in PrPSc. Clearly, more work will be required to determine the precise mechanism by which branched polyamines destroy PrPSc.

The in vitro assay described here may find more general application in the search for drugs that effectively treat as well as prevent a number of degenerative and inherited diseases, where the accumulation of proteins seems to mediate the pathogenesis of these illnesses. By simulating lysosomes, where proteases hydrolyze proteins under acidic conditions, we were able to evaluate rapidly the efficacy of a variety of polyamines to induce degradation of PrPSc. It may be possible to develop similar screening assays with wild-type and mutant proteins like the amyloid precursor protein, alpha-synuclein, superoxide dismutase, tau, amylin, and transthyretin, found to accumulate in some of the disorders mentioned below.

Mutational Analysis of Het-s Prion Analog of Podospora anserina

Genetics, Vol. 153, 1629-1640, December 1999
Virginie Coustou, Carole Deleub, Sven J. Saupe, and Joël Bégueret 
The het-s locus is one of nine known het (heterokaryon incompatibility) loci of the fungus Podospora anserina. This locus exists as two wild-type alleles, het-s and het-S, which encode 289 amino acid proteins differing at 13 amino acid positions. The het-s and het-S alleles are incompatible as their coexpression in the same cytoplasm causes a characteristic cell death reaction.

We have proposed that the HET-s protein is a prion analog. Strains of the het-s genotype exist in two phenotypic states, the neutral [Het-s*] and the active [Het-s] phenotype. The [Het-s] phenotype is infectious and is transmitted to [Het-s*] strains through cytoplasmic contact.

Het-s and het-S were associated in a single haploid nucleus to generate a self-incompatible strain that displays a restricted and abnormal growth. In the present article we report the molecular characterization of a collection of mutants that restore the ability of this self-incompatible strain to grow. We also describe the functional analysis of a series of deletion constructs and site-directed mutants. Together, these analyses define positions critical for reactivity and allele specificity.

We show that a 112-amino-acid-long N-terminal peptide of HET-s retains [Het-s] activity. Moreover, expression of a mutant het-s allele truncated at position 26 is sufficient to allow propagation of the [Het-s] prion analog.

Comment (webmaster): The protein is not available at GenBank under het-s but rather as small s and big S. Their previous publication, PNAS 1997 Sep 2;94(18):9773-8 is relevent; sequencing was done in Genetics, Vol 135, 45-52.

The proteins bare no resemblance to other proteins with prion-like properties:



This case need a great deal more work to bring it up to a par with work on yeast prion-like proteins. It is sometimes touted as the only natural use of cross-beta aggregation but the fungus is never studied in the wild, the phenomenon has never been demonstrated to occur under natural conditions, and the encounter is lethal (how useful is that?). Podospora anserina is a little-studied organism with only 258 publications on medline, only 9 of which were published in the last year.

Prominent psychiatric features and early onset in an inherited prion disease with a new insertional mutation in the prion protein gene

Brain, Vol. 122, No. 12, 2375-2386, December 1999
Jean-Louis Laplanche, Khalid Hamid El Hachimi, Isabelle Durieux,...Jean-François Foncin and
Alain Destée
This 8x is different from previously reported 1 2 2 3g3 2 2 2 2...2 2 3 4 and 1 2 2 3 2 2 2 2 2...2 2 2a4. The new total is 63 genetic events in the prion gene. Mutations reported here are forwarded to SwissProt and OMIM mutational database curators. prion point mutations
prion repeat insertions
prion repeat deletions

"In five generations of the French M-E kindred, 11 members are now known to be or have been affected by a form of spongiform encephalopathy previously recorded as Gerstmann­Sträussler­Scheinker disease. Mean age at onset was 28 years (range 21­34 years).

In six instances, these patients were hospitalized in psychiatric institutions with various diagnoses, the most frequent being mania or mania-like symptoms. Dementia occurred progressively after a lengthy course. Histological studies showed atrophy of the cerebellar molecular layer, which contained kuru and multicentric plaques labelled with anti-prion protein antibodies. Spongiosis was not prominent and remained largely limited to the periphery of plaques; it was more marked in the thalamus, where plaques were scarce.

A 192 base pair (bp) insert (eight extra repeats of 24 bp) in the octapeptide coding region of the prion protein gene (PRNP) within a codon-129 methionine allele was found in four symptomatic subjects. Early age at onset, the prominence of psychiatric symptoms and the long course of the disease are noticeable clinical features in this family with an inherited prion disease due to a new insertional mutation in PRNP.

