MISFOLDING OF PARTICULAR PrP SPECIES AND SUSCEPTIBILITY TO

PRION INFECTION

By

Muhammad Qasim Khan

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of BiochemistryUniversity of Toronto

© Copyright M.Q. Khan 2010

ABSTRACT

TITLE: Misfolding of Particular PrP Species and Susceptibility to Prion Infection

Muhammad Qasim Khan, M. Sc.

Department of Biochemistry

University of Toronto

2010

Pathogenesis of prion diseases in animals is associated with the misfolding of the cellular

prion protein PrPC to the infectious form, PrPSc. We hypothesized that an animal’s

susceptibility to prions is correlated with the propensity of an animal’s PrPC to adopt a β-

sheet, PrPSc-like, conformation. We have developed a method which uses circular

dichroism (CD) to directly calculate the relative population of PrP molecules that adopt a

β-sheet conformation or the ‘β-state’, as a function of denaturant concentration and pH.

We find that the PrP from animals that are more susceptible to prion diseases, like

hamsters and mice, adopt the β-state more readily than the PrP from rabbits. The X-ray

crystal structure of rabbit PrP reveals a helix-capping motif that may lower the propensity

to form the β-state. PrP in the β-state contains both monomeric and octameric β-

structured species, and possesses cytotoxic properties.

ii

ACKNOWLEDGMENTS

First off, a big thanks to my supervisor Avi Chakrabartty. I appreciate everything

that he has done for me. He has taught me a great deal about proteins and how they

behave, and has also taught me a lot of life lessons, and how to apply protein behaviour

to our own lives – global minimum energy and the Lamm equation (he knows what I’m

talking about).

I like to thank everyone who has supported me in Toronto and all those back in

Ottawa. Y’all know who you are.

A special mention to my guitar, my running shoes and my basketball, for allowing

me to escape and rock out when I’m not crunching my brain in the lab.

Yeah.

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TABLE OF CONTENTS

ABSTRACT ii

ACKOWLEDGMENTS iii

CHAPTER 1: INTRODUCTION………………………………………………….. 1The Protein-Only Hypothesis………………………………………………………. 1Biological, Biochemical and Structural Characteristics of the cellular prion protein (PrPC) and its scrapie isoform (PrPSc)………………………………………………... 2Prion Replication Models……………………………………………………………. 7Creutzfeldt-Jakob Disease and other Human Prion Diseases………………………... 8Neuropathology of Prion Diseases…………………………………………………... 9Prion Transmission…………………………………………………………………... 10Primary Amino Acid Sequence and Species Barriers to Prion Transmission……….. 12Kinetic and Equilibrium Intermediates of recombinantPrP………………………….. 16Rationale, Hypothesis and Objectives……………………………………………….. 19

Chapter 1 References………………………………………………………………… 24

CHAPTER 2: Prion disease susceptibility tracks with β-structure folding of the prion protein in golden hamsters, mice, and rabbitsIntroduction………………………………………………………………………….. 38Resutls……………………………………………………………………………….. 41

Low pH, urea-unfolding of GHaPrP 90-231…………………………….. 41Monomer-Octamer equilibrium …………………………………………. 42Model-free method to quantify fractional concentration of β-state……… 44pH and urea-unfolding of GHaPrP 90-231 monitored at 2 wavelengths… 46Interspecies comparison of β-state PrP fraction………………………….. 48Detection of monomeric, β-state PrP species…………………………….. 53Cytotoxicity of β-state PrP………………………………………………... 54Three-dimensional structure of RaPrP 121-230…………………………... 55

Discussion…………………………………………………………………………….. 62

Material and Methods………………………………………………………………… 68Protein Expression and Purification……………………………………… 68Circular Dichroism (CD) Spectroscopy………………………………….. 69Urea-unfolding of PrP 90-231 monitored by Circular Dichroism……….. 69Urea-refolding of PrP 90-231 monitored by Circular Dichroism………... 69Size Exclusion Chromatography…………………………………………. 70Sedimentation Equilibrium Ultracentrifugation………………………….. 70Determination of the Fraction Octamer…………………………………... 71

