Chapter 1 Prions

Chapter 1: Prions

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Chapter 1 Prions

Chapter 1: Prions

This thesis has two introductory chapters. In Chapter 1 I first provide a general

overview of the prion field. Understanding of the impact and significance of prion

diseases, prion concept, and features of mammalian prion protein is a necessary basis

for understanding the evolution and elusive normal function of prion protein gene,

which is the main goal of this thesis.

1.1 Prion Diseases

Prion diseases in humans and animals (Table 1.1) are disorders of protein conformation.

A common feature of infectious, inherited and sporadic forms of prion diseases is

aberrant metabolism of prion protein (PrP) (Prusiner, 1998). During pathogenesis of

these fatal neurodegenerative diseases, a cellular isoform of prion protein (PrPC; C is for

cellular) adopts pathogenic conformation (PrPSc; Sc is for scrapie), accumulates in cells

and causes disease (Chapter 1.2). Spongiform degeneration and reactive gliosis in brain

make a neuropathologic footprint in prion diseases. Human prion diseases typically

manifest as dementia and animal prion diseases manifest as ataxia. Whereas prions,

proteinaceous infectious particles whose only known component is PrPSc (Prusiner,

1982), cause infectious forms of prion diseases, pathogenesis of inherited prion diseases

is triggered by mutations in the prion protein gene (PRNP) and etiology of the sporadic

prion diseases is not well understood.

Apart from scrapie, emergence and spread of infectious prion diseases is mediated by

human practice and mistakes that occurred, paradoxically, in the most primitive

societies and in the most developed societies. A common critical trigger for prion

expansion was usage of tissues from dead humans or animals.

Kuru was propagated via ritualistic endocannibalism among the tribes in tropical

highlands of Papua New Guinea (Gajdusek, 1977). There were more than 2700 disease

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Chapter 1 Prions

Table 1.1: Prion diseases

Disease Host Mechanism of pathogenesis Kuru Fore tribe, PNG Infection through ritualistic cannibalism

Iatrogenic Creutzfeld-Jakob disease (iCJD)

Humans Infection from prion-contaminated HGH, dura mater grafts, etc.

Variant Creutzfeld-Jakob disease (vCJD)

Humans Infection from bovine prions

Familial Creutzfeld-Jakob (fCJD)

Humans Germ-line mutations in PRNP gene

Gerstmann-Straussler-Sheinker disease (GSS)

Humans Germ-line mutations in PRNP gene

Fatal familial insomnia (FFI)

Humans Germ-line mutations (D178N, M129) in PRNP gene

Sporadic Creutzfeld-Jakob disease (sCJD)

Humans Somatic mutation or spontaneous conversion of PrPC into PrPSc

Fatal sporadic insomnia (FSI)

Humans Somatic mutation or spontaneous conversion of PrPC into PrPSc

Scrapie Sheep Infection in genetically susceptible sheep Bovine spongiform

encephalopathy (BSE) Bovine amyloidotic

spongiform encephalopathy (BASE)*

Cattle

Cattle

Infection with prion-contaminated MBM Infection or spontaneous conversion of

PrPC into PrPSc

Transmissible mink encephalopathy (TME)

Mink Infection with prions from sheep or cattle

Chronic wasting disease (CWD)

Mule deer, elk Unknown

Feline spongiform encephalopathy (FSE)

Cats Infection with prion-contaminated bovine tissues or MBM

Exotic ungulate encephalopathy

Greater kudu, nyala, oryx

Infection with prion-contaminated MBM

PNG, Papua New Guinea; HGH, human growth hormone; MBM, meat and bone meal. Prusiner, 1998; *, Casalone et al., 2004.

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Chapter 1 Prions

cases recorded in the time period from 1957 to 2003 (Will, 2003). Iatrogenic CJD in the

Western world, on the other hand, is a consequence of use of prion-contaminated

human tissues and their derivatives, and prion-contaminated medical instruments. In the

time period from 1974 to 2003, 312 disease cases were caused by usage of the

contaminated human growth hormone (162), dura mater transplants (136), human

gonadotrophin (5), contaminated corneas (3), neurosurgical instruments (4) and depth

electrodes (2) (Will, 2003).

The most abundant and perhaps best known infections prion disease is the zoonosis

BSE. It manifests mainly as ataxia and hyperaesthesia. The main spreading pathway of

the BSE panzootic has been BSE-infected meat and bone meal, a widely used

supplementary feedstuff rich in proteins that is produced by the rendering process

(Prusiner, 1998). An outcome of “industrial cannibalism”, this disease has had a

worldwide influence on cattle trade, beef consumption, animal health and public health,

and has caused enormous economic damage. BSE was diagnosed worldwide, and

between 1986 and 2003 more than 180000 cases were diagnosed just in the UK cattle

(Smith and Bradley, 2003). This number is likely to have been under-reported in the

past, as detection of BSE cases increased in the EU with the introduction of compulsory

epizootiological surveillance in 1998 and use of new tests (Report on BSE;

http://europa.eu.int/comm/food/fs/bse/index_en.html). Mathematical modelling

indicated that more than three million cattle could have been infected with BSE prions,

most of which entered the human food chain in a subclinical phase (Donnely et al.,

2002). The BSE prions are very promiscuous and they were transmitted to a range of

species both naturally (human, cats, greater kudu, nyala, oryx) and experimentally

(cattle, sheep, mice, pig, mink) (Prusiner, 1998). For instance, parenteral inoculation

(intracranially, intravenously and intraperitoneally) of the BSE prions to pigs caused

disease after 69-150 weeks (Wells et al., 2003). Bovine amyloidotic spongiform

encephalopathy (BASE) is a new form of cattle prion disease (Casalone et al., 2004)

with different prion features (distribution of PrPSc glycoforms, size of proteinase-

resistant PrPSc fragment, and regional PrPSc distribution in brain; Chapter 1.2).

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Chapter 1 Prions

vCJD was caused by oral exposure of humans to the bovine prions (Hill et al., 1997).

There were 146 diagnosed cases of vCJD in the UK by March 2004 (Incidence of vCJD

disease onset and deaths in the UK; http://www.cjd.ed.ac.uk/vcjdq.htm), and 6 cases in

France and 1 case in Ireland, Italy, Canada and the USA by May 2003 (Will, 2003).

Classical CJD, a progressive dementia, is usually detected in older people, but vCJD

occurs mainly in younger people and manifests as a psychiatric disorder. In vCJD, PrPSc

was found in lymphoreticular tissues (tonsil, appendix, spleen, lymph nodes), but these

tissues contain no PrPSc in other forms of human prion disease. Whereas no evidence of

transmission of sporadic CJD by blood transfusion is reported, vCJD could be

transmissible through blood transfusion (Llewelyn et al., 2004).

Scrapie in sheep is an archetypal prion disease that has been known for more than 250

years, yet it remains the most mysterious. Neither the number of affected sheep is

known, nor how it spreads from sheep to sheep (Hunter, 2003). The clinical signs of

scrapie include extreme nervous reactions to stimuli, ataxia and pruritis. Both genetic

factors and infectious agent determine its spread (Chapter 1.2.4).

In humans, genetic causes account for approximately 10 % of all cases of prion diseases

(Prusiner and Scott, 1997). These are at the same time both genetic and transmissible

diseases, as mutant PrPScs may transform normal PrPC into the pathogenic form

(Chapter 1.2). For example, the GSS prions were transmitted from humans to

nonhuman primates (Masters et al., 1981), and the fCJD and FFI prions were

transmitted from humans to transgenic mice (Telling et al., 1996). A large number of

reported mutations in human PRNP include 24 missense point mutations, 27 mutations

in the repeat PrP region (Figure 1.1), and two nonsense mutations (Gambetti et al.,

2003).

Sporadic human prion diseases comprise 75 % of all cases (Kübler et al., 2003). They

are induced by spontaneous transformation of normal form of prion protein to its

pathogenic form (Prusiner, 1998; Legname et al., 2004). Approximately one in 106

people develop sCJD (Prusiner, 1991). The sporadic form of prion diseases shows a

phenotypic heterogeneity, which has made the recognition of prion diseases difficult

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Chapter 1 Prions

(Gambetti et al., 2003). The six sCJD phenotypes segregate with the PRNP genotype at

PrP codon 129 and PrPSc features (Chapter 1.2). Of note here is that the sporadic prion

disease could occur in any mammal expressing PrP (Legname et al., 2004).

Prion diseases are typically detected upon manifestation of clinical symptoms but a

definitive diagnosis is possible only post mortem after neuropathologic assessment

(Kübler et al., 2003). There is no definitive diagnosis in living individuals and there is

no method for detection of preclinical cases at present. Prion titer can be determined

using the bioassays (infection of experimental animals such as mice and transgenic

mice). The only molecular marker specific for prion diseases is altered form of prion

protein (Chapter 1.2): detection of its proteinase K-resistant fragment is a basis for

current “rapid” ELISA- or Western blot-based assays for BSE detection. These valuable

diagnostic tools are used routinely in the EU countries for surveillance of cattle entering

the human food chain (Report on BSE; http://europa.eu.int/

comm/food/fs/bse/index_en.html).

There is no therapy for prion diseases. At the molecular level, potential drugs should

interfere with interactions between normal and altered form of prion protein and inhibit

pathogenic transformation (Chapter 1.4.2), induce increased clearance of altered from of

prion protein, and ameliorate prion-induced neurotoxicity (reviewed by Dormont,

2003). For example, many different molecules inhibit prion replication when

administered with PrPSc, including anthracyclines, porphyrins, diazo dyes, quinacrine

and bisacridines (reviewed by Cohen and Kelly, 2003). Further, clearance of PrP using

antibodies may slow or cure prion disease, and small molecules or antibodies may be

designed to inhibit contact between the two PrP isoforms. Major obstacles for

development of such drugs are their toxicity and inability to pass brain-blood barrier.

Discovery of other host molecules that facilitate prion replication (Chapter 1.2.4) is

essential for development of therapeutics against prion disease.

With no cure or vaccine at present, the only mode of action against prion diseases is

general prophylaxis. For instance, kuru declined after the cessation of cannibalism.

Similarly, after the ban of feeding MBM to sheep and cattle in July 1988, the number of

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Chapter 1 Prions

BSE-infected cattle in the UK decreased (Prusiner, 1998). However, over 44000 BSE

cases in the UK were reported in the animals that were born after the ban (Smith and

Bradley, 2003).

Europe has been a focal point for emergence and spread of BSE with the highest

number of detected cases. The recent rate of BSE infection in the EU is 1/1400 among

the emergency slaughtered cattle and cattle showing clinical symptoms, and 1/35000 in

healthy animals. The EU developed a 4.7 billion Euros-strategy to prevent BSE prions

entering animal feed or human food between 1998 and 2001 (Report on BSE;

http://europa.eu.int/comm/food/fs/bse/index_en.html). Overall implementation of this

strategy has been problematic due to inappropriate delays in its adoption and execution

by the agro-feed industry: examples include contamination of feed with MBM, and

improper labelling of feed containing MBM.

In Australia there were 27,215,000 heads of cattle in 2003 (FAOSTAT;

http://faostat.fao.org/) producing approximately 2 million tonnes of beef per year worth

about 4.4 billion dollars (Meat and Livestock Australia; http://www.mla. com.au). By

exporting some 66% of its total beef production, Australia is the world’s largest

exporter of beef and one of the world’s leading producers of cattle. Australia does not

have BSE or scrapie (Australian Government, Department of Agriculture Fisheries and

Forestry; http://www.affa.gov.au/). Back in 1966, as a preventive measure against

spread of anthrax, Australia banned imports of stockfeed of animal origin; this has

luckily kept the main source of BSE away from the continent. In 2003, 464 cattle and

438 sheep were tested negative (Animal Health Australia; http://www.aahc.com.au/).

Prion diseases are threat to human and animal health, and also have profound and

devastating economic and social impact. Unanswered questions such as the therapy,

vaccine and reliable diagnosis in vivo remain to be solved.

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Chapter 1 Prions

1.2 The Prion Concept

Stanley Prusiner’s Nobel Prize-winning prion concept (Prusiner, 1998; Table 1.2) in

summary is:

(a) Prions are proteinaceous infectious particles that lack nucleic acid. They can be

defined also as infectious proteins. In a broader sense, prions are elements that impart

and propagate conformational variability.

(b) Prions are composed solely of the PrPSc.

(c) The cellular PrPC is converted into PrPSc through a refolding of part of its α-helices

and loops into β-helix (Govaerts et al., 2004). PrPSc acts as a template upon which PrPC

is refolded into nascent PrPSc. This transformation results in different physicochemical

properties of two isoforms. It is facilitated by another, unknown, protein (protein X).

(d) Efficiency of templating (species barrier) is determined by the difference in PrP

sequences between prion donor and recipient, strain of prion and species specificity of

protein X.

(e) Prions encipher their strain-specific properties in the tertiary structure of PrPSc. The

amino acid sequence of PrPSc is encoded by the PRNP gene of the host in which it last

replicated.

1.2.1 Unusual Nature of Prions

Prions have properties that are unusual and very different from those of conventional

infectious agents.

The infectious peptide, defined by transmission, showed unusual behaviour when

probed by various laboratory methods modifying either proteins or nucleic acids

(Prusiner, 1982). The scrapie agent was readily inactivated by the methods that

hydrolize or modify proteins: proteinase digestion (proteinase K), chemical

modification (diethyl pyrocarbonate), detergents (sodium dodecyl sulphate), chaotropic

salts (guanidinium thiocyanate), phenol and urea. In contrast, it was resistant to

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Chapter 1 Prions

Table 1.2: Evidence for the identity of prions (Prusiner, 1998). 1. PrPSc and scrapie infectivity co-purify by biochemical and immunologic procedures. 2. The unusual properties of PrPSc mimic those of prions. Many different procedures that

modify of hydrolize PrPSc also inactivate prions. 3. Levels of PrPSc are directly proportional to prion titres. Non-denatured PrPSc has not been

separated from scrapie infectivity. 4. No evidence exists for a virus-like particle or a nucleic acid genome. 5. Accumulation of PrPSc is invariably associated with the pathology of prion diseases,

including PrP amyloid plaques that are pathognomonic. 6. PRNP mutations are genetically linked to inherited prion diseases and cause formation of

PrPSc. 7. Overexpression of PrPC increases the rate of PrPSc formation, which shortens the

incubation time. PRNP knock-out eliminates the substrate necessary for PrPSc formation and prevents both prion disease and prion replication.