Comment (webmaster): The sequence in the paper above from Laplanche et al is indeed a novel repeat insertion mutation, R12232222a22234, that differs from two previous 8x repeats, namely R1223g322222234 and R122322222222a4. Codon 129 is methionine on the mutant strand.

This makes for 27 different mutations of this type. While the same repeat has on occasion been found in seemingly different kindred (ie, different founders), on the whole nearly every replication slippage event has a different outcome. Once the DNA polymerase gets off track, there is no predicting what it will do before it recovers. However, the very narrow set of accompaning point mutations that sometimes occur in the initial proline and penultimate glycine and the continuing absence of R1 and R4 among the repeated octapeptides are manifestly process constraints.

They reportedly found further missense and silent mutations. Clearly the list of pathogenic mutations is nowhere near saturated. It is equally clear that a lot of alleles were missed in previous years and a lot of CJD was misdiagnosed as sporadic. (It is not that unusual even in 1999 for researchers to just take a cheap shot at 129, 102, 178, 200, and a few other sites.)

 P   H   G   G   G   G   W   G   Q

How to improve the clinical diagnosis of CJD

Brain, Vol. 122, No. 12, 2345-2351, December 1999
Sigrid Poser, Brit Mollenhauer, ... Hans A. Kretzschmar and Klaus Felgenhauer
This paper describes a prospective follow-up of 364 patients initially notified as suspected CJD to a Surveillance Unit in Göttingen, Germany. Six patients were diagnosed as having genetic prion disease by blood analysis and were excluded from the study. After examination and review of the remaining 358, 193 were classified as probable Creutzfeldt­Jakob disease.

However, autopsy revealed that five of the 193 did not have CJD (four cases, Alzheimer's disease; one case, cerebral lymphoma). Of the 54 patients classified as possible CJD, 10 had another diagnosis made at autopsy.

Two of the 111 cases originally classified as having other diseases were found to have CJD on autopsy. Autopsy evidence, together with follow-up of the patients still living and those who died without autopsy, revealed a broad range of other diagnoses. In the younger age groups, the commonest were chronic inflammatory diseases including Hashimoto encephalitis, whilst rapidly progressive Alzheimer's disease was most common in the older age groups.

The presence of 14-3-3 protein in the CSF discriminated better between CJD and other rapidly progressive dementias than did the EEG pattern or the MRI. The inclusion of this CSF protein in the criteria of Masters and colleagues (Ann Neurol 1979; 5: 177­88) improves the accuracy and confidence in the clinical diagnosis of CJD.

Increased levels of epsilon and gamma isoforms of 14-3-3 proteins in cerebrospinal fluid in patients with CJD

Clin Diagn Lab Immunol 1999 Nov;6(6):983-5
Takahashi H, Iwata T, Kitagawa Y, Takahashi RH, Sato Y, Wakabayashi H,
Takashima M, Kido H, Nagashima K, Kenney K, Gibbs CJ Jr, Kurata T
We established four hybridoma cell lines producing monoclonal antibodies (MAbs) against 14-3-3 proteins. Immunoblot analysis revealed that epsilon and gamma isoforms were specifically increased in premortem cerebrospinal fluid samples from patients with sporadic CJD. Furthermore, dot immunoblot analysis showed that MAbs were more specific for native antigen than polyclonal antibodies were.

Isoform pattern of 14-3-3 proteins in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease

J Neurochem 1999 Dec;73(6):2485-90
Wiltfang J, Otto M, Baxter HC, Bodemer M, Steinacker P, Bahn E, Zerr I,
Kornhuber J, Kretzschmar HA, Poser S, Ruther E, Aitken A
Two-dimensional polyacrylamide gel electrophoresis of CSF has been used in the diagnosis of CJD. One of the two diagnostic protein spots was identified as isoform(s) of the 14-3-3 family of abundant brain proteins. This has led to the development of one-dimensional 14-3-3 sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot, which is currently used to support the diagnosis of CJD. In the present study employing western blot analysis, we have identified the panel of 14-3-3 isoforms that appear in the CSF of 10 patients with CJD compared with 10 patients with other dementias.

The results clearly show that the 14-3-3 isoforms beta, gamma, epsilon, and eta are present in the CSF of patients with CJD and can be used to differentiate other dementias. 14-3-3eta also gave a baseline signal in all patients with other dementias, including six patients with Alzheimer's disease. The presence of 14-3-3eta in the CSF of a patient with herpes simplex encephalitis was particularly noteworthy. This study has determined that isoform-specific 14-3-3 antibodies against beta, gamma, and epsilon should be considered for the neurochemical differentiation of CJD from other neurodegenerative diseases.