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Sulforhodamine B Cytotoxicity Assay…………………………………… 72Crystallization of RaPrP 121-231 and Data Collection…………………... 72

Data Processing, Refinement and Model Building………………………………….. 73

Acknowledgements…………………………………………………………………... 73

Chapter 2 References………………………………………………………………… 74

CHAPTER 3: Conlusions and Future Work…………………………………………. 84

List of Figures for Chapter 2:

Figure 1. Urea-unfolding and circular dichroism spectra of GHaPrP 90-231.. 42Figure 2. Sedimentation equilibrium data, and size exclusion

chromatography of GHaPrP90231………………………………… 44Figure 3. pH-dependent, urea-unfolding of GHaPrP 90-231……………….... 47Figure 4. Sedimentation equilibrium analysis of Mo and RaPrP 90-231…….. 48Figure 5. pH-dependent, urea-unfolding of MoPrP 90-231………………….. 49Figure 6. pH-dependent, urea-unfolding of RaPrP 90-231…………………... 50Figure 7. Reversible, equilibrium unfolding/refolding……………………….. 51Figure 8. Interspecies comparison of β-state fraction………………………... 52Figure 9. Determining fractional concentrations of octamer…………………. 53Figure 10. Comparison of β-state and octamer fraction……………………….. 54Figure 11. Toxicity of β-State PrP……………………………………………... 55Figure 12. Dimeric arrangement in the asymmetric unit of wild-type

RaPrP 121-230 crystal lattice……………………………………….. 57Figure 13. Omit FO-FC electron density of the β2-α2 loop……………………... 59Figure 14. Long rang contacts between the β2-α2 loop and helix-3……………. 59Figure 15. Comparison of residues 170-174 of the rigid loop from

RaPrP 121-230 structures and the lowest energy structures from the hamster and mouse PrPC

NMR structure ensembles………. 61

List of Tables for Chapter 2:

Table 1. Crystallographic data collection and model refinement statistics…… 56

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INTRODUCTION

The Protein-Only Hypothesis

Up until the early 1980’s, the infectious agent that caused transmissible

spongiform encephalopathies (TSE) was not yet isolated nor characterized. Some authors

presumed TSEs involved ‘slow’ or ‘unconventional’ viruses – ‘slow’ describing the long

incubation period of the disease [1, 2]. However, Stanley Prusiner demonstrated that

TSEs were not caused by viruses or other DNA-containing entities, rather they were

caused by the misfolding of host-encoded protein, and replication of its aberrant form [3].

Thus, TSEs could be regarded as ‘conformational diseases’, as manifestation of the

disease depends upon a change in protein conformation.

Upon successfully isolating the infectious agent, there were numerous indications

that the infectious agent, also referred to as scrapie, consisted of protein. UV irradiation

experiments reveal that scrapie was more easily inactivated at a wavelength of 237 nm,

rather than 256 nm [4]. Additionally, the UV irradiation spectrum of scrapie was

comparable with that of the protein trypsin and differed from the spectrum of a

bacteriophage [5]. Furthermore, scrapie was capable of resisting formalin treatment,

whereas viruses were inactivated [6]. Infectivity of the scrapie agent using mice

bioassays were measurably lower when scrapie was treated with chemicals that modify

proteins, including urea, SDS, diethylpyrocarbonate and treatment with proteases, and yet

scrapie infectivity was unaltered when treated with nucleases [3]. Thus, Prusiner

proposed the ‘protein-only hypothesis’ and coined the term prions as proteinaceous

infectious particles that are resistant to most procedures which modify nucleic acids.

Although some skeptics remain, to date the protein-only hypothesis is commonly

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accepted among scientist in the prion research field, and states that prion diseases are

manifested when cellular prion protein (PrPC) converts to the scrapie, infectious isoform

(PrPSc).