8. Species variations in the PrP sequence are responsible, at least in part, for the species barrier that is evident when prions are passaged from one host to another.

9. PrPSc preferentially binds to homologous PrPC, resulting in formation of nascent PrPSc and prion infectivity.

10. Chimerism and partial deletions of PRNP change susceptibility to prions from different species and support production of prions with novel properties that are not found in nature.

11. Prion diversity is enciphered within the conformation of PrPSc. Strains can be generated by passage through hosts with different PRNPs. Prion strains are maintained by PrPC / PrPSc interactions.

12. Human prions from fCJD (E200K) and FFI patients impart different properties to chimeric MHu2M PrP in transgenic mice, which provides a mechanism for strain propagation.

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Chapter 1 Prions

procedures that degrade nucleic acids: pH (low pH), nucleases (ribonucleases,

deoxyribonucleases), UV irradiation (254 nm of 42000 J/m2), divalent cation hydrolysis

(Zn2+), psoralen photoreaction and chemical modification (hydroxylamine). This

strongly suggested that scrapie agent is a novel infectious entity with characteristics of a

protein.

1.2.2 Pathogenic Transformation of Prion Protein

Prion protein is the first protein known to exist in two different active conformations,

which have very different properties despite the same primary structure.

Bolton et al. (1982) discovered PrP in the protein purifications from scrapie-infected

Syrian hamster brains. In the 125I-labelled sucrose gradient fractions enriched for

infectious agent, a diffuse 27-30 kDa protein band appeared that was resistant to limited

proteolysis by proteinase K (100 µg/ml; 30 min at 25 °C), and absent from normal

brains. This protein band (PrP 27-30; Figure 1.1) was the first molecular marker

specifically associated with prion infections. The purification protocol enriched scrapie

agent preparations from 100- to 1000-fold with respect to cellular protein (Prusiner et

al., 1982) and it was estimated that there are 104-105 molecules of PrP 27-30 per ID50

unit. The same PrP 27-30 was identified after labelling protein fractions with [14C]

diethyl pyrocarbonate.

In contrast, after 5’-end labelling with [γ-32P]ATP, no significant differences in nucleic

acids content between the scrapie-infected and normal Syrian hamster brains were

found. Further, in the most purified sample fraction aggregates composed of amorphous

material were found using electron microscopy, but not viruses. Among other

alternatives, it was hypothesized that changes in conformation of the agent may

modulate its susceptibility to proteolysis, and that the agent could be devoid of nucleic

acids.

The concentration of PrP 27-30 was shown to be directly proportional to the titer of

infectious agent (McKinley et al., 1983). Further, the kinetics of proteolytic digestion of

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Chapter 1 Prions

Figure 1.1: Bar diagram shows the isoforms and artificial constructs of prion protein. (A) The Syrian hamster PrP polypeptide consists of 254 aa and has proximal repeats (Repeats), middle hydrophobic region (H), two signal peptides (S1 and S2), two glycoslylation sites (N), two cysteines forming disulphide bridge (S-S) and a glycosyl-phosphatidyl inositol attachment site (GPI). (B) After removal of the signal peptides, the mature PrPC has 209 amino acids. (C) The PrPSc has 209 amino acids as well. (D) After limited proteolysis with proteinase-K, the N-terminus of PrPSc is truncated to form PrP 27-30 of approximately 142 amino acids (Prusiner, 1998). (E) Deletion of the mouse PrP N-terminal region between residues 32-93 permits prion propagation (Flechsig et al., 2000). (F) Deletion of the mouse PrP regions between residues 23-88 and 141-176 also permits prion propagation (Suppattapone et al., 1999). ∆, deletion. Ruler at the bottom indicates primary sequence amino acid coordinates.

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Chapter 1 Prions

PrP 27-30 was undistinguishable from that of prion, suggesting that PrP is a structural

component of prions. Both PrP 27-30 and infectious prions were purified and sequenced

(Prusiner et al., 1984), and the same N-terminal amino acid sequence was determined

showing that PrP indeed is the constituent of prions. The UV spectra demonstrated

absence of nucleic acid covalently linked with PrP 27-30.

Using this N-terminal amino acid sequence, Chesebro et al. (1983) synthesized an

oligonucleotide probe and found a related cDNA in the scrapie-infected mouse brain.

This mouse cDNA was later detected in both infected and normal brains, indicating that

PrP could also have some normal role (Chapter 2.5). Using the scrapie-infected hamster

brain cDNA library, Oesch et al. (1985) also isolated a mRNA encoding PrP. It was

expressed in both infected and normal brain at the same level, but also in a range of

other normal hamster tissues (heart, lung, pancreas, spleen, testes, kidney; Chapter 2.3).

This study also indicated that a single gene (PRNP) encodes PrP in hamster, mouse and

human (Chapter 2.2). The mRNA for PrP was not found in infectious prions.

An amino acid sequence translated from mRNA showed that the PrP 27-30 was derived

by proteolysis from a larger molecule. The antibodies raised against PrP 27-30 detected

a larger molecule of 30-33 kDa in both normal (PrPC) and scrapie-infected brains

(PrPSc). The PrP 27-30 was the result of partial proteinase K digestion of PrPSc from

scrapie-infected brains but the PrPC from normal brains was completely digested

(Figure 1.1). Further, the PrPC, but not the PrPSc, was solubilized in nondenaturing

detergents (Meyer et al., 1986).

The first complete amino acid sequence of PrP was translated from the Syrian hamster

genomic DNA sequence and a cDNA containing the complete open reading frame

(ORF) (Basler et al., 1986). This study showed that there is only one gene encoding

PrP, that there is no difference between cDNAs from healthy and scrapie-infected

hamster brain and that both PrPC and PrPSc have the same primary structure (Figure

1.1). Their different properties could be attributed to different tertiary or quaternary

structures of the protein.

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Chapter 1 Prions

There were found to be remarkable differences between the PrPC and PrPSc secondary

structure content (Pan et al., 1993). Fourier infrared (FTIR) spectroscopy of PrPC

indicated high α-helix (42 %) and low β-sheet (3 %) content. In contrast, PrPSc had an

α-helix content of 21 % and a β-sheet content of 43%, indicating that the formation of

PrPSc may involve transformation of α-helices from PrPC into β-sheets (Chapter 1.3).

This conversion is the fundamental event underlying prion propagation, and the primary

lesion in prion diseases: despite having the same primary structure, PrPC and PrPSc have

different properties. The predominantly α-helical PrPC is soluble in nondenaturing

detergents and readily degraded by proteases, but PrPSc with its high β-sheet structure is

insoluble in nondenaturing detergents, it has proteolytically stable core (PrP 27-30) and

accumulates in brain and causes disease.

The transition of PrPC to PrPSc is therefore accompanied by a profound conformational

change. Peretz et al. (1997) raised antibodies against diverse PrP epitopes in order to

determine similarities and differences between the conformations of PrPC and PrP 27-

30. One epitope at the C terminus (residues 225-231) was accessible in both isorforms.

Two epitopes in the middle of the protein (amino acids 95-104 and 152-163), were

accessible in PrPC but buried in PrP27-30. However, after denaturation of PrP 27-30

with 3M guanidium thiocyanate (GdnSCN) both epitopes became accessible, indicating

that the major conformational change in PrP occurred in the middle part of the protein.

This was in line with other studies (Muramoto et al., 1996; Telling et al., 1996) which

all indicated an essential role for this middle conserved region (Chapter 2.1) in PrP

transformation.

Analysis of folding and unfolding of a 142 amino acid peptide corresponding to Syrian

hamster PrP 27-30 (Zhang et al., 1997) indicated that under different conditions it can

adopt either α-helix- or β-sheet-enriched conformations, both containing intramolecular

disulphide bond and with various stable intermediate structures. Increased temperature

induced a cooperative thermal denaturation and transition from an α-helical- to β-sheet-

enriched structure which was more thermodynamically stable (Chapter 1.4.1). The

authors concluded that this conformational plurality indicates the intrinsic plasticity of

the PrP sequence and its propensity to undergo to structural alterations.

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Chapter 1 Prions

Paramithiotis et al. (2003) found increased solvent accessibility of tyrosine after

induction of β-sheet structures in recombinant PrP. Antibodies raised against the motif

Tyr-Tyr-Arg recognized PrPSc but not PrPC. These are the first antibodies that recognize

PrP isoforms selectively. Exposure of this PrPSc-specific and saturable epitope is a

consequence of the conformational transformation. Such antibodies could have

application in development of diagnostic methods, therapy and prophylaxis against

prion diseases.

Plastic prion protein may adopt two distinct active conformations after the major

conformational change that occurs in the conserved middle part of the protein. The

normal PrP isoform (PrPC) is predominantly α-helical, but the pathogenic PrP isoform

(PrPSc) is rich in β-sheet, accumulates in brain and causes disease.

1.2.3 Species Barrier

Passage of prions between species is regularly characterized by a prolonged incubation

time in the new host; this prolongation is called the “species barrier”. During the second

passage in the new host incubation time shortens, and it remains constant upon

subsequent passages. Prion passages through different hosts may cause significant

changes in the disease phenotype (Pattison and Jones, 1968).

Crossing the species barrier is a slow and inefficient stochastic process. For instance,

when the hamster-adapted scrapie prions were inoculated in mice, few mice developed

disease, and the incubation times were very long (more than 500 days) (Figure 1.2A).

The species barrier was shown to depend on the difference between endogenous and

infectious PrP (Scott et al., 1989). Two lines of transgenic mice (TgShaPrP) were

constructed by introduction of a transgene encoding hamster PrP. After inoculation with

the hamster-adapted scrapie prions all infected mice developed disease, and the

incubation times were dramatically reduced to roughly 75 days (similar to incubation

time when hamsters are inoculated with hamster prions) and 170 days, respectively. The

species barrier between mouse and hamster, maintained by differences in PrP between

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Chapter 1 Prions

host and donor, was abrogated. Neuropathology in these infected transgenic mice was

more hamster-like than mouse-like (e.g. they had abundant amyloid plaques in brain

which are usually rare in mouse) indicating that the host PrPC determines susceptibility,

incubation time, neuropathology and prion species tropism after prion infection.

Prusiner et al. (1990) demonstrated an inverse relationship between incubation time and

host PrPC level when transgenic mice expressing different levels of Syrian hamster PrPC

were challenged with either hamster or mouse prions (Carlson et al. (1994) also showed

inverse correlation between the Prnp gene dosage and incubation time). Incubation time

also depended on the dose of prions. Next, amino acid sequence of the PrPSc in the prion

inoculum dictates de novo prion synthesis: hamster prions were produced after

inoculation with hamster-adapted prions, mouse prions were generated upon infection

with mouse-adapted prions (Figure 1.2A). The prion inoculum also determines

neuropathology, since the distribution of spongiform change and formation of amyloid

plaques differed between transgenic mice inoculated with hamster prions and mouse

prions. Thus, the PrPSc could act as a template upon which PrPC is transformed in a

similar nascent molecule, and the conformation of template may determine properties of

prion strains. The minimal size of the PrPSc/ PrPC complex appeared to be a

heterodimer.

There are 16 amino acid differences between the Syrian hamster and mouse PrPs. In

order to test their importance in maintaining the species barrier, Scott et al. (1993)

designed two lines of transgenic mice expressing chimeric mouse/Syrian hamster PrPCs:

Tg(MH2MPrP) line expressing MH2MPrPC with Syrian hamster substitutions at the

residues 108, 111, 138, 154 and 169 and Tg(MHM2PrP) line expressing MHM2PrPC

with Syrian hamster substitutions of the residues 108 and 111. By changing a few

residues in PrPC it was possible to manipulate prion properties, disease phenotype and

host susceptibility. Whereas the Tg(MH2MPrP) mice were susceptible to both the

Syrian hamster- (Sc237 and 139H) and mouse-adapted (RML) scrapie prions, the

Tg(MHM2PrP) mice were as resistant to the Syrian hamster-adapted prions as were

normal mice (Figure 1.2B). Substitutions at the residues 138, 154 and 169 determined

the species barrier for transmission of the hamster-adapted scrapie prions to mice. After

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Chapter 1 Prions

Figure 1.2 (page 12a): Experiments which support the prion concept. (A) Expression of the hamster PrPC in the Tg(ShaPrP) transgenic mice abrogates species barrier between mouse and hamster, demonstrating that differences in the PrP sequence between host and inoculum determine species barrier. Amino acid sequence of the PrPSc in inoculum dictates de novo prion synthesis: mouse prions were generated after inoculation with the mouse-adapted scrapie prions, and hamster prions were generated after inoculation with the hamster-adapted scrapie prions. (B) The Tg(MH2MPrP) mice were susceptible to both the mouse- and hamster-adapted scrapie prions. However, the Tg(MHM2PrP) mice were susceptible to the mouse-adapted scrapie prions, but they were resistant to the hamster-adapted scrapie prions, although the two chimeric PrPs differ in only three residues. Therefore, the homotypic interaction between PrPC (substrate) and PrPSc (template) determines prion propagation. Normal mice were susceptible to the chimeric prions that originated from the hamster-adapted scrapie prions to which normal mice are resistant. (C) The Prnp0/0 mice were resistant to the mouse-adapted scrapie prions. Prion-induced pathology is strictly dependent on the presence of cellular PrP (substrate). (D) Normal mice and the Tg(HuPrP) mice expressing both mouse and human PrPC were resistant to the human CJD prions. The Tg(MHu2MPrP) transgenic mice expressing chimeric mouse-human PrPC were susceptible to the human CJD prions, indicating involvement of an additional host factor (protein X) which interacted with the mouse PrPC and MHu2MPrPC, but not with the human PrPC. (E) The Tg(HuPrP)Prnp0/0 mice were susceptible to human prions but not the hemizygous Tg(HuPrP)Prnp0/+ mice. Therefore, the mouse PrPC titrated protein X, and prevented it to interact with the human PrPC. (F) Two different human prion strains templated transformation of the same chimeric MHu2MPrPC into two nascent MHu2MPrPScs with different conformations. Thus, the prion strain properties are enciphered in the conformation of PrPSc (see references in the text).

a few passages in Tg(MH2MPrP) mice, the artificial MH2M(Sc237) prions had a

unique host range: they were transmissible to Tg(MH2MPrP) mice, Syrian hamster and

normal mice whereas normal mice were resistant to inoculation with the Sc237 prions

passaged in Syrian hamster! The Tg(MH2MPrP) mice were more susceptible to

MH2M(Sc237) prions than Syrian hamster and normal mice. The preference for

homologous PrP showed that a direct, homotypic interaction between PrPC (substrate)

and PrPSc (template) is essential for prion propagation.