Comment (webmaster): This sounds like a significant improvement in specificity (fewer false positives) for this diagnostic test, which as a practical matter is fairly widely used in the early stages of suspected CJD. 10 cases are not a lot given the diversity of this disease but a start. The Wiltfang paper used a 1D immunoblot, apparently polyclonal.

It would be good if labs offering the 14-3-3 test would set up an expert consensus web page. Why not have a place online that tells the family or regional neurologist where they can get this test done with a minimum of false positives, explains issues with sample-taking and sending, and pulls together upcoming developments? Right now it seems to be word of mouth; some neurologists know, others do not.

The 14-3-3 family of proteins exert various influences on the signal transduction pathways but has only tangential direct involvement in CJD, serving as a proxy for disease. Some 14-3-3 family members are related to protein kinase C inhibitory protein (KCIP) and activators of tyrosine and tryptophan hydroxylases and are expressed in many tissues. Human zeta 14-3-3 protein mediates phospholysis. Human 14-3-3 gamma (YWHAG) is on chr 7q11.23 and identical to rat. 14-3-3 has proved a very unfortunate name, one that is very unsatisfactory for search engines. Click to pull up the Swiss prot entry on 14-3-3 for humans.

>sp|P31946|143B_HUMAN 14-3-3 PROTEIN BETA

P101L in mouse alters TSE transmission time

The EMBO Journal Vol. 18,pp. 6855-6864, 1999
Jean C. Manson, Elizabeth Jamieson, Herbert Baybutt, ... David W. Melton, James Hope, Cr Bostock 
A mutation equivalent to P102L in the human PrP gene associated with GSS has been introduced into the murine PrP gene by gene targeting. Mice homozygous for this mutation (101LL) showed no spontaneous transmissible spongiform encephalopathy (TSE) disease, but had incubation times dramatically different from wild-type mice following inoculation with different TSE sources. Inoculation with GSS produced disease in 101LL mice in 288 days.

Disease was transmitted from these mice to both wild-type (226 days) and 101LL mice (148 days). In contrast, 101LL mice infected with ME7 had prolonged incubation times (338 days) compared with wild-type mice (161 days). The 101L mutation does not, therefore, produce any spontaneous genetic disease in mice but significantly alters the incubation time of TSE infection. Additionally, a rapid TSE transmission was demonstrated despite extremely low levels of disease-associated PrP

Yeast Hsp70-related chaperone Ssb in formation, stability, and toxicity of psi prion.

Mol Cell Biol 1999 Dec;19(12):8103-8112 
Chernoff YO, Newnam GP, Kumar J, Allen K, Zink AD
Propagation of the yeast protein-based non-Mendelian element [PSI], a prion-like form of the release factor Sup35, was shown to be regulated by the interplay between chaperone proteins Hsp104 and Hsp70. While overproduction of Hsp104 protein cures cells of [PSI], overproduction of the Ssa1 protein of the Hsp70 family protects [PSI] from the curing effect of Hsp104.

Here we demonstrate that another protein of the Hsp70 family, Ssb, previously implicated in nascent polypeptide folding and protein turnover, exhibits effects on [PSI] which are opposite those of Ssa. Ssb overproduction increases, while Ssb depletion decreases, [PSI] curing by the overproduced Hsp104.

Both spontaneous [PSI] formation and [PSI] induction by overproduction of the homologous or heterologous Sup35 protein are increased significantly in the strain lacking Ssb. This is the first example when inactivation of an unrelated cellular protein facilitates prion formation. Ssb is therefore playing a role in protein-based inheritance, which is analogous to the role played by the products of mutator genes in nucleic acid-based inheritance. Ssb depletion also decreases toxicity of the overproduced Sup35 and causes extreme sensitivity to the [PSI]-curing chemical agent guanidine hydrochloride.

Our data demonstrate that various members of the yeast Hsp70 family have diverged from each other in regard to their roles in prion propagation and suggest that Ssb could serve as a proofreading component of the enzymatic system, which prevents formation of prion aggregates.

Molecular basis of a yeast prion species barrier (not yet published)

8 Dec 99 Genbank
Santoso,A., Chien,P. and Weissman, J.S.
Comment (webmaster):
These are new GenBank entries for sup35 prion proteins for various fungi. One can guess from the article title and range of species what they are up to.

No function has ever been assigned to the N-terminal repeat domain of yeast; this will be severely delimited by the chaotic development of this domain across fungi as a group.

As far as amyloid disease analogy goes, they resemble polyglutamine repeat disorders more than AD or CJD types. The chaperone story has also proved very instructive in yeast but it is unclear what if anything carries over to unrelated amyloidogenic genes in mammals.