Biological, Biochemical and Structural Characteristics of the cellular prion protein

(PrP C ) and its scrapie isoform (PrP Sc )

PrP C

In humans, the cellular prion protein (PrPC) is encoded by the PRNP gene, located

on chromosome 20 [7]. PrPC is expressed by most tissues ; however, higher expression

levels are achieved in neuronal cells [8]. Human PrPC is first expressed as a 253 amino

acid polypeptide and is relocated from the cytoplasm to the rough endoplasmic reticulum

(ER) where the N-terminal signal is cleaved. Oligosaccarides are linked to two

asparagine residues (Asn 174, Asn 191) and after entering the Golgi apparatus, the

oligosaccarides undergo modifications to become complex-type chains that consist of

sialic acid that are resistant to endoglycosidase H [9, 10]. The two only cysteine residues

(Cys 179, Cys 214) form a disulfide bond [11]. The cleavage of the C-terminal sequence

signals the addition of a glycosyl-phosphatidylinositol (GPI) anchor [12]. PrPC is

expressed on the outer-leaflet of the plasma membrane as either a di-, mono- or

unglycosylated protein [13].

PrPC is sorted to caveolae-like domains (CLDs) or lipid rafts which are

specialized domains of the cell membrane that contain caveolae, cholesterol and

glycosphingolipids [14, 15]. PrPC re-enters the cell either through clathrin-mediated

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endocytosis [16] or non-clathrin coated invaginations forming endocytic vesicles [14].

PrPC can then either recycle back to the plasma membrane or be degraded. In the latter

case, endocytic vesicles mature to become early and late endosomes and eventually

deliver PrPC to lysosomes for degradation [10].

In cell cultures, pulse-chase experiments determined the half-life of PrPC to be ~

4-6 hours [10]. The time between initial appearance and re-appearance of PrPC at the

plasma membrane is ~ 1 hour, with 1-5 % of molecules undergoing endoproteolysis [17].

Metalloproteases (ADAM 104, 107) cleave PrPC on the cell membrane between residues

110 and 111 to generate C1 and N1 fragments, the latter fragment can be detected in cell

culture media [18]. A second cleavage event is mediated by calpains, which are calcium-

activated cysteine proteases, which cleave at residue 88 to generate a C2 fragment [19].

Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy

analysis reveal that secondary structure of PrPC is mainly α-helical with little β-sheet

content [20]. The structure of E.coli-recombinantly expressed human PrP (recPrP)

determined by nuclear magnetic resonance (NMR) reveals that the protein consists of a

largely disordered domain (N-terminal domain) and a structured C-terminal domain [21].

Within the C-terminal domain, three α-helices are present, as well as 2 short, anti-parallel

β-sheet structures. The disulphide bond links α-helices 2 and 3 together, and the two Asn

residues that are linked to complex sugar-moieties in PrPC, are also present in the C-

terminal domain. In human PrP, the N-terminal domain contains 5 ‘octapeptide repeats’

of the sequence - PHGGGWQQ. Working in tandem, these sequences have the capacity

to coordinate Cu (II) ions [22]. NMR solution structures of both brain-derived, bovine

PrPC and recombinantly-expressed bovine PrP show little deviations in the overall fold of

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the protein [23]. Furthermore, the comparison of recombinantly-expressed PrP from a

number of species including humans, cows, mice, hamsters, sheep, pigs, cats, dogs, bank

voles and wallabies, show very small deviations in the folded, structural domains [21, 24,

25, 26, 27, 28, 29]. Both NMR and X-ray crystallography studies of the C-terminal

region from a variety of species yield the same structural architecture [30, 31].

The function of PrPC still remains unknown. PrPC knock-out mice (Prnp 0/0)

reveal no major phenotypic or physiological effects [32]. Proposed roles for PrPC

include: neuroprotectant, a free-radical scavenger, Cu metabolism, cell signaling,

adhesion and differentiation (see review [33]). Interestingly, KO mice are incapable of

contracting prion disease when inoculated with the infectious PrPSc agent [32, 34]. Thus,

in order for an animal to succumb to prion disease, it must express the normal cellular

form - PrPC.