Development of prion-induced pathology is strictly dependent on the presence of host

PrPC, and incubation time and disease progression are proportional to the level of PrPC.

This was shown by constructing a Prnp knock-out mice (Prnp0/0) with disrupted ORF

(Bueler et al., 1992). The mice with inactivated Prnp gene (Prnp0/0) are resistant to

infection with mouse-adapted prions (Bueler et al., 1993) (Figure 1.2C). Heterozygotic

mice (Prnp0/+) showed protracted incubation time and disease progression after

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Chapter 1 Prions

inoculation. After introduction of the Syrian hamster PrP transgene, the Prnp0/0 mice

became more susceptible to the hamster-adapted scrapie prions than to the mouse-

adapted scrapie prions.

Therefore, differences between PrP from inoculum and host determine species barrier,

and the conformation of PrPSc determines properties of prion strains. During the

homotypic interaction, the PrPSc templates pathogenic transformation of a substrate,

PrPC, into a nascent PrPSc molecule. There is no prion replication without PrPC.

1.2.4 Auxilliary Host Factor Required for Prion Propagation: Molecular

Chaperone Protein X

Experimental transmissions of prions indicated involvement of an additional host factor,

unknown protein X, which supports the PrP transformation.

Telling et al. (1994) inoculated transgenic mice with the human CJD prions.

Surprisingly, the mice overexpressing human PrPC (TgHuPrP) were partially resistant to

human prions, showing long incubation time (590 - 840 days) and a transmission rate

(8.3% of 196 mice) similar to that of normal mice (10.3% of 58 mice) (Figure 1.2D).

The transgenic mice Tg(MHu2MPrP) expressed a chimeric MHu2MPrPC that contained

nine human substitutions in the mouse PrP region from 96 to 167 amino acids. These

mice were susceptible to the human CJD prions (transmission rate 100% of 24 mice)

showing incubation time of approximately 200 days. Taking this into account together

with results of Scott et al. (1993), it was determined that the region from 96 to 167

amino acids is a domain where PrPC interacts with PrPSc (Prusiner, 1998). After

infection with the mouse RML scrapie prions (transmission rate 100% of 24 mice),

average incubation time was 178 days. Following inoculation with human prions only

the MHu2MPrPSc was generated, and after inoculation with mouse prions only the

mouse PrPSc was found. Regional distributions of MHu2MPrPSc and mouse PrPSc

differed as well as the patterns of spongiform change. Difference in susceptibility to

human prions between the TgHuPrP and TgMHu2MPrP mice indicated that a mouse

molecule (e.g. chaperone; “protein X”) could catalysed transformation of PrP by

14

Chapter 1 Prions

interacting with the MHu2MPrPC and human PrPSc but not with the HuPrPC and human

PrPSc. Alternatively, the N-terminal or/and C-terminal sequences of human PrPC,

different from those of MHu2MPrPC, could inhibited production of human PrPSc in

mouse cells.

Following up on the study above, Telling et al. (1995) introduced human PrPC in the

Prnp0/0 background. The Tg(HuPrP)Prnp0/0 mice were susceptible to human prions.

However, the hemizygous Tg(HuPrP)Prnp0/+ mice expressing also wild type mouse

PrPC were as resistant to human prions as the Tg(HuPrP) mice, indicating that the wild

type host mouse PrPC inhibited formation of human prions in mice, even in excess of

human PrPC (Figure 1.2E). This may occur by binding and titration of the protein X, as

mouse PrPC could exhibit higher affinity for it than human PrPC. A binding site for

protein X was mapped to the C-terminal part of PrP. In the region bounded by residues

167-231 there are amino acid differences between human and mouse, but not between

mouse and Syrian hamster (the Tg(ShaPrP) mice became susceptible to hamster prions).

Protein X was predicted to be either a molecular chaperone, a scaffolding protein to

provide a milieu for PrP isoforms to interact, or a modifier of PrPC. Dynamic PrPC may

exist in more than one physiological state, and its transformations could be facilitated

by protein X even without PrPSc or mutation in PRNP gene (Chapter 1.2.6).

Telling et al. (1995) also showed that the chimeric MHu2MPrPSc was successfully

passaged to both the Tg(MHu2M) and Tg(MHu2M)Prnp0/0 transgenic mice, but not to

normal mice. Thus, the PrP region from 96 to 167 amino acids is a domain where PrPC

interacts with PrPSc and it facilitates prion propagation. In this region, the mutation of

human residue 102 (P102L mutation causes GSS in human) and the polymorphism of

residue 129 (M or V) but not the mutation of residue 200 (E200K mutation causes fCJD

in humans) affected prion transmission. A methionine homozygosity of the residue 129

in humans could increase susceptibility to the vCJD: all tested cases of vCJD in the UK

are 129M homozygotes (Will, 2003). Further, this residue affects prion disease

phenotype. The M129/N178 mutation pair causes FFI in humans, and the V129/N178

combination triggers fCJD in humans (Chapter 1.3.1).

15

Chapter 1 Prions

A number of other studies also support the involvement of molecular chaperones in

prion propagation. Glycerol and trimethylamine N-oxide, cellular osmolytes, and

organic solvent dimethylsulfoxide are “chemical chaperones”, compounds that protect

proteins from thermal denaturation. When applied to the scrapie-infected mouse ScN2a

neuroblastoma cells, these reagents interfered with transition of PrP reducing the rate

and extent of PrPSc formation (Tatzelt et al., 1996). The effect of glycerol was both dose

and time dependent. In the presence of high concentrations of this polyol, protein-

solvent interactions would be expected to increase; proteins would then counter this

increase in relative hydration by means of tighter packing of their domains. Therefore,

the chemical chaperones could stabilized the α-helical conformation of PrPC and

prevented it from transforming to the β-sheet-rich isoform.

A two-hybrid screen in S.cervisiae was employed to identify proteins interacting with

PrP (Edenhofer et al., 1996). This study showed that a chaperone Hsp60 interacts with

PrP region between the residues 180-210. This interaction was confirmed in vitro,

indicating direct interaction between the two proteins in a cell. GroEL, a bacterial

homologue of mammalian Hsp60 family, also interacted with PrP.

Using the ScN2a cells transfected with different constructs expressing chimeric

mouse/human PrPC, Kaneko et al. (1997) delineated a discontinous epitope on PrPC to

which protein X binds. This epitope consists of side chains of the mouse residues 214

and 218 on helix three (Chapter 1.3), and residues 167 and 171 on the loop connecting

beta sheet and helix two. Acting as “dominant negatives”, basic substitutions of these

residues had a protective effect on prion infection by binding protein X tightly and

rendering it unavailable for prion propagation (Chapter 1.4.2). The protein X interacts

firstly with PrPC and this complex subsequently binds PrPSc. Evolution had already

explored protective effect of these substitutions in the human (K219) and sheep (R171)

PrPs. For instance, sheep with the genotype A136-R154-R171 are resistant to natural

scrapie. This was a basis for breeding of the scrapie-resistant sheep and eradication of

disease (Hunter, 2003). These sheep were also resistant to experimental transmission of

BSE prions (Baron et al., 2000). Thus, a strategy for eradication of prion diseases could

be introduction of the dominant negative PrPs to germ-line of domestic animals. Perrier

16

Chapter 1 Prions

et al. (2002) constructed transgenic mice expressing prion protein with the Q167R (this

position corresponds to residue 171 in sheep PrP) or Q218K (this position corresponds

to human PrP residue 219 which heterozigosity E/K protects from CJD) mutation. The

Tg(MoPrP,Q167R)Prnp0/0 mice remained healthy >550 days after inoculation with the

RML prions. The Tg(MoPrP,Q167R)Prnp+/+ mice expressing both mutant and wild type

PrP did not show signs of disease, but their brains had low levels of PrPSc, and signs of

vacuolation and astrocytosis were also found. Both Tg(MoPrP,Q218K)Prnp0/0 and

Tg(MoPrP,Q218K)Prnp+/+mice remained healthy >300 days after inoculation.

Therefore, the dominant-negative inhibition of PrPSc production could occur, but it does

not prevent prion formation completely.

DebBurman et al. (1997) investigated the influence of bacterial and yeast chaperones on

PrP transition in a cell-free system (Chapter 1.2.7). None of chaperones induced PrP

transformation without PrPSc. A bacterial chaperone GroEL promoted transformation

templated by untreated PrPSc. After PrPSc was partially denatured with urea, the

bacterial chaperones GroEL and GroES and a yeast chaperone Hsp104 all promoted PrP

transformation. None of the chaperones inhibited PrP transition. Thus, the PrP

transformations were mediated by chaperones, suggesting a role for chaperones in this

process in vivo. Analysis of circular dichroism spectra showed that the GroEL and

Hsp104 interact directly with PrP (Schirmer and Lindquist, 1997). Hsp104 also

interacted directly with a yeast prion Sup35 (Chapter 1.5) and with a β-amyloid peptide,

suggesting that interaction with chaperones could be a common feature of

amyloidogenic proteins. Molecular chaperones may be also involved physiologically in

a control of certain types of conformational switches.

Bosque and Prusiner (2000) developed a method to derive subclones of N2a cell lines

that were highly susceptible to RML prions and in which every cell was infected. This

study showed a heterogeneity of N2a cells in prion susceptibility, ranging from highly

susceptible to totally resistant cells. The difference in susceptibility existed even when

PrPC levels were similar between cells indicating that other factors also influence prion

replication. Comparison between susceptible and resistant cells is a strategy to detect

cellular factors involved in prion propagation.

17

Chapter 1 Prions

In summary, investigations with chimeric transgenes indicated that the PrPC interacts

with PrPSc within a central domain bordered by residues 96-169. Inoculation of the

transgenic mice with prions indicated involvement of the unknown host factor in PrP

transformation. This auxiliary factor facilitating transformation of PrP is a molecular

chaperone “protein X”.

1.2.5 Regions of Prion Protein Involved in Pathogenic Transformation

Which regions of PrP are essential for pathogenic transformation, and which regions are

dispensable?

Increased number of the N-terminal PrP octarepeats (two, four, five six, seven, eight or

nine in addition to the normal five otarepeats) leads to inherited CJD (Prusiner and

Scott, 1997). However, the Prnp0/0 mice expressing PrP with deletions at its N-terminus

were susceptible to infection by scrapie prions and capable of allowing efficient prion

replication (Fischer et al., 1996). Although PrP transition was slightly less efficient,

deletions bordered by residues 68-85 and 31-81 did not affect prion propagation upon

infection, indicating that these regions are dispensable for pathogenic transformation.

The size of PrP 27-30 derived from truncated PrPs was identical to that derived from

wild type PrP. Deletion of the larger region between residues 32-93 also permitted prion

propagation (Figure 1.1E; Flechsig et al. 2000) showing that the N-terminal PrP

octarepeats are dispensable for prion propagation. However, deletion up to the residues

121 or 134 (32-121, 32-134) induced ataxia and neuronal cell death in the granular cell

layer of the cerebellum per se when this truncated PrP was expressed in Prnp0/0 mice

(Shmerling et al., 1998). This indicated functional importance for the middle

hydrophobic region of PrP (Chapter 2.1).

Of note here is that the peptide corresponding to the PrP residues 106-126 (PrP106-126)

has been used in analysis of the neurotoxic mechanisms underlying the prion diseases.

The PrP106-126 forms amyloid fibrils in vitro, it is partially resistant to proteolysis and

induces apoptosis in primary cultures of cortical, hippocampal, and cerebellar neurons.

18

Chapter 1 Prions

Brown (2000) showed that the optimal neurotoxic peptide contains human residues 112-

126: MAGAAAAGAVVGGLG. This sequence was necessary but not sufficient for

neurotoxicity. However, twofold excess of the AGAAAAGA peptide selectively

blocked the neurotoxic effect of PrP106-126, possibly by binding to PrPC. Indeed,

Kaneko et al. (1995) showed binding to PrPC of the peptide corresponding to the

regions between residues 90/109-145 in Syrian hamster PrPC containing both the

PrP106-126 and AGAAAAGA. The anti-PrP monoclonal antibody 3F4 that binds

residues 109-112 and the anti-PrP monoclonal antibody 13A5 prevented this

interaction. Together with investigations using chimeric transgenes, these results

confirmed that PrPC and PrPSc interact within the central domain bordered by residues

96-169 (Chapter 1.2.4).