Zygosaccharomyces rouxii

 Saccharomycodes ludwigii

Pichia pastoris

Kluyveromyces marxianus

Kluyveromyces lactis

Candida albicans

Saccharomyces cerevisiae (previously determined)

Oral transmission and early lymphoid tropism of chronic wasting disease PrPres in mule deer fawns.

J Gen Virol 1999 Oct;80 (Pt 10):2757-64 
Sigurdson CJ, Williams ES, Miller MW, Spraker TR, O'Rourke KI, Hoover EA
Mule deer fawns (Odocoileus hemionus) were inoculated orally with a brain homogenate prepared from mule deer with naturally occurring chronic wasting disease (CWD), a prion-induced transmissible spongiform encephalopathy.

Fawns were necropsied and examined for PrPres, the abnormal prion protein isoform, at 10, 42, 53, 77, 78 and 80 days post-inoculation (p.i.) using an immunohistochemistry assay modified to enhance sensitivity. PrPres was detected in alimentary-tract-associated lymphoid tissues (one or more of the following: retropharyngeal lymph node, tonsil, Peyer's patch and ileocaecal lymph node) as early as 42 days p.i. and in all fawns examined thereafter (53 to 80 days p.i.). No PrPres staining was detected in lymphoid tissue of three control fawns receiving a control brain inoculum, nor was PrPres detectable in neural tissue of any fawn.

PrPres-specific staining was markedly enhanced by sequential tissue treatment with formic acid, proteinase K and hydrated autoclaving prior to immunohistochemical staining with monoclonal antibody F89/160.1.5. These results indicate that CWD PrPres can be detected in lymphoid tissues draining the alimentary tract within a few weeks after oral exposure to infectious prions and may reflect the initial pathway of CWD infection in deer.

The rapid infection of deer fawns following exposure by the most plausible natural route is consistent with the efficient horizontal transmission of CWD in nature and enables accelerated studies of transmission and pathogenesis in the native species.

Prion Protein Glycosylation Is Sensitive to Redox Change.

J Biol Chem 1999 Dec 3;274(49):34846-34850 
Capellari S, Zaidi SI, Urig CB, Perry G, Smith MA, Petersen RB
The conversion of soluble prion protein into an insoluble, pathogenic, protease-resistant isoform is a key event in the development of prion diseases. Although the mechanism by which the conversion engenders a pathogenic event is unclear, there is increasing evidence to suggest that this may depend on the function of the prion protein in preventing oxidative damage.

Therefore, in this study, we assessed the interrelationship between redox-sensitive cysteine, glycosylation, and prion metabolism. Cells were treated with a thioreductant, dithiothreitol, to assess the effect of the cellular oxidation state on the synthesis of the prion protein. This change in redox balance affected the glycosylation of the prion protein, resulting in the sole production of glycosylated forms.

The role of the single disulfide bridge in mediating this effect within the prion protein was confirmed by mutating the cysteine residues involved in its formation. These data suggest that conditions that increase the rate of formation of the disulfide bridge favor formation of the unglycosylated prion protein. Thus, since the presence of glycans on the prion protein is protective against its pathogenic conversion, a change in the redox status of the cell would increase the risk of developing a prion disease by favoring the production of the unglycosylated form.

Subcellular trafficking abnormalities of a prion protein with a disrupted disulfide loop.

FEBS Lett 1999 Oct 22;460(1):11-6
Yanai A, Meiner Z, Gahali I, Gabizon R, Taraboulos A
The single disulfide loop (Cys178-Cys213) of the prion protein (PrP) may stabilize the conformation of this protein by bridging the C-terminal alpha-helices. The substitution mutant Cys178Ala fails to form the prion isoform PrPSc when expressed in scrapie-infected neuroblastoma ScN2a cells (Muramoto et al., Proc. Natl. Acad. Sci. USA 93, 15457-15462).

To investigate the reasons for this failure, we introduced the C178A substitution in the full length mouse PrP gene as well as in its N-terminally truncated delta23-88 version. The resulting mutants (C178A and deltaC178A, respectively) were transiently expressed in N2a and CHO cells. Wild-type PrP, wild-type delta23-88 and the point mutant E199K served as controls in these experiments.

Compared to the wild-type controls, the C178A mutants were markedly resistant to proteolysis and they were also vastly insoluble in sarcosyl. Studying the metabolic fate of the C178A mutants, we found that in contrast to control PrP molecules, these mutants (i) remained sensitive to the diagnostic endoglycosidase EndoH, (ii) failed to reach the cell surface and (iii) congregated in large juxtanuclear spots. We surmise that these severe trafficking abnormalities may contribute both to the spontaneous aggregation of the C178A mutants and to their reported inability to form PrP(Sc).

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