PrP Sc

The process of conversion from PrPC to PrPSc is based solely on a protein

conformational event, as studies report no indications of covalent modifications when the

protein assumes the scrapie conformation [35]. Furthermore, the disulfide bond remains

intramolecular during conversion from PrPC to PrPSc [36]. The CD spectra of PrP27-30

yields a β-sheet structure [37]. FTIR analysis of the secondary structural composition of

purified hamster scrapie reveal higher β-sheet content, with considerable α-helical

content [20, 38]. Whereas PrPC shows sensitivity to limited proteinase K (PK) digestion,

PrPSc generates a 27-30 kDa band (referred to as PrP27-30) as well as two lower bands

(from ~18 kDa to 24 kDa) when SDS-PAGE electrophoresis is performed on PK-

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digested, PrPSc samples [39]. Amino acid sequencing reveals that the bands represent

amino-terminal truncated (90-231), di-,mono- and unglycosylated forms of PrPSc [40].

Re-inoculation of PK-treated PrPSc (referred to as PrP-res, ‘res’ for resistant) yield

infectivity in animals, demonstrating that the C-terminal structured domain (121-231) and

a portion of the disordered N-terminal domain (90-120) represent the infective, protease-

resistant core of PrPSc [41].

Cell culture studies demonstrate that conversion from PrPC to PrPSc (either

spontaneous or exogenously-induced by foreign PrPSc molecules) occurs after post-

translation modification and expression of PrPC on the plasma membrane [13, 42].

However, the exact sub cellular location of where the conversion event occurs remains

elusive. Some authors suggest the conversion process takes place within the lipid and

cholesterol-rich, microenvironment of the cell membrane, as cell fractionation

experiments reveal that infectious PrPSc can be isolated from detergent-resistant

microdomains (DRM) of the cell membrane [15, 43]. Alternatively, the endosomal

pathway may play a role in conversion as cell imaging experiments reveal that PrPSc

aggregates accumulate in acidic lysosomes, and that cell to cell infection may occur

through secreted vesicles called exosomes [44, 45]. Also, endosomal recycling

compartments were shown to house greater percentages of PrPSc in comparison to other

subcellular sites, indicating that endosomal re-shuttling of PrPC may provoke the change

[46]. Thus, spontaneous conversion of PrPC to PrPSc or conversion induced by foreign

PrPSc can occur in any subcellular site(s) between the initial expression of PrPC on the cell

membrane, to when PrPC is shuttled back to the cell membrane, or when PrPC is targeted

for degradation.

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Purified preparations of PK-treated PrPSc isolated from animals brains show rod-

shape and amorphous aggregate morphology when viewed with electron microscopy

[47]. The ‘prion rods’ exhibit characteristics of amyloid structure. The rods show

fibrillar or ribbon-like morphology, and display birefringence when bound to Congo red.

X-ray diffraction studies gave measurements indicating a cross-beta core arrangement

with H-bond and intersheet distances matching those found in other amyloid structures

[48]. Immunostaining of diseased-animal and human tissues showed that amyloid

plaques were composed of PrPSc molecules [49, 50]. However, prion amyloid fibrils are

not a requirement for infection, and the formation of prion rods were found to be induced

by proteases and detergents involved in the purification protocol [51, 52]. Recent studies

have shown that lower molecular weight particles or oligomers of PrPSc are better

initiators of prion diseases than large aggregates or fibrils [53].

Achieving high-resolution, data on the structure of PrPSc using NMR and X-ray

crystallography has not been possible due to the tendency of PrPSc to aggregate in

solution. Nevertheless, a number of models on the structure of PrPSc have been proposed,

mostly from in silico studies. Secondary structure prediction methods from FTIR

analysis of PrPSc contend that only α-helix 3 remains in tact, whereas both α-helix 1 and 2

convert to either β-sheet or turn structures [38]. The spiral-protofibril model was derived

from molecular dynamics (MD) simulations of the conversion of hamster PrP 109-219

with the mutation D174N, to the scrapie isoform at low pH [54]. Rather than direct

conversion of the α-helical regions to β-sheet, this model predicts that the original β-

sheets become extended, and the α-helical regions are unaffected. Multimerization into a