Meyer et al. (2000) showed that the native bovine PrPC could exist as dimer. Formation

of dimer was inferred from three lines of evidence (ELISA, cross-linking experiments,

size exclusion chromatpgraphy). Therefore, a fraction of PrPC may exist in a monomer-

dimer equilibrium. Tompa et al. (2002) noted that dimerization is the most ancient and

most common step in the evolution of oligomeric proteins. In an isologous dimer, two

identical subunits interact, one from each monomer. In an heterologous dimer, two

different binding surfaces from each monomer interact, giving rise to elongated or

cyclic structures. Thus, the isologous PrP homo- and hetero-dimers dimers could be

formed by interaction via the central domain bordered by residues 96-169.

Corsaro et al. (2003) showed that the PrP106-126 induces apoptosis in SH-SY5Y cells

through the activation of caspase-3 and p38 MAP kinase. Both amyloidogenic and

mutant (G114A and G119A) non-amyloidogenic form of the PrP106-126 induced

apoptosis in vitro by activating the same pathway. Moreover, the mutant peptide was

even more potent inducer that the wild type PrP106-126. The PrP106-126 signalling in

SH-SY5Y cells did not involve activation of the MAP kinases ERK1/2 (Chapter 2.5.2).

The ERK1/2 and p38 pathways affect cells in opposite ways, promoting cell

proliferation and degeneration, respectively.

19

Chapter 1 Prions

PrP transformation was assessed in ScN2a cells transfected with PrP deletion mutants

(Muramoto et al., 1996). First, deletion of the residues 23-88 did not influence PrP

transformation but transformation of PrP was prevented when it was coupled with

deletions of the regions between amino acids 95-107, 108-121, 122-140, 177-200 or

201-217, or with mutation C178A. On the other hand, PrP was still capable of

transformation after a deletion of the residues 141-176 was combined with the deletion

of amino acids 23-88. This PrP molecule of 106 amino acids (PrPC106) was converted

into a proteinase K resistant but nondenaturing detergent soluble PrPSc106. This

deletion analysis showed that the disulphide bond was necessary, but the N-terminus

containing repeats, the first α-helix (residues 141-154) and the second strand of β-sheet

(amino acids 161-164) were all dispensable for PrP transformation in the cell culture.

The transgenic mice Tg(PrP106)Prnp0/0 expressing PrPC106 (Figure 1.1F) but no wild

type PrP were susceptible to the mouse-adapted RML scrapie prions (Suppattapone et

al., 1999), developing disease after approximately 300 days. The incubation time

shortened drastically to approximately 66 days after second and third passage of newly

formed prions. This artificial prion transmission barrier (induced by artificial PrP

sequence) was equivalent to the species barrier. The PrP106Sc from RML106 prions was

resistant to proteinase K digestion and insoluble in the nondenaturing detergents.

However, in hemizygous Tg(PrP106)Prnp0/+ mice inoculated with RML prions

incubation times of approximately 165 days were observed, showing that the wild type

mouse PrP acted in trans and facilitated production of RML106 prions. This study

confirmed that the residues between 23-88 and 141-176 amino acids are dispensable for

formation of PrPSc in vivo, transmissibility of prions or pathogenesis of prion disease.

Roughly half of mature PrPC residues are dispensable for prion propagation and prion

disease pathogenesis, including the N-terminal octarepeats, the first α-helix, and the

second β-sheet (Chapter 1.3). Chimeric prions with these deletions could be generated,

as well as artificial species barrier. Yet, deletions that included the conserved middle

hydrophobic sequence were neuropathogenic per se, indicating functional and/or

structural importance of this PrP region (Chapter 2.1).

20

Chapter 1 Prions

1.2.6 Features of Prion Strains are Enciphered in PrPSc Conformation

The features of prion strains, such as incubation times and distribution of

neuropathologic lesions, are all enciphered in the conformation of PrPSc.

Patients carrying mutations in the PRNP gene generate prions de novo. In a FFI patient

with a D178N mutation and methionine at the position 129, the size of the proteinase K

resistant deglycosylated PrPSc fragment was 19 kDa, but in a fCJD patient with an

E200K mutation it was 21 kDa (Telling et al., 1996; Prusiner, 1998). Two different

PrPSc conformations, reflected by the two sizes of proteinase K resistant PrPSc cores,

could be expected: they are induced by the two different PRNP mutations. Following

the transmission of the two prions into the same line of transgenic mice

(Tg(MHu2M)Prnp0/0) expressing MHu2MPrPC, the sizes of newly formed proteinase K

resistant deglycosylated MHu2MPrPSc fragments remained 19 kDa and 21 kDa despite

the same primary amino acid sequence, and incubation times were 200 days and 170

days, respectively (Figure 1.2F). On the second passage in Tg(MHu2M)Prnp0/0 mice

sizes of the proteinase K resistant deglycosylated MHu2MPrPSc fragments remained the

same, but incubation times were now 130 days and 170 days, respectively, implying

that the species barrier was now abrogated. Therefore, the same host PrPC

(MHu2MPrPC) adopted two different pathogenic conformations that were templated by

two different human PrPScs. These were faithfully propagated after passages. The

neuropathologic phenotypes also differed between mice infected with two prions. For

instance, in the mice inoculated with FFI prions MHu2MPrPSc accumulated mostly in

the thalamus and the rostral part of the corpus callosum. In contrast, in the fCJD prion-

infected mice, MHu2MPrPSc accumulated diffusely in many brain regions. Thus, two

different prion strains templated transformation of the same host PrPC into two different

nascent PrPSc and determined two different prion disease phenotypes. The features of

prion strains are determined by the conformation of PrPSc.

21

Chapter 1 Prions

Two mouse-adapted scrapie strains, Me7 and RML, have very similar incubation times

after their passage in mice but showed different incubation times after their transmission

to the Tg(MH2MPrP) mice, indicating that PrP sequences in both host and donor

determine the properties of prion strains (Scott et al., 1997). Prion strains can indeed

change their conformations during prion propagation, as the primary structure of PrP

presumably restricts the number of possible PrPSc conformations. It is known, for

example, that many different laboratory mouse- and hamster-adapted scrapie strains

were derived from the same source; the only difference is in their passage history.

In order to discriminate features of PrPSc molecules from different prion strains, Safar et

al. (1998) used a conformation dependent immunoassay. By plotting the ratio of

denatured and native PrPSc as a function of PrPSc concentration before and after

proteinase K digestion, eight Syrian hamster-adapted prion strains (HY, DY, 139H,

Sha(Me7), Me7-H, Sc237, MT-C5 and SHa(RML) showed distinct and specific

patterns, indicating that all have different spectra of PrPSc conformations. The fraction

of PrPSc sensitive to proteolysis (sPrPSc) plotted as a function of the incubation time

showed a linear relationship. Thus, the concentration of sPrPSc is proportional to

incubation time.

Two strains of Syrian hamster-adapted prions, Sc237 and DY, contain PrPScs with the

same primary amino acid structure but with different conformations. Inoculation of the

Tg(MH2M)Prnp0/0 mice with the Sc237 strain produced a prolonged incubation time

and a neuropathology different from that in Syrian hamster (Peretz et al., 2002). The

new MH2M(Sc237) prion strain had the same size of deglycosylated-proteinase K

resistant fragment and glycosylation pattern as original Sc237 strain, but the

conformational stability measurements following GdnHCl denaturation and proteinase

K digestion showed a marked difference, indicating a change in PrPSc conformation that

accompanied the emergence of a new prion strain. In contrast, after inoculation with the

DY strain there was no difference between Syrian hamsters and Tg(MH2M)Prnp0/0

mice. Different host responses after inoculation with prions strains differing only in

their conformations indicated that the conformation of PrPSc determines disease-causing

prion strain properties, and that it may modulate interspecies prion transmissibility. The

22

Chapter 1 Prions

long incubation times characteristic of the species barrier probably reflect selection of a

small subpopulation of PrPSc that is preferentially replicated in the new species, from a

heterologous PrPSc population pre-existing in the prion inoculum.

Glycosylation may influence protein conformation. DeArmond et al. (1997) mutated

two glycosylation consensus sites in Syrian hamster PrP (Figure 1.1A) and assessed the

influence of these mutations on prion passage in transgenic mice. The first mutation

(T183A) modulated intracellular trafficking of PrPC in hippocampus and prevented

prion infection. The second mutation (T199A) did not influence trafficking of PrPC in

hippocampus, it allowed passage of Sc237 strain and influenced its neuropathologic

phenotype. However, it did not allow passage of 139H strain. Glycosylation may

modify conformation of PrPC and, by stabilizing PrPSc-binding domain, change its

affinity for particular PrPSc species and strain, and determine selective neuronal

targeting of prions in brain.

A mouse PrP peptide (residues 89-143) containing the GSS-like mutation P101L was

synthesized using solid phase peptide synthesis and folded under conditions which

favour β-structure (Kaneko et al., 2000). This peptide induced de novo synthetic prion

formation in the transgenic mice (Tg(MoPrP,P101L)196/Prnp0/0) overexpressing PrPC

with the GSS-like mutation P101L. This was the case only when the peptide was folded

into a β-rich structure, due to conformational specificity of prion propagation. The

newly formed synthetic prions could be further passaged in the same transgenic mice

(Tremblay et al., 2004), but low levels of protease resistant PrPSc (rPrPSc) could be

found only after samples were concentrated by ultracentrifugation. However,

severalfold higher concentration of PrP that can be precipitated using sodium

phosphotungstate precipitation (PTA) was found in the synthetic prion-infected mice in

comparison with controls. Further, after cold proteinase K digestion (25 µg/ml of PK

for 1 h at 4°C), a 22-24 kDa fragment (PrP 22-24) was detected that was absent from

controls. The cold PK-resistant signal was stronger after PTA precipitation. The

neuropathologic phenotype in both spontaneously ill mice and those infected with the

synthetic prions was similar to the GSS phenotype in humans. Thus, P101L mutation is

required for a PrPSc conformation that enciphers the GSS disease characteristics.

23

Chapter 1 Prions

Legname et al. (2004) showed that prions are indeed infectious proteins. PrP amyloids

could represent a subset of β-rich PrPs, some of which could be infectious. The seeded

and unseeded amyloid fibrils from recombinant mouse recMoPrP(89-230) were

produced in vitro and inoculated into transgenic Tg(MoPrP,∆23-88) mice expressing

high levels of MoPrP(89-230)C. The mice developed disease between 380-660 days

after inoculation. The brains of mice inoculated with seeded amyloid had more protease

resistant PrPSc than brains of those inoculated with unseeded amyloid, and the

neuropathologic footprints were also different. Prions from the brains of mice

inoculated with seeded amyloid (mouse synthetic prion strain 1; MoSP1) were

inoculated into wild type mice (FVB) and into transgenic mice overexpressing wild type

mouse PrPC and caused disease after 154 and 90 days, respectively. Thus, PrP is

necessary and sufficient for infectivity. Glycans and GPI-anchor are dispensable for the

infectivity as MoPrP(89-230) contains neither of these posttranslational modifications,

and variations in PrP glycosylation are not required for prion diversity. Thus, biological

information carried by prions is determined by PrPSc conformation and spontaneous

prion formation could occur in any mammal expressing PrPC.

Prion strains properties are enciphered in PrPSc conformations. Yet, prion strains could

change their conformations and properties during prion propagation, as the primary

structure of host PrP presumably restricts the number of possible PrPSc conformations.

The species barrier could reflect selection of a small subpopulation of PrPSc that is

preferentially replicated in the new species, from a spectrum of PrPScs pre-existing in

the prion inoculum. No exogenous agent is required for prions to form in any mammal

expressing PrPC.

1.2.7 Cell-Free in vitro PrP Conversion

PrP may be transformed into protease-resistant forms in vitro using different templates,

different substrates and different conditions.

24

Chapter 1 Prions

Conversion of PrPC to a proteinase K-resistant form similar to PrPSc was shown in vitro

in a cell-free system (Kocisko et al., 1994). For transformation of a partially denaturated

PrPC into protease resistant form, partially denaturated preexisting PrPSc was required in

50 times excess, indicating that the specific interactions between PrPC and PrPSc are

sufficient for conformational change of PrPC. A seeding polymerisation mechanism,

with a nucleus formation as rate limiting step, was proposed as model for PrP

transformation (Chapter 1.4.2).

Kocisko et al. (1995) used the cell free conversion reaction to investigate the “species

specificity” in PrPC-PrPSc interactions. Whereas hamster PrPC was transformed into the

protease K-resistant forms when incubated with both the mouse-adapted (Chandler) and

hamster-adapted (263K) scrapie prion strains, mouse PrPC was resistant to

transformation by the hamster-adapted prion strain, as shown in vivo (Chapter 1.2.2).

This confirmed that the species specificity depends on interactions between PrPC and

PrPSc. The profiles of protease resistant bands were different after transformations of

hamster PrPC with mouse and hamster prions, indicating different conformations of the

newly formed proteinase K-resistant molecules.

To test whether the inheritance of strain characteristics is determined by stable

differences in PrPSc structure, Bessen et al. (1995) used two hamster-adapted mink TME

strains: hyper (HY) and drowsy (DY). These two prion strains show different

incubation times, clinical symptoms and neuropathologic profiles in both mink and

hamster. In the cell-free assay, these two prion strains converted the same hamster PrPC

into two protease-resistant forms with distinct characteristics indicating “strain

specificity”. For example, sizes of proteinase-resistant fragment differed by 1 kDa. This

was maintained under various conditions, showing that the structure of PrPSc stably

determines structure of nascent PrPScs.

To investigate whether the proteinase-K resistant PrP variant generated in vitro is

infectious, Hill et al. (1999) used the hamster-adapted scrapie strain Sc237 (not

transmissible to wild type mice) to transform the chimeric mouse-hamster PrP

(MH2MPrP) in vitro. Normal mice were susceptible to the MH2MPrPSc generated in

25

Chapter 1 Prions

vivo (Chapter 1.2.3), but no transmission was detected after inoculation of normal mice

with the MH2MPrPSc generated in vitro, suggesting that the acquisition of proteinase K-

resistance in vitro is not sufficient for production of infectivity. However, the authors

did not determine a ratio between initial Sc237 PrPSc and newly formed MH2MPrPSc,

nor clearly demonstrated that the MH2MPrPSc is major component of inoculum.

The effect of various endogenous glycosaminoglycans on PrP transformation was tested

in the cell-free system in the absence of denaturants (Wong et al., 2001). Whereas

heparan sulphate (HS) and pentosan polysulphate (PPS) stimulated reaction, chondroitin

sulphate had only a minor effect and keratan sulphate had no effect at all. In addition,

the PPS at higher temperature stimulated PrP conversion, possibly by increasing the

probability of overcoming the energy barrier (Chapter 1.4.2). PPS also increased the

rate of PrPSc formation, and abrogated the species barrier between hamster and mouse.

A decrease in α-helical content was found after PPS bound PrPC, possibly lowering the

activation energy in a way that favours formation of PrPSc. Of note here is that

contradictory reports about both positive and negative effects of glycosaminoglycans on

prion propagation in vivo and in vitro are also known.

Denaturation of PrPSc was not required when the CHO cell line lysates containing PrPC

were incubated with 6 times molar excess of the purified PrPSc from ME7- or 139A-

infected mouse brain (Saborio et al., 1999). However, it was not possible to transform

purified PrPC, showing that other cellular factor(s) are required for successful PrP

transformation.

PrPC and PrPSc co-localize in the detergent-resistant membranes of infected brains

(DRMs or “rafts”; Chapter 2.4). An effect of the cell membrane on PrP transition was

investigated in the cell-free system (Baron et al., 2002). As a source of PrPC, DRMs

were purified from the 5E4E neuroblastoma cells. As a source of PrPSc, brain

microsomes were isolated from the 87V prion strain-infected mice. The transformation

was optimal at pH 6-7, indicating that it occurs on the cell surface/extracellular space

and/or in the early endosomes. Simply mixing the two components was insufficient to

initiate PrP conversion, but transformation occurred after release of PrPC by cleavage of

26

Chapter 1 Prions

the GPI-anchor using the phosphatidyl-inositol-specific phospholipase C (PI-PLC).

Transformation also occurred when PrPC and PrPSc were present on the same

membrane, after fusing two membrane components using the 30% polyethylene glycol.

Thus, both the GPI-containing and GPI-depleted PrPC were transformed, but the

membrane-bound PrPC was substrate for conversion only when PrPSc was present on the

same membrane. Transfer and insertion of PrPSc responsible for spread of prions

(Chapter 2.4) may occur by the uptake of membrane particles (Mack et al., 2000),

exchange of membrane components (Batista et al., 2001) and GPI-directed insertion

into membrane (Medof et al., 1996).

Minute amounts of PrPSc (6-12 pg) were amplified using the protein-misfolding cyclic

amplification (PMCA) strategy (Saborio et al., 2001). PrPSc was replicated and

amplified by incubating the brain homogenates from prion-infected hamsters and

healthy hamsters during several cycles of incubation and sonication. The sonication

presumably broke PrPSc oligomers into smaller units, each of which was capable of

inducing further prion amplification. PrPSc, PrPC and unknown catalysts from brain

were required for this reaction.

Deleault et al. (2003) used a modified PMCA method to assess which cellular factors

other than PrPC might be involved in the pathogenic transformation. PrPSc amplification

was inhibited when mixtures of normal and scrapie infected hamster brains were treated

with the RNases that degrade single strand RNA (pancreatic RNase A, Rnase T1,

microccocal nuclease, benzoase), but not using the RNases that degrade double strand

RNAs or chimeric RNAs, DNAses and heparinases. Treatment of PrPC alone had no

effect. Purified RNAs from uninfected hamster and mouse brains showed stimulatory

catalytic activity but not RNAs from bacteria, yeast, worm and fly: thus the specific,

host-encoded RNAs with sizes of >300 bp were cellular cofactors for PrPSc formation.

The same method was used to determine which specific membrane subset contains all

necessary factors for PrP transformation, and to facilitate discovery of cofactors

involved (Nishina et al., 2004). Components of purified synaptic plasma membrane

preparations were sufficient to sustain PrP transformation, indicating that this process

27

Chapter 1 Prions

occurs on the cell membranes. Membrane attachment of PrPC was not necessarily

required for the process suggesting that prions might spread through central nervous

system from cell to cell. The requirement for a protein cofactor in PrP transitions was

also shown.

May et al. (2004) questioned the biological relevance of the in vitro conversion reaction

products on the grounds that these systems differ: substrates may be either recombinant

proteins or purified PrPC from brain, they may be seeded and non-seeded, dependent or

not-dependent on specific reaction conditions and prion strains used are different. The

proteinase K resistant PrPSc could shield PrPC from proteinase K degradation upon its

binding, producing a very modest rates of PrP transformation in vitro. These new

proteinase K resistant cores may even not be composed of PrPSc per se, as they contain

no α-helices typical of PrPSc.

1.2.8 Role of Disulfide Bond in Prion Propagation

Prion proteins contain a disulphide bond that stabilizes the folded globular C-terminal

region (Chapter 1.3), which may influence the pathogenic transformation. For instance,

the reduction of the disulphide bond may render the PrPC flexible for transformation or,

alternatively, free-thiolates may be required for polymerisation mediated by the

disulfide-shuffling.

Welker et al. (2002) showed that the monomers of purified PrPSc from scrapie-infected

hamsters are not linked by intermolecular disulphide bonds. Furthermore, in the cell-

free reactions, breakage and re-formation of disulphide bond were not required for PrP

conformational transition.

In contrast, recombinant hamster PrP could be converted to a new form, PrPRDX, by an

in vitro redox process (Lee and Eisenberg, 2003). The PrPRDX is prone to

oligomerization and seeded conversion. An amyloid-like structure of polymers suggests

a domain-swapping model for oligomerization, based on formation of intermolecular

disulfide bonds.

28

Chapter 1 Prions

In order to preserve cellular protein factors, Lucassen et al. (2003) modified the PCMA

strategy by omitting the use of sonication and non-denaturing detergent SDS. The

optimal pH range for PrP transition was between 6 and 8, suggesting that this process

occurs either on the cell membrane or within cytoplasm. Divalent cations were not

required for PrP transformation but a thiol-containing factor was required either in the

PrPC, PrPSc or in another molecule in synaptic membrane preparations, indicating that

reduction and re-formation of the intramolecular disulphide bond may occur during

conversion of PrPC to PrPSc.

Due to these contradictory reports, the role of disulphide bond in PrP transformation is

unclear at present.

1.2.9 Prions and Immune System

Prions accumulate in the lymphoreticular organs of mice after peripheral challenge.

However, replication of prions in brain does not depend on the lymphoreticular system.

Prions propagate in brain and overcome the species barrier even in absence of PrPC

expression in lymphoid and other nonneural tissues (Race et al., 1995). On the other

hand, prions accumulate in the lymphoreticular system of mice after peripheral

administration (Weissmann et al., 2001). Infectivity appears in spleen 1 week after

inoculation and reaches its maximum after 3-7 weeks (Montrasio et al., 2000). In

general, it is thought that stimulation of the host immune system increases susceptibility

to prions, and immunosupressive treatments reduce susceptibility. However, stimulation

of innate immunity by CpG oligodeoxynucleotides delayed onset of experimental prion

disease (Sethi et al., 1999).

One scenario is that prions after oral infection pass the gastrointestinal mucosa, possibly

through M cells, and accumulate in follicular dendritic cells (FDC) in the Peyer’s

patches (Weissmann et al., 2001). They then make their way (unclear) to the

lymphoreticular organs (enteric lymph nodes and spleen) where they replicate. In the

29

Chapter 1 Prions

lymphoid organs there is a “neuroimmune synapse”, communication between the

immune and lymphoreticular systems. Both T and B cells secrete nerve growth factors,

and nerve endings secrete molecules that stimulate immune cells. Following the

replication, prions access the central nervous system via the peripheral nervous system.

B cells, but not T cells or FDC, were first regarded as crucial for neuroinvasive (prion

transfer from the periphery to the central nervous system) scrapie (Klein et al., 1997).

Disruption of B cells in the Rag-2-, Rag-1- and Agr-deficient mice, and in the µMT

mice prevented peripheral RML prion propagation in most of infected mice. On the

other hand, the RML prion strain propagated in absence of functional T cells in the

CD4-deficient mice. The prions also propagated in the tumour-necrosis factor receptor-

1-deficient mice, which contain very few FDCs but have functional B and T cells. It

was speculated that B cells could carry prions.

However, expression of PrPC on B cells is not required for neuroinvasion but the

presence of B cells (Klein et al., 1998). Repopulation of the Rag-1 deficient mice with

fetal liver cells from either PrP-expressing or PrP-deficient mice enabled prion

replication. Of note here is that the B cell-deficient mice showed symptoms of scrapie

when high titer of prions in inoculum was used even without repopulation. Furthermore,

full infectivity was shown for brain extracts from the asymptomatic Rag-2-deficient and

µMT mice peripherally challenged with RML prions (Frigg et al., 1999). This ruled out

possibility that B cells may be sites for prion replication and transport.

FDCs, which express large amounts of PrPC, accumulate PrPSc extensively in the

lymphoid tissues of vCJD patients, sheep with natural scrapie and experimentally prion-

infected mice (Brown et al., 1999). Replication of the ME7 mouse-adapted scrapie

strain in spleen depended on PrP-expressing FDC, but not on lymphocytes. Maturation

of FDC in the severe combined immunodeficiency (SCID) mice, which show profound

deficiency of B- and T-lymohocytes, can be induced by signals from grafted B cells.

The spleens of SCID/PrP+/+ mice grafted with either PrP+/+ or PrP-/- bone marrow had

high titers of infectivity after both intracerebral and peripheral challenge. On the other

hand, the spleens from SCID mice expressing no PrP grafted with either PrP+/+ or PrP-/-

30

Chapter 1 Prions

bone marrow contained no infectivity, or only traces of infectivity. Thus, FDCs

expressing PrP may be critical for peripheral prion replication. Most of mice with

inactivated TNF-α or TNFR1, which are critical for FDC maturation, failed to develop

scrapie after peripheral challenge.

Treatment of mice with soluble lymphotoxin-β receptor causes disappearance of mature

FDCs from spleen (Montrasio et al., 2000). This treatment (during 8 weeks) retarded

neuroinvasion after peripheral challenge of mice with RML prions, indicating that

FDCs are essential for prion propagation in spleen. Mabbott et al. (2000) also showed

that a single treatment of mice with lymphotoxin-β receptor fused with human

immunoglobulin immediately before or after prion challenge interfered with the ME7

prion strain-induced pathogenesis, extending incubation time. This treatment had no

effect after intracerebral challenge.

Ablation of the chemokine receptor CXCR5 caused rearrangements within the spleen

microcompartments (Prinz et al., 2003). The CXCR5-/- knockout mice showed slightly

decreased incubation time after peripheral challenge with low doses of the RML strain.

It was assumed that relative positioning of FDCs and nerves may control the efficiency

of peripheral prion infection.

In contrast, Manuelidis et al. (2000) showed that the FU CJD prion strain propagated

after peripheral inoculation of mice with inactivated lymphotoxin β, which have no

FDCs. Prions propagated even when very low titres were used. Macrophages were

proposed as the cell type to disseminate prions, because infectivity associated with the

macrophages from prion-infected wild type mice was preserved after their propagation

in vitro.

The FU strain of CJD prions also replicated after peripheral challenge of mutant mice

that either lacked B cells, had B cells unable to secrete Ig, or could secrete only IgM

(Schlomchik et al., 2001). Therefore, neither B cells nor FDC, which depend on B cells,

were required for neuroinvasion from the periphery. An intact immune system could

increase prion uptake and delivery, but this is not essential condition.

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Chapter 1 Prions

A role of immune system in the peripheral prion disease pathogenesis is unclear.

Different results were reported using different prion strains. Immune system is not

conditio sine qua non for prion neuroinvasion, but it may either assist it or retard it.

1.2.10 Challenges to the Prion Concept

Some authors regard the prion biology as a poorly understood phenomenon and as an

open question.

Chesebro (1998) regarded the nature of the causative agent of prion diseases as an

enigma. Several arguments were raised against the prion concept. For example, it is

unknown if infectivity is generated in the cell-free conversion and there are too many

mouse-adapted scrapie strains (20). There was no prion transmission to the Prnp0/0 mice

maybe because PrPC may be involved as an agent cofactor, or as a virus receptor. As an

alternative to the prion concept, it was suggested that a virus could be the causative

agent of prion diseases. The absence of a virus detection by now could be just because

they are very difficult to find.

Chesebro (1999) indicated that neither the protein-only nor the viral model were proved

or disproved. Proof for the protein-only hypothesis requires generation of infectious

agent in a cell-free system (see Legname et al. (2004), Chapter 1.2.6), and proof for the

virus hypothesis requires isolation of a candidate virus. Prusiner’s arguments were

challenged by pointing out that infecting agents could be shielded during the UV

irradiation by other molecules in the mixture. Further, the more penetrant X rays might

break the agent’s nucleic acid, but it could re-assemble from small fragments during

replication. The mutant PrPs might be more efficient receptors, or susceptibility factors,

for a ubiquitous viral agent. Conversely, wild type PrP would be much less efficient so

the incidence of sporadic prion diseases is low. The prion purifications used in

experiments do contain nucleic acids and possibly some other factors that might

facilitate disease development. Prion transmissibility is a major difference between

prion diseases and amyloid diseases: what makes it so different?

32

Chapter 1 Prions

Weissmann (1999) noted that the experimental data support the hypothesis that prion is

derived partly or entirely of a PrP-derived molecule. However, as the ratio of infectivity

and PrPSc molecules is 1:100000, it remains possible that the PrPSc molecule associated

with infectivity is a separate entity designated PrP*. A number of questions about the

prion concept still remained to be solved. Among others, the mechanism and

requirements for PrP conversion, transport of prions from periphery to central nervous

system and mechanism of pathogenesis.