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protofilbril occurs as hexameric units associate and build in parallel to the fiber axis. A

second model, the β-helix model, was based on threading analysis from experimental data

gathered from 2D-crystals formed from two prion strains [55]. The β-structure model

that best agreed with the constraints of the crystallographic data was that the region 89-

175 formed left-handed, β-helices, which are capable of associating into trimers that can

stack perpendicular to the fibril axis. The intermolecular disulfide bond, as well as the N-

glycosylation oliogosaccarides are unaffected and lay outside the β-core. The model

upholds the percentages of β-sheet and α-helical content determined from FTIR analysis

of PrPSc [20, 38]. Lastly, the in-register β-sheet model was derived from experimental

data involving site-directed, spin-labeling and electron paramagnetic resonance (EPR)

spectroscopy on amyloid prepared from recombinant PrP [56]. The model predicts that

both α-helix 2 and 3 are converted into a β-structured core. Although the core structure,

mapped to residues 169-221, is consistent with hydrogen-deuterium (H/D) exchange

experiments performed by the same authors [57], the model is inconsistent with antibody

binding studies. The 90-121 region that is accessible to antibodies in PrPC is inaccessible

in PrPSc, yet is accessible in the amyloid form of recombinant PrP [58].

Prion Replication Models

Although the exact mechanism which governs prion replication is unknown, there

are two theoretical models which describe how PrPC is converted to PrPSc.

Heterodimer or Template-Assisted Model [59, 60]

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This model states that PrPSc is more thermodynamically stable than PrPC;

however, a large kinetic barrier prevents spontaneous conversion from PrPC to PrPSc.

Thus, the rate-limiting step or the slow phase of this reaction is the conversion from PrPC

to PrPSc. Once formed spontaneously, or introduced exogenously, PrPSc binds to PrPC in a

heterodimer complex and templates the conversion of PrPC to PrPSc. The newly formed,

homodimeric PrPSc complex may dissociate and template further reactions. PrPSc

oligomers and aggregates form as a result of PrPSc accumulation.

Seeded Nucleation or Nucleation-Dependent Polymerization Model [61, 62]

In the nucleation-dependent polymerization model, both PrPC and PrPSc are in

reversible equilibrium and PrPC is the more thermodynamic stable conformation. The

rate limiting step is the formation of an oligomeric PrPSc seed, which is favored with the

introduction of exogenous PrPSc. Once oligomeric or aggregated PrPSc reach a ‘critical

concentration’, conversion from PrPC to PrPSc becomes spontaneous. The lag phase of

this process (i.e. the time required to reach critical concentration) can be overcome by

seeding the reaction.

Creutzfeldt-Jakob Disease and other Human Prion Diseases

Creutzfeldt-Jakob Disease (CJD) accounts for the majority of human prion

diseases and can be manifested through one of three etiologies: familial, infectious, and

sporadic. Familial or genetic CJD arises due to point mutations in the coding region of

PrPC that result in amino acid substitutions or the production of stop codons, which result

in the expression of truncated PrPs [63, 64, 65]. These mutations are inherited in an

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autosomal-dominant fashion. There may also be insertions of extra octapeptide repeat

sequences, in addition to the five that are normally expressed [66, 67]. Infectious and

iatrogenic CJD occurs through physiological exposure of foreign prions. Tainted

neurosurgical instruments [68], human corneal transplants [69], tissue grafts [70], human

pituitary-derived hormones [71] and the consumption of prion-infected meat [72] are

modes by which foreign prions may cause infectious CJD to be manifested. Variant CJD

(vCJD) is a special class of infectious prion diseases, as it is an illness specific to the

consumption of processed meat from prion-infected cattle by humans. Sporadic CJD

accounts for nearly 85% of all CJD cases. Sporadic CJD occurs through unknown

mechanisms that cause host-encoded PrPC to convert spontaneously to PrPSc, which leads

to the disease phenotype. Although the sources that cause sporadic CJD are unclear,

somatic mutations within PrPC and/or prions in the environment are possible origins for

the manifestation of sporadic CJD [73].

Other human prion diseases include Gertsmann-Straussler Scheinker (GSS)

Syndrome, Fatal-Familial Insomnia (FFI) and Kuru. Like familial CJD, GSS and FFI are

diseases that are due to point mutations within the coding region of PrP and show

autosomal-dominant inheritance patterns [74]. Kuru is specific to the Fore people of

New Guinea. It was discovered in the 1950’s that the disease occurred through the

practice of ritual cannibalism of deceased tribe members [75]. Once the practice was

halted, the number of Kuru cases declined [76].

Neuropathology of Prion Diseases

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The clinical and pathological symptoms of prion disease can vary depending on

the host species and the particular type of prion disease contracted or inherited. Some

clinical symptoms that are manifested and that intensify during the late phases of the

disease include: dementia, ataxia, behavioral disturbances, involuntary movements and

dysphasia [68, 74]. In general, the main pathological features of prion diseases are

neuronal vacuolation and spongiform degeneration accompanied with astrocytic gliosis

[77]. The histopathological disease marker is the accumulation of PrPSc in the form of

amyloid plaques, which can be detected by PrPSc-specific antibodies [49, 50]. Different

prion diseases can vary in their relative quantity of amyloid deposits and PK resistant

PrP, as well as affect different areas of the brain. For example, in variant CJD cases the

amyloid plaques are surrounded by areas of spongiosis resulting in ‘daisy’ or ‘florid’

plaques [72, 78]. These plaques can be found within the molecular layer of the cerebral

cortex, with greater concentration of plaques appearing in the occipital lobe, the granule

cell layer of the cerebellum, the basal ganglia and the thalamus [78].

Prion transmissibility to laboratory animals

In order to study prion diseases in more detail, prions can be passaged into

laboratory animals. CJD, kuru and GSS isolates have all been successfully transmitted to

nonhuman primates; however, long incubation times were recorded [79, 80, 81]. To

achieve a more efficient animal model, prion isolates have been transmitted to smaller

laboratory animals such as hamsters and mice [82, 83]. Prion isolates can be transmitted

intracerebrally, intraperitoneal, subcutaneous, intravenous or orally. The intracerebral

route is the most efficient mode for prion transmission [84]. The prion titer in the

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original sample, the LD50 value can be calculated by performing endpoint titration

experiments, where the original inoculum is sequentially 10-fold diluted and

administered to a group of animals. The animals are monitored for clinical symptoms of

the disease and the time required for disease onset or death are recorded – the incubation

time [85]. There have been a few interesting discoveries made with prion transmission

experiments. First, when prions are isolated from one species, and administered into

another species, a species barrier exists that is evident in the long incubation period and

atypical histopathological characteristics of the disease. Upon isolation of the prions

from that species and re-introduction into the same animals causes the incubation to

become shorter in comparison to first passage, and with subsequent passages, the

incubation period as well as the clinical, histopathological and biochemical features of

the disease become constant and predictable. Second, animals display variable

susceptibility to prion isolates. For example, both Syrian hamsters and CD-1 mice are

susceptible to the scrapie isolate from Cheviot sheep [82, 83]. After successive rounds of

passages, the incubation time and histopathological features became predictable. The

Sc237 (hamster-adapted scrapie) and the Chandler-strain (mouse-adapted scrapie) yielded

steady incubation times of ~60 and 120 days when administered to hamsters and mice,

respectively. Because mice exhibited longer incubation times, hamsters became the

choice for animal bioassays and endpoint titrations as they replicated prions with faster

kinetics and produced higher titers of prions in their brains. Additionally, both animals

have demonstrated susceptibility to a number of prion isolates including: CJD, Kuru,

BSE (bovine spongiform encephalopathy), mouse-adapted prions and TME

(transmissible mink encephalopathy) [82, 83, 86, 87, 88, 89, 90], although mice show

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lower susceptibility to hamster and mink-adapted prions. Prions have not been

successfully transmitted to rabbits, as rabbits display no susceptibility to CJD, Kuru,

sheep or mouse-adapted prions [86, 91].