Prions differ from other amyloidoses by being able to travel from gut to brain

(Weissmann et al., 2002). There is the question why prions occur, and what drives

prions to destruct their own organism? Perhaps the “misfolded” variant of PrP may have

originated as a sort of “messenger” protein with a malignant potential that becomes

prominent in the postreproductive age. Alternatively, prions might be derived from an

ancient pathogen that integrated in genome and adopted physiological role. Prions could

also developed from a natural propensity of proteins to assume β-sheet rich

conformation and a failure of organism to prevent their formation.

Aguzzi and Polymenidou (2004) regarded the biology of prions as poorly understood, in

spite of spectacular advances in last few decades. They noted that a physical nature of

the prion disease-causing agent is unclear. Various hypotheses state that it may be

congruent, partially overlapping or different from the protease-resistant PrP isoform.

Neither PrP conversion nor prion replication is understood; also, what auxiliary factors

are involved of in these processes, what is the essence of different prion strains? What

are the mechanisms of neuroinvasion after prion infection and neurodegeneration in

brain? What is the normal function of PrP?

1.3 PrPC and PrPSc: Conformational Promiscuity

Two isoforms of PrP have different structures that determine their different physico-

chemical properties.

33

Chapter 1 Prions

1.3.1 Structures of PrPC

Structures of PrPC were determined using the NMR spectroscopy and X-ray

crystallography.

The first experimentally determined conformation of PrP was NMR structure of the

mouse PrPC C-terminal domain (residues 121-231; Riek et al., 1996; Figure 1.3A). A

globular fold contained a few elements of secondary structure: one anti-parallel β-sheet

(amino acids 128-131 and 161-164) and three α-helices (residues 144-154, 179-193 and

200-217). A disulphide bond connecting the first turn of the second helix and the last

turn of the third helix was highly shielded in a hydrophobic core of the peptide. The two

helices formed a twisted V-shaped scaffold onto which the first helix and β-sheet were

attached. The overall fold was stabilized by hydrophobic interactions in the

hydrophobic core between the side chains of residues from all secondary structure

elements and loops. The surface had a dipolar character, with a positive surface facing

the cell membrane side and with a negative surface containing two glycoslylation sites

facing the solvent. Residues associated with the inherited prion diseases were all located

in or adjacent to the secondary structure elements, suggesting that they might

destabilize the fold or influence its ligand-binding properties. NMR analysis of the full-

length mature mouse PrPC (residues 23-231) showed that the globular structure

described above is preserved in a longer peptide (Riek et al., 1997). The rest of the

protein (amino acids 23-120) was flexibly disordered, assuming a random-coil like

conformation. A PrP region between the residues 90-120 was indicated as a possible

major site in transition from the native to the pathogenic conformation.

The solution structure of the peptide corresponding to Syrian hamster PrP 27-30

(residues 90-231) showed that it has a globular fold similar to that of mouse PrP(121-

231) (James et al., 1997). The fold consisted of three helices (amino acids 144-156,

172-193 and 200-227) and an irregular antiparallel β-sheet (residues 129-131 and 161-

163). All three helices were longer than those of mouse, and a loop between the second

β-sheet strand and second helix was structured, unlike that in the mouse structure. This

analysis indicated an unusual dynamic structural feature of a region enriched with

34

Chapter 1 Prions

Figure 1.3: Structures of PrPC. (A) NMR structure of the mouse PrP(121-231). Ribbon diagram shows positions of the helices (yellow), antiparallel β-sheet (sky blue), connecting loops (green if defined, otherwise magenta) and disulphide bond (white) (copied from Riek et al., 1996). (B) NMR structure of the Syrian hamster PrP(90-231). The three helices (orange) are labelled A, B and C. The strands of β-sheet are labelled S1 (green) and S2 (blue). The red region is a cluster of hydrophobic amino acids 113-128 (copied from Liu et al., 1999). (C) NMR structure of the human PrP(23-230). The helices α1, α2 and α3 (orange), β-sheet (cyan), regions of nonregular secondary structure (yellow) and flexibly disordered tail of residues 23-121 (yellow dots) are shown (copied

34a

Chapter 1 Prions

from Zahn et al., 2000). et al., 2004). (D) NMR structure of the bovine PrP(23-230). Ribbon representation shows the three helices α1, α2 and α3 (green), β-sheet (cyan), regions of nonregular secondary structure (yellow) and flexibly disordered tail of residues 23-121 (yellow dots) (copied from Lopez Garcia et al., 2000). (E) Crystal structure of the human PrP dimer. Stereo view ribbon diagram indicates the two peptide chains (green and pink) with their helices three swapped and the N- and C-termini labelled. The intermolecular disulphide bridges are shown as ball-and-stick structure (copied from Knaus et al., 2001). (F) Crystal structure of the sheep PrP(123-230). Stereo view shows the elements of secondary structure (H1, H2, H3, β1, β2) and intramolecular disulphide bridge. The colours denote experimentally determined thermal factors (copied from Haire et al., 2004).

34b

Chapter 1 Prions

glycines and hydrophobic amino acids (residues 113-125; Chapters 1.2.5 and 2.1) that

potentially permitted alternative conformations. This cluster interacted with the first

strand of the β-sheet, constituting a domain with marginally stable polymorphic

structure. It was hypothesized that this conformational flexibility permits PrP

transitions. The structure also revealed a shape of the protein X-binding epitope

(Chapter 1.2.4).

The NMR structure of the full length Syrian hamster PrPC (amino acids 29-231)

showed, again, a folded globular domain containing three helices (residues 144-156,

172-193, 200-227) and possibly one short antiparallel β-sheet (160-163, 137-140

(Donne et al., 1997). The rest of the protein (residues 29-125) was highly flexible. No

major differences were observed between this structure and that of Syrian hamster PrP

27-30 (James et al., 1997), apart from stabilization of the distal end of the second helix

(residues 187-193), caused possibly by transient interactions with the flexible N-

terminus. The flexible region could provide the plasticity required for transformation of

PrP (Peretz et al., 1997); the energy barrier for the formation of a β-sheet in PrPSc will

be much lower from an initial “random coil” than an already stable structure. Although

deletions of the residues 68-85, 31-81 (Fischer et al., 1996) or 32-93 (Flechsig et al.,

2000; Figure 1.1E) did not prevent pathogenic transformation of PrP (Chapter 1.2.5),

the PrPs lacking residues 32-121 or 32-134 caused ataxia and neurodegeneration when

expressed in transgenic mice (Shmerling et al., 1998).

A refined NMR structure of the mouse globular domain (residues 121-131) revealed

somewhat different boundaries of the α-helices two and three (residues 175-193, 200-

219), and an additional short helix-like structure at the C terminus (amino acids 222-

226) (Riek et al., 1998). This analysis also showed dynamic plasticity of the β-sheet,

defined hydrophobic core and hydrogen bonding patterns. Analysis of the residues

involved in hereditary human prion diseases contradicted previous assumptions (Riek et

al., 1996) by showing that there may be differences in affecting stability of the globular

fold, ranging from very little deviations to major destabilizations. For example, the

mutation D178N would remove strictly conserved D178-R164 salt bridge. Depending

on the nature of residue 129 (M or V), the hydrogen bonding network that involves

35

Chapter 1 Prions

R164, Y128 and D178 would be affected differently. The M129/N178 mutation pair

causes FFI, and the V129/N178 combination triggers fCJD.

A refined structure of the Syrian hamster PrPC (residues 90-231) showed a defined β-

sheet and shorter first helix (amino acids 144-154) (Liu et al., 1999). This analysis also

showed a partial structure of the hydrophobic region between residues 113-128

(Chapter 2.1) and its interactions with the β-sheet and second helix (Figure 1.3B). No

regular secondary structure element was determined but this region manifested one or

more metastable, partially structured states. The flexibility of this region and its

conformational heterogeneity, as well as that of the adjacent irregular β-sheet, is a

hallmark of prion protein. Its characteristics resemble short-lived conformations

existing at the intermediate stage of protein folding that need additional impetus to form

a stable structure. Conformational promiscuity of the hydrophobic cluster may be a key

for the transition of PrPC to PrPSc: different members among ensemble of its

conformations may assist PrPC or PrPSc to adopt different conformations. In favour of

this hypothesis argue findings that the hydrophobic region is the best conserved

sequence across mammalian proteins (Wopfner et al., 1999; Chapter 2.1), it is the only

protein region that is always present in the prion protein protease resistant fragments,

and its mutation A117V causes GSS.

Zahn et al. (2000) showed that the human PrPC (residues 23-230) also adopted the

common mammalian fold in solution (Figure 1.3C). It had the folded C-terminal

globular domain (amino acids 125-228) and flexible N-terminal tail (residues 23-124).

Three α-helices (residues 144-154, 173-194, 200-228) and a β-sheet (residues 128-131,

161-164) comprised the fold. Interactions between the flexible tail and C-terminal

domain slightly stabilized disordered ends of helices two (amino acids 187-193) and

three (residues 219-226).

Despite overall conservation of the PrPC fold architecture, two regions showed some

discrepancy: the C-terminal end of the helix three was less defined in mouse, and the

loop between the β-sheet and helix two were well defined only in Syrian hamster.

Calzolai et al. (2000) introduced into human PrP C-terminal region (residues 121-230)

36

Chapter 1 Prions

mutations resembling either mouse PrP (M166V, R220K) or Syrian hamster PrP

(S170V). The mouse-like substitutions changed the structure of helix three into a

mouse-like structure. The Syrian hamster-like substitution increased definition of the

loop 166-170, typical for the Syrian hamster fold. Thus, difference in a single amino

acid may cause conformational variations in PrP.

The NMR structure of the bovine PrPC (residues 23-230 and 121-230) showed a typical

mammalian PrPC fold (Lopez Garcia et al., 2000). The three helices (amino acids 144-

154, 173-194, 200-226) and a β-sheet (residues 128-131, 161-164) were determined in

the folded C-terminus (amino acids 122-227), with a flexibly disordered N-terminal part

of the protein (Figure 1.3D) indicating that conformational transitions between PrPC and

PrPSc follow the same pathway in all the species. The bovine structure was more like to

the human fold than the mouse and Syrian hamster structures; the most prominent

differences were in the helices one and three and in the loop 166-172. Local structure

differences affect PrP conversion so the species barrier between human and bovine may

be more relaxed than that between bovine and mouse or Syrian hamster. The only

difference between human and bovine structures was the distribution of surface charges.

The structure of the human PrPC dimer (residues 119-126; Figure 1.3E) was the first

crystal structure of PrP (Knaus et al., 2001). During the process of crystallization PrP

was truncated and dimerized. Dimerization was the result of a profound conformational

change involving, first reduction of the intramolecular disulfide bonds, and then

swinging out, swapping and packing against the other half of the dimer of the helices

three, and finally re-formation of now intermolecular disulphide bridges. The overall

structure of the monomers in the dimer was still very similar to the solution-determined

structures. The differences were swapping of helix 3 and formation of an interchain β-

sheet region from a switch region between helices two and three. In contrast to the

NMR structures, a part of the hydrophobic region between amino acids 119-124 was

stabilized by packing against helix two, and the surface electrostatic potential was much

lower. The mutations causing familial prion diseases all mapped to the swapped helix

three, neighbouring helix two and switch region. The transition from monomer to dimer

may be important both for normal and pathogenic role (Chapter 1.2.5).

37

Chapter 1 Prions

The structure of the sheep PrPC C-terminal domain (residues 123-230) monomer was

also determined by X-ray crystallography (Haire et al., 2004). This structure showed a

globular fold corresponding to the NMR-determined structures (Figure 1.3F) with three

helices (residues 143-154, 172-194, 200-225) and an antiparallel β-sheet (amino acids

129-131, 161-163). In contrast with the human dimer crystal structure, the hydrophobic

region (residues 124-137) showed a high thermal factor. This analysis indicated two

possible regions where a β-sheet could be propagated from pre-existing β-strands. The

first region (L1) was the antiparallel β-sheet: it was associated with a crystallographic

dyad that generated a four-stranded intermolecular β-sheet as a lattice structure. The

second such region (L2) was a polythreonine rich end of the helix two and adjacent loop

in which α to β transition may occur due to its propensity to form a network of

hydrogen bonds. Of note here is that this region is conserved in all mammalian PrPs,

and that a number of other proteins contain this sequence motif as well (Chapter 6.3). A

model of PrP tetramers was generated involving L1 and L2 regions as initiators of

noncovalent molecular associations, forming potential oligomeric nucleating units for

the PrP aggregation.

The NMR and crystal structures of the normal isoform of PrP indicated that the N-

terminal part of the protein is unstructured, and that the C-terminal region has the

globular fold. The flexibility and conformational heterogeneity of the conserved middle

hydrophobic region is a hallmark of the prion protein. Different spectra of

conformations of this region may enable PrPC or PrPSc to adopt different conformations.

1.3.2 Models of PrPSc Structure

Experimental determination of the PrPSc structure using NMR or X-ray crystallography

poses a problem due to its insolubility in the non-denaturing detergents.

Combining computational techniques and experimental data, Huang et al. (1996)

constructed a model of PrPSc structure (residues 108-218; Figure 1.4A). Previous

circular dichroism and Fourier transform infrared measurements indicated that roughly

38

Chapter 1 Prions

Figure 1.4: Models of PrPSc structure and PrP transformation. (A) Model of human PrPSc with a four-strand mixed β-sheet and two helices packed against one face of the β-sheet. Some residues implicated in the species barrier (N108, M112, M129, A133) are shown in the ball-and-stick model clustering on the PrPC-PrPSc interface. The loop involved in the species barrier is labelled yellow (adapted from Huang et al., 1995). (B) Transformation of PrP. Structure of the PrP 27-30 contains the left-handed parallel β-helical fold. The α-helices (red) are labelled A,B, and C and the two strands of β-sheet (green) are labelled S1 and S2. The model indicates refolding of the PrPC region involving the β-sheet, helix A, N-terminal part of helix B and connecting loops into the parallel β-helical fold in PrP 27-30 (Wille et al., 2002). The model representing PrP transformation was downloaded from http://www.cmpharm.ucsf.edu/cohen/welcome. html and is reprinted with permission from The Cohen Group, University of California San Francisco.

38a

Chapter 1 Prions

50% of α-helices in PrPC adopt β-sheet structure in PrPSc. Taking this into account and

using the combinatorial packing approach for α/β-rich proteins, from initial 106

structures models were selected representing 6 topology families. These models

predicted a 4-stranded β-sheet structure with one face covered with two α-helices. The

region of PrP between residues 90-145 postulated as PrPC-PrPSc interface (Chapter 1.2)

adopted β-sheet structure in the model.

Two dimensional crystals were discovered in the preparations of PrP 27-30 and

miniprion PrP106Sc with high infectivity titers (Wille et al., 2002). Electron

crystallography analysis at the low resolution of 7Å indicated a structure with three-fold

symmetry of crystals (three dimers) and with sugar chains located toward the outside of

oligomers. A model predicted that both PrP 27-30 and PrP106Sc adopted a parallel β-

helical fold (Figure 1.4B) with the α-helices and sugars on the periphery of the

oligomer. The planar faces permitted stacking of oligomers along the fibril axis with the

β- helices providing flat sheets for lateral assembly into oligomers and filamentous

assemblies. The β-helix is a simple and very stable fold, and it is found in proteins

exposed to harsh environments. The PrP transition may involve a stabilization of a

proto-β-helical motif by adjacent PrPSc molecule with subsequent extension to form the

complete β-helix.

Additional molecular modelling and improved electron crystallographic data indicated

that the structure of PrPSc is best described as a parallel left-handed β-helical fold

(Govaerts et al., 2004). This structure may form trimers, with the residues from 89-175

adopting β-helical structure, and with the rest of the C-terminal region (residues 176-

227) retaining the disulphide-linked α-helical conformation. These trimers are the

fundamental unit of PrPSc structure. An oriented fibril could be assembled by stacking

of the units in the head to tail fashion. Of course, the full length PrPSc does not

polymerize into fibrils until the N-terminal resides 23-88 are cleaved using proteinase K

and detergent is added.

39

Chapter 1 Prions

Combination of the low-resolution experimental data and molecular modelling

indicated refolding of the PrPC region involving the β-sheet, helix A, N-terminal part of

helix B and connecting loops into the parallel, left-hand β-helix in PrPSc.

1.4 Conformational Transformation of Prion Protein

What is the mechanism for pathogenic conformational transformation of PrP?

1.4.1 Protein Folding and Misfolding

I describe firstly the general principles of protein folding and misfolding.

Protein folding is an example of biological self-assembly. Evolution has invented

numerous highly specific and highly selective protein structures whose key functional

groups are brought to close proximity by the folding process (reviewed by Dobson,

2003). The folding process represents a stochastic search of many conformations

possible for a polypeptide chain in a given conditions, driven by fluctuations in the

conformations of unfolded and partially folded polypeptides (Figure 1.5). The native-

like amino acid interactions are more stable and more persistent than those of non-

native ones. By a process of trial end error, a polypeptide chain can find its structure

with lowest energy.

Evolution has enabled proteins to fold rapidly and efficiently by exploring just a small

number of all possible conformations during transition from an initial randomly-coiled

structure to its native conformation. The interactions involving small number of key

residues force the polypeptide chain to adopt a rudimentary native-like architecture.

Once this transition state is achieved, proteins almost invariably adopt their final native

conformation, condensing around the folding nucleus. Formation of the overall chain

topology may be even more important that formation of helices and sheets because it

was shown experimentally that a stochastic search process will be more time-

consuming if the nucleus-forming residues are further away from each other in protein

sequence.

40

Chapter 1 Prions

Figure 1.5: An energy landscape for protein folding: its surface “funnels” the denatured starting conformations to the final unique native structure. The surface was derived by computer simulation of a simplified small protein model folding. The structures superimposed on the schematic surface correspond to different stages of folding process. At the top, the three unfolded species are depicted representing three different starting points for folding. The simplified folding trajectories for each species are shown on the surface. The saddle point (transition state) is the barrier all conformers must pass in order to fold to the native state. The transition state ensemble was calculated using computer simulations taking into account experimental data from mutational studies of acylphosphatase. The yellow spheres in the depicted transition state are the three “key residues” in the structure: once they have formed their native-like contacts the overall native-like fold topology is established. The native structure is shown at the bottom of the surface (copied from Dobson, 2003).

40a

Chapter 1 Prions

Due to the stochastic nature and complexity of protein folding, inevitable mistakes in

this process lead to protein misfolding. Life has invented a number of strategies to cope

with this problem; for example, molecular chaperones prevent residues exposed in

unfolded proteins, but otherwise buried, from inappropriate interactions. Catalysts such

as peptidylprolyl isomerases and protein disulphide isomerases accelerate protein

folding. Extracellular proteins fold in the endoplasmic reticulum (ER). Few chaperones

exist outside cell, but quality-control mechanisms (molecular chaperones and enzymes

in the ER) trim proteins before their export.

Amyloidoses are diseases associated with accumulation of a specific protein, which

occur when the housekeeping mechanisms cannot cope with protein misfolding. These

disorders are frequent in older age, when the tendency for protein misfolding and

related damage increases and the efficiency of chaperones and response mechanisms

decreases.

In contrast to highly individualistic protein structures, the amyloid aggregates all have

very similar fibrillar morphology. They bind Congo red (a sugar stain, therefore

“amyloid”) and show a characteristic “cross-β” x-ray diffraction pattern indicative of a

hydrogen bonded, β-sheet-rich structure with units arranged perpendicularly to the axis.

Hydrogen bonds forming the core structure are from the main polypeptide chain. Since

the main chain backbone is present in every protein, it is not surprising that every

protein could be amyloidogenic under certain conditions. Fändrich et al. (2001) showed

that even an ordinary α-helical globular protein such as myoglobin can assume an

amyloidogenic conformation if conditions are right (e.g. incubation of apomyoglobin

(protein without the haem group) in 50mM sodium borate, pH 9.0 at 65°C).

Evolution has selected against protein sequences that would favour the amyloid β-sheet-

rich structure. However, mutations leading to amyloidoses may increase the population

of partially folded intermediates, may affect proteolysis and processing of proteins and

may increase the propensity for protein aggregation by increasing protein

hydrophobicity or decreasing protein charge.

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Chapter 1 Prions

Cohen and Kelly (2003) discussed the kinetics and thermodynamics of protein folding,

misfolding and aggregation (Figure 1.6). Proteins usually fold into their native states

because they can easily overcome the free energy of activation separating the unfolded

and transition states. Under misfolding conditions, misfolded oligomeric state of

aggregation-prone proteins could be more stable that the native folded state. However,

the kinetic barrier separating the folded and aggregation competent states is big and

usually insurmountable over a biologically relevant time frame. Furthermore, intrinsic

clearance mechanisms operate more efficiently than misfolding processes. Yet, under

misfolding conditions such as ageing and mutations, a misfolded aggregation-competent

state could be preferred to the native state, due to the interplay between sequence and

environment. Changes in environment may be caused by intracellular and extracellular

alterations. The environments enabling amyloid formation are highly selective, as they

must allow the formation of noncovalent polypeptide interactions, but, at the same time,

they must be specifically unfavourable to protein folding (Fändrich et al., 2003).

Of particular note here is that amyloid can also be a natural product (Kelly and Balch,

2003). First, the synthesis of pigment granules in mammalian melanocytes and retinal

epithelial cells includes formation of amyloid-like fibrils from a glycoprotein Pmel17.

Second, a bacterial Curli protein forms fibrous matrix on the outside cell wall in its

amyloid form. Therefore, the amyloid-like fibrils could also exist naturally as

quaternary protein nanostructures.

The stability of native structure of soluble proteins depends on the intramolecular

protection of hydrogen bonds from water attack. Fernandez et al. (2003) defined a

structural characteristic indicating amyloidogenic propensity: a density of backbone

hydrogen bonds exposed to water attack in monomeric structure. In the folded C-

terminus of PrPC, 55% of the hydrogen bonds are “underwrapped”, insufficiently

shielded from water attack. For instance, in the first helix of PrPC, 100% of hydrogen

bonds are underwrapped. Thus, the first helix is particularly vulnerable to water attack

and prone to rearrangement, and it indeed undergoes α-helix to β-strand transformation

(Figure 1.4). PrP mutations that cause familial prion disease may either cause a

42

Chapter 1 Prions

Figure 1.6: The thermodynamics of protein folding (copied from Cohen and Kelly, 2003). The free energy of activation (∆G‡) separates the unfolded and transition states. The difference in free energy between the unfolded and folded states (∆G) favours the folded state and dictates the relative proportions of unfolded and folded proteins. The kinetic barrier (∆G‡*) separating the native and misfolded states is big, but the monomeric misfolded state is more favourable than the folded state (∆G*) under misfolding conditions. Aggregation of the monomeric misfolded proteins is dependent on concentration.

42a

Chapter 1 Prions

reduction in desolvation destabilizing the structure (e.g. F198S increasing flexibility of

the helix 2-helix 3 junction; Chapter 1.3), or improve the packing of the β-sheet bonds

(e.g. mutations stabilizing the 134,159 β-strand hydrogen bond) and stabilize the region

in which pathogenic transformation is initiated. Binding of protein X may provide some

protection of underwrapped hydrogen bonds at the C-terminus of PrP. The PrPC fold

contains the highest concentration of underwrapped hydrogen bonds of 2811 PDB

proteins sampled. This feature could be related with the dynamic intrinsic character of

PrP (Chapter 1.4.2).

Aberrant proteins occur sporadically or as a consequence of mutations, unbalanced

subunit synthesis and damaging conditions (e.g. oxidation). A mechanism for primary

quality control in the ER discriminates between non-native and native proteins

(reviewed in Sitia and Braakman, 2004). Exposure of hydrophobic residues, unpaired

cysteins and immature glycans in the non-native protein structures induces binding of

ubiquitous folding sensors (ER-resident chaperones, lectins, glycan-processing

enzymes, peptidylprolyl isomerases and oxidoreductases).

A secondary quality control is mediated by cell-specific factors that facilitate export of

certain cell-specific proteins. The heat shock-response is activated when aberrant

proteins accumulate in the cytosol, and the unfolded protein-response (UPR),

coordinated synthesis of the ER-resident chaperones and enzymes, is activated upon

accumulation of misfolded proteins in the ER. There are three stress-sensors in the

mammalian ER: ATF6 (Chapter 5.5.6), Ire1 and PERK. ATF6 is a transmembrane

protein and interacts via its luminal domain with BiP, an abundant ER chaperone (for

example, BiP is chaperoning the folding of PrP (Jin et al., 2000). When unfolded

proteins titrate BiP from ATF6, the ATF6 is released from the ER membrane and

translocates to the nucleus. In the nucleus it stimulates the transcription of genes

involved in UPR. For instance, transcription of the gene encoding XBP-1 is initiated.

The transcription factor XBP-1 (regulated post-translationally by Ire-1) in turn induces

the transcription of genes encoding factors that facilitate ER-associated degradation

(ERAD; PrPC is also degraded by the ERAD-proteasome pathway; Chapter 2.4). PERK

transiently inhibits translation by phosphorylating eIF2α. Homeostasis in the ER is

43

Chapter 1 Prions

maintained by the translocation of terminally misfolded proteins to the proteasomes in

the cytosol. When the ER cannot cope with load of misfolded proteins (ER stress),

apoptosis is triggered.

Cells constantly have to cope with a heavy load of misfolded proteins. Although the

general error rate in DNA synthesis is only about 1 in 10 billion incorporated

nucleotides (10-10) the error rate in RNA synthesis (10-5) is million times higher and the

error rate of protein synthesis (above 10-4) is even higher (Radman et al., 1999). Next, a

number of intracellular conditions damage cell proteins: increased temperature, reactive

small molecules causing oxidation, deamidation, glycation or nitrosylation, high salt

concentrations favouring dissociation of multimers, fatty acids acting like detergents

and, finally, other “sticky” unfolded proteins. Multiple changes such as the deamidation

of glutamines and isomerization also occur in proteins spontaneously over time.

Incomplete proteins, missense proteins, free subunits of multimeric complexes and post-

synthetically damaged proteins will be rapidly degraded in cells (reviewed in Goldberg,

2003). Although only changes that significantly perturb protein folding (e.g. mutation of

key residues, large indels) will lead to their rapid clearance, 30% of newly sythesized

proteins are rapidly degraded.

The basic function of the cell degradative machinery is elimination of misfolded or

damaged proteins that would otherwise harm the cell. Key components of this system in

eukaryotic cells are the molecular chaperones, 26S proteasomes and ubiquitination. The

chaperones mediate ubiquitin conjugation, triggering rapid degradation by the 26S

proteasome. Misfolded proteins may also be degraded by proteasome without

ubiquitination. Once production of misfolded proteins exceeds the cell degradative

capacity, the aberrant proteins will begin to accumulate and aggregate into intracellular

inclusions; they may also damage the cell and trigger apoptosis.