Prion tansmissibility in the field

Bovine spongiform encephalopathy (BSE) is a prion disease that affects cattle and

gained notoriety in the late 1980’s and 1990’s as the causative agent of the United

Kingdom mad cow crisis [92]. From 1985 to 2000, over 180 000 cattle were affected

with BSE. The rapid spread of the disease occurred through the consumption of meat and

bone meal (MBM), which contained recycled brain and spleen tissue from dead cows.

The ban on MBM and selective culling of cattle helped significantly to diminish the

population of infected cattle.

BSE contaminated meat entered human and animal food supplies. Humans that

consumed BSE-contaminated meat developed variant CJD (vCJD). As of August 2002,

there were 129 cases of vCJD in Great Britain [93]. Zoo animals of the bovidae and

felidae families which include antelope, bisons, cheetahs, tigers and lions were also

affected through consumption of BSE-contaminated meat [92]. Also, 93 cases of FSE

(feline spongiform encephalopathy) were confirmed in household cats, and strikingly,

there were no reported cases of any household dogs affected with BSE-contaminated pet

foods, indicating that only cats, not dogs, were susceptible to BSE [92, 94].

Primary Amino Acid Sequence and Species Barriers to Prion Transmission

One key factor that plays an important role in species barriers and the success of

prion transmission is the sequence of the host-encoded PrPC. For a given species, the

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shortest incubation time is seen with 100% sequence identity between the PrPC of the host

animal and the foreign scrapie source that is injected intracerebrally into that animal [85].

The amino acid of PrP can influence prion disease susceptibility and incubation times.

For example, two polymorphisms in the mouse Prnp gene encoding the L108F and

T189V polymorphisms in mouse PrP can render longer incubation times, given one or

both of the polymorphisms are present [95, 96]. In sheep PrP, polymorphisms at codons

136, 154 and 171 play a role in sheep susceptibility to scrapie [97]. In most cases,

homozygotes with Q or R at position 171 leads to scrapie susceptibility or scrapie

resistance, respectively. All 129 cases of vCJD that resulted from ingesting BSE-

contaminated foodstuff in the UK were homozygous for methionine at codon 129 [98].

The M/V polymorphism at codon 129 also determines the type of genetic prion disease

inherited. An M or V at codon 129 in cis with the mutation D178N leads to either FFI or

CJD, respectively [74]. Other factors such as the prion dose, the level of PrPC expression,

different prion strains and differences in host-animal physiology may also influence

species barriers and prion susceptibility [85].

When prions are transmitted between different species, longer incubation times

and inefficient attack rates are indications that a species barriers to prion transmission

exist. Using transgenic (Tg) mice, studies have shown that the amino acid PrP sequence

of the host species plays a significant role in species barriers [99, 100]. When Tg mice

expressing hamster PrP are challenged with hamster scrapie, the mice exhibit very similar

clinical symptoms and pathological characteristics to hamsters infected with hamster

scrapie. Also, when Tg mice expressing hamster PrP are challenged with mice prions,

longer incubation times and inefficient prion transmission were observed. However, in

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the latter case, the species barrier can be overcome by substituting key amino acid

polymorphisms of the mouse PrP sequence into the hamster PrP expressed in Tg mice.

Subsequently, when these Tg mice expressing chimeric mouse-hamster sequences were

challenged with mice scrapie, lower incubation times and greater efficiency of prion

transmission were observed [101].

Probing species barriers in an in vivo context can also be performed with in vitro

conversion assays [102]. The cell-free conversion assay probes species barriers by

mixing purified, 35S-labelled PrPC from one animal with unlabelled PrPSc from another

animals. If resistant PrP (PrP-res) remains after proteinase K (PK) digestion, then

conversion has occurred. The authors found that while hamster PrPC was capable of

being converted by PrPSc adapted in both hamsters (263K strain) and mice (Chandler

strain), mouse PrPC was only capable of being converted with only mouse PrPSc and not

hamster PrPSc [103]. In order to successfully convert mouse PrPC with hamster PrPSc, the

mouse PrPC sequence must express the hamster segment of the PrP sequence between

residues 139 to 170, which contains three residues that differ between the PrP sequence

of hamsters and mice. Thus, this region of mouse PrPC plays an important role in species

barrier to hamster scrapie. Another assay used to assess the effects of mutations on

species barriers includes using scrapie-infected mouse neuroblastoma cells (Sc+-MNB).