Many different proteins can misfold and form aggregates within or outside the cell that

induce cell dysfunction (reviewed in Selkoe, 2003). The systemic amyloidoses are

characterized by protein accumulation in a number of tissues; for example, light

immunoglobulin chain is deposited in multiple tissues during the primary systemic

44

Chapter 1 Prions

amyloidosis. The organ-limited amyloidoses result from protein accumulation in

specific tissues. Neurodegenerative diseases are characterized by the accumulation of

aberrant proteins in brain: the post-mitotic milieu of the mature neuron is particularly

sensitive to protein misfolding and accumulation. The most common such diseases are

Alzheimer disease (extracellular aggregation of amyloid β-peptide and intracellular

aggregation of tau protein), Parkinson disease (intracellular aggregation of α-

synuclein), Huntington disease (intracellular aggregation of proteins with polyglutamine

repeats) and prion diseases (intracellular accumulation of PrPSc). For example, the A30P

and A53T α-synuclein mutant forms bind strongly to the receptor for chaperone-

mediated autophagy (CMA) on lysosomes and impair the degradation of other CMA

substrate proteins (Cuervo et al., 2004). This disturbs normal rates of protein

degradation and, by promoting aggregation, favours toxic gain-of-function.

Virtually every protein has propensity to fold and misfold, and the cell has to deal with

constant load of aberrant proteins. Once capacity of the cells, and of the organism, to

clear misfolded proteins is overwhelmed, the diseases of protein folding occur.

1.4.2 Models of Prion Protein Transformation

Two competing models were proposed for the mechanism of pathogenic PrP

conformational transformation.

1.4.2.1 Template Assisted Polymerisation Model

Assuming that the causative agent of scrapie is a protein, Griffith (1967) proposed three

ways in which its replication may occur. Firstly, the infectious protein may induce

expression of an otherwise repressed gene. This gene may not be repressed with

absolute efficiency so the protein may occasionally appear causing sporadic forms of

disease. Secondly, the infectious protein may exist in two different forms

(conformations) that would self-replicate by dimerization. The reactive form of protein

may dimerize more readily than its stable form. However, reactive molecules are not

45

Chapter 1 Prions

normally available due to a large energy barrier for transition from stable to reactive

form. If the kinetic barrier between protein forms were not absolute, spontaneous

appearance of the disease would occasionally occur. The already existing dimers may

act as a template (“condensation nuclei”) converting stable protein forms into a novel

dimer. If the dimers could penetrate the cell, they may act as infectious agent. Existence

of reactive forms with different conformations may explain the existence of different

clinical forms of scrapie. Third, the agent may replicate by a mechanism similar to

production of antibodies; the induced antibodies must be identical with the agent.

Cohen et al. (1994) proposed a conformational model for prion replication. A basis for

PrP transformation was stochastic fluctuation in the structure of PrPC resulting in a

partially unfolded monomer PrP*, intermediate in the formation of PrPSc. The PrP*

would either revert to PrPC, be degraded or converted to PrPSc. The PrPSc, which is

normally formed in insignificant amounts, would promote conversion of PrP* to PrPSc.

In the infectious forms of prion diseases it could be exogenous. Mutations in the PRNP

gene causing genetic prion diseases would induce greater stochastic fluctuations in the

structure of mutant PrPC, promoting its conversion to PrP* and increasing the likelihood

for PrPSc formation. Sporadic forms could be explained by chance accumulation of

excess of PrP* on rare occasions, or by somatic mutations that destabilize PrPC

structure, promoting the transformation. It was suggested that oligomerization is

required for the formation of PrPSc, but no amyloid aggregates of prion protein were

experimentally observed in prion-infected brains.

Prusiner et al. (1998) introduced into the model an additional component, protein X,

which preferentially binds PrP* (Figure 1.7A). They suggested that in normal cells PrPC

exists in equilibrium in its proteinase-sensitive monomeric α-helical state, or bound to

protein X. Protein X binding precedes productive PrPSc interactions: the PrP*/protein X

complex will bind PrPSc and form a replication-competent assembly which is required

for transformation. The smallest infectious particle of PrPSc may be as small as dimer or

trimer of parallel left-handed β-helices (Govaerts et al., 2004), since inactivation of

prions by ionizing radiation indicated a target size of approximately 55 kDa (Bellinger-

Kawahara et al., 1988). A fraction of nascent infectious dimers (or trimers) dissociate

46

Chapter 1 Prions

Figure 1.7: Models of prion replication. (A) Template assisted polymerisation model. PrPSc is thermodynamically more stable than PrPC, but it is kinetically inaccessible. PrPC transforms to PrP* and binds protein X in the first step. The PrP*/protein X complex then binds a PrPSc molecule, and PrP* is transformed into nascent PrPSc. Finally, protein X is released and a PrPSc dimer remains. A number of PrPSc dimers will dissociate enabling more replication cycles but the most of dimers will accumulate increasing prion titre during the incubation time. Stoichiometry of this process is unknown (Prusiner et al., 1998). (B) Noncatalytic seeded polymerisation model. The PrPC and PrPSc are in equilibrium that favours PrPC. The PrPSc is stabilized upon interaction with the pre-formed seed. The seed formation is unfavourable but once it is present rapid addition of PrPSc molecules occurs. Fragmentation of aggregates increases the number of seeds (Caughey, 2001).

46a

Chapter 1 Prions

into uninfectious monomers that prime new replication cycles, but the majority of

dimers (or trimers) accumulate, resulting in an increase in prion titer during the

incubation period.

Observations supporting this model showed that the prion amyloid rods observed by

electron microscopy in PrP 27-30 preparations are in fact an artefact of limited

proteolysis of PrPSc in the presence of nondenaturing detergent. These rods are not an

infectious entity, despite showing amyloid properties, because infectivity was not

affected when the prion rods were dissociated using a mixture of phospholipids and

nondenaturing detergent. The liposomes containing two to four PrP 27-30 molecules

generated by dispersion of PrP 27-30 aggregates retained scrapie infectivity (Gabizon et

al., 1987). Also, using organic solvents Wille et al. (1996) showed that the PrPSc forms

amyloid polymers only after it is converted to PrP 27-30, that the β-sheet-rich structures

required for infectivity and amyloid formation are different, and that amyloid formation

is not required for the PrPSc synthesis and prion propagation.

The folding of PrP to the α-helical isoform is very rapid, with a half-life of 170 µs at

4°C (Wildegger et al., 1999). However, the conformational transformation from the α-

helical to the thermodynamically more stable β-sheet rich PrP isoforms is opposed by a

large energy barrier that is associated with unfolding (Baskakov et al., 2001; Figure

1.6). The calculated energy barrier of 35-45 kcal/mol is sufficient to prevent the

transformation over the functional lifetime of the protein. Because of the size of the

energy barrier PrP is kinetically trapped in the α-helical form, and the transformation

occurs slowly. Under partially denaturing conditions it is possible to avoid the kinetic

trap that leads to the normal cellular isoform, PrPC, and fold the protein into the non-

native β-sheet rich PrP isoforms.

Unglycosylated recombinant PrP corresponding to PrP 27-30 may adopt in vitro two

different non-native abnormal β-sheet-rich isoforms, a β-oligomer (octamer) and an

amyloid fibril (Baskakov et al., 2002). These two distinct isoforms co-exist under

certain conditions, but experimental conditions dictate preference for forming either

form: acidic pH favours formation of β-oligomer, neutral pH favours formation of

47

Chapter 1 Prions

amyloid. Whereas the β-oligomer has proteinase K-resistant core, the amyloid was

degraded by proteinase K.

Baskakov et al. (2004) studied the biophysical nature of transition state to PrPSc. The

kinetic of in vitro denaturation of the folded α-helical recombinant human PrP 90-231

to amyloid fibrils showed a change in thermodynamic character of the native ensemble

under partially denaturing conditions. Thus, the variable thermodynamic character of

the native ensemble mirrors the intrinsic ability of PrP to adopt different abnormal

conformations under pathologic conditions.

1.4.2.2 Seeded Polymerisation Model

Alternatively, transformation of PrP may be induced by the contact between PrPC and

PrPSc polymer (“seed”; Figure 1.5B) (Kocisko et al., 1994; Caughey, 2001), in which

case the mechanism of pathogenic transformation of PrP could be similar to those of

other amyloidogenic proteins. However, this does not explain the main difference

between prion diseases and other amyloidoses, which is the infectivity associated with

PrPSc. According to this model, the PrPC and PrPSc are either in equilibrium

(noncatalytic polymerisation) or PrPC interacts directly with the PrPSc seed

(autocatalytic or templated polymerisation). In both cases the PrPSc is stabilized only

when it is added to a pre-existing crystal-like seed or aggregate of PrPSc. The stable

aggregate must contain a minimal number of molecules and its spontaneous formation

would be a rare event, but once it is present it will trap PrPC at much faster rate

(Weissmann, 1999). Fragmentation of the aggregates (secondary seeding) would

increase number of seeds and enable exponential aggregation. The replication of yeast

prions (Tuite and Cox, 2003; Chapter 1.5.1) argues in favour of this model.

Based on the seeded polymerisation model, Hall and Edskes (2004) developed a two-hit

model of amyloid formation and infection. Potentially infectious amyloid may be

present in healthy individual in a quiescent state. A change in the state of host or a

transmission to a more susceptible host will initiate propagation of the prion/amyloid

form.

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Chapter 1 Prions

1.5 Prions in Other Systems

In a broad sense of the term, prions are elements that propagate conformational

variability (Prusiner, 1998) and need not necessarily be pathogenic entities. Findings of

yeast prions therefore showed the generality of this epigenetic phenomenon, raising

questions whether similar mechanisms may exist normally also in higher organisms. It

was recently demonstrated for the first time that a neuronal isoform of the cytoplasmic

polyadenylation element binding protein (CPEB) from California sea hare Aplysia

californica does exhibit prion-like properties in the yeast model system.

1.5.1 Yeast Prions

Three prion-mediated phenotypes were discovered in the yeast Saccharomyces

cerevisiae (reviewed in Tuite and Cox, 2003): the [PSI+] phenotype determined by the

prion form of Sup35 protein, the [URE3] phenotype determined by the prion form of

Ure2 protein, and the [RNQ+] phenotype determined by the prion form of Rnq1 protein.

In the filamentous fungus Podospora anserina the [Het-s] phenotype determined by the

amyloid form of HET-s protein was also found.

The [PSI+] cells show increased suppression of nonsense mutations due to insertion of

serine instead of the UAA-mediated ochre stop signal. This is a consequence of the

aggregation of translation termination factor Sup35. The [URE3] cells may utilize

ureidosuccinic acid in the presence of ammonium ions as a consequence of the

conversion of Ure2 to its prion form. The prion form of Rnq1 in the [RNQ+] cells allows

induction of the other yeast prion phenotypes.

A common feature of yeast prions is the presence of prion-forming domains (PrDs)

enriched with the polar amino acids (Gln, Asn). These domains function as transferable

prion-forming modules and are required for both induction and propagation of the prion

state. The PrD from Sup35 contains five protein repeats (PQGGYQQYN) that modulate

prion phenotype. Vertebrate prion proteins also contain repeats (Chapters 1.2.5 and 2.1).

49

Chapter 1 Prions

The normal forms of Sup35 and Ure2 may be converted into their prion states in vitro

by “seeding” with the prion aggregates. This transformation occurs by the nucleated

polymerisation. Fragmenting seeding material by sonication reduces the lag-time before

the appearance of fibrillar forms (see also Figure 1.7B). During this process transient

oligomeric units are first formed. Sup35 oligomers contain between 20 and 50

molecules andUre2 oligomers contain between 4 and 6 molecules. These undergo slow

conversion to the stable nucleating units (seeds). The prion form of Ure2 might retain

functional activity, but this is not clear for the Sup35 prion.

Bona fide protein-only transmission was first demonstrated using yeast prions. Yeast

prion strains have different phenotypes. For instance, [PSI+] strains show differences in

suppression efficiency reflected in different aggregate morphology (e.g. size, curvature,

x-ray diffraction pattern) and different colour of the ade1 mutant colonies. King and

Diaz-Avalos (2004) isolated the [PSI] particles from yeast cells harbouring the [VH],

[VK] and [VL] prion strains and used them to transmit their properties to the Sup35(1-

61)-GFP-Strep(II) chimeric proteins in vitro. The infectious amyloid fibres nucleated in

vitro faithfully propagated the strain-specific properties of prion seeds, indicating that

the basis for prion strain difference is structural.

Tanaka et al. (2004) also demonstrated that the Sup-NM (recombinant Sup35 fragment)

amyloids have distinct conformations leading to different [PSI+] strains. The amyloids,

artificial prion strains generated in vitro at different temperatures, had different

physical-chemical properties (e.g. melting temperature range and structure determined

by the electron paramagnetic resonance spectroscopy). When yeast cells were infected

with these strains, different phenotypes (e.g. transmission efficiencies and suppression

efficiencies) were observed. Different conformations of the single infectious protein

determined variation in prion strains.

1.5.2 An Animal Protein Shows Prion-like Properties in Yeast

The normal neuronal variant of the California sea hare Aplysia californica CPEB has

similar characteristics to the yeast prions. Its N-terminal domain is rich in Gln and is

50

Chapter 1 Prions

predicted to be conformationally flexible (Si et al., 2003b). Like the yeast prions, this

region was able to confer epigenetic changes when it was fused either to the green

fluorescence protein or the rat glucocorticoid receptor and expressed in Saccharomyces

cerevisiae. The full-length protein also existed in distinct conformational states. Unlike

the yeast prions, the dominant, self-perpetuating prion state of this protein increased

CPEB function, as reflected in the increased stimulation of CPEB-activated mRNAs.

Although it was dispensable for CPEB activity, the N-terminal prion-like domain

increased the rate at which CPEB assumed active state, perhaps maintaining the

catalytic C-terminal domain in active state.

The prion state may therefore sustain increased translation locally at the activated

Aplysia californica synapses. In the context of long-term memory storage and in an

analogy with the posttranslational modifications of proteins, CPEB may be transformed

into the prion state after synaptic stimulation, and it could increase activation of the

mRNAs required for the long-term forms of synaptic plasticity. The self-perpetuating

prion state is energetically “cheap” as it does not require continued signalling by

kinases or phosphatases, and it can be easily reversed. Similar mechanisms may work in

other biological contexts as well (e.g. transcription, differentiation). Thus,

conformational variability as part of the normal function and action could be utilized in

many other prion-like proteins.

51