These cells are persistently infected with RML mouse scrapie, express mouse PrPC and

accumulate mouse PrPSc [104]. When PrPC sequences from other animals are transduced

into these cells, the result can either be interference or conversion. Interference means

that the foreign PrPC binds to mouse PrPSc and prevents the conversion of mouse PrPC to

PrPSc. Conversion means the foreign PrPC is converted and enhances levels of PrPSc

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compared to control cells. Using this assay, hamster PrPC was transduced into these cells

and interfered with mouse PrPC conversion to mouse PrPSc. However, upon introducing

the mouse PrP sequence segment from residue 139--170 into the hamster PrPC sequence,

conversion occurred readily. Thus, Tg mice, cell-free and cell conversion studies

demonstrated that the region between 139 to 170 of the PrPC amino acid sequence plays

and important role in governing species barriers between hamsters and mice (Figure 1.1).

Rabbits

When rabbit PrPC was transfected into SC+-MNB cells, they interfered with

normal levels of conversion [105]. Additionally, chimeric constructs of rabbit-mouse PrP

sequence revealed that any portion of the rabbit PrP sequence caused interference

compared to normal conversion levels. Expressing mouse PrPC sequences with single

rabbit mutations (N99G, N107S, N173S) lead to interference as well, demonstrating that

single amino acid polymorphisms interfered with scrapie propagation. Thus, the authors

concluded that the rabbit PrP sequence contains multiple amino acid differences that

affect the overall structural characteristics making it less amenable to conversion with

mouse PrPSc (Figure 1.1).

Another useful method of exploring species barriers is by generating PrPSc using

the PMCA method [106]. PMCA stands for protein misfolding cyclic amplification and

it involves reacting PrPSc from one animal with an excess of PrPC from the brains of

another animal. With each binding and converting event, the products are subjected to

sonication, which breaks apart the newly formed scrapie material into seeds that act to

convert additional PrPC. When injected into animals, the PrPSc that results from PMCA

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show incubation times, clinical symptoms, histopathological features, electrophoretic

mobility of protease-resistant bands, guanidine-hydrochloride denaturation and brain

lesion profiles similar to a prion strain that has been adapted to that animal – essentially,

the PMCA method allows the resultant scrapie source to faithfully be replicated and

adapted to an animal’s PrPC [107, 108, 109]. Using mouse PrPSc as the seed, and rabbit

PrPC as the reactant, the PMCA method was able to amplify and produce a prion strain

adapted to rabbit PrPC, albeit a large number of PMCA cycles were required to produce

enough detectable scrapie (unpublished results). When rabbit-adapted-scrapie was

injected into rabbit, so far there have been no reports of rabbit succumbing to the disease

(J. Castillo, personal communication). The authors have commented that rabbits are

intrinsically difficult to inoculate. Furthermore, the PMCA method was not able to

amplify PrPSc when scrapie seeds were reacted with either brain-derived dog or horse

PrPC (unpublished results). Thus, PMCA investigates the differences in the ‘converting

ability’ of the PrPC from a variety of animals. The PrPC from animals such as hamster

and mice can convert to the PrPSc form using PMCA, and the products are infectious

when introduced into these animals. Rabbit PrPC can be converted; however, the

resultant PrPSc have not been shown to be infectious. Dog and horse PrPC were not

converted using the PMCA reaction.

Kinetic and Equilibrium Intermediates of recombinant PrP

Protein misfolding diseases are a class of diseases that arise from the misfolding

of otherwise benign proteins to altered, toxic forms. One hypothesis as to how these

diseases come about is that partially folded, and/or partially unfolded intermediate states

- 16 -

may play a role in reassembling into higher-order oligomeric, or fibrillar amyloid states

[110, 111]. The intermediate state can be thought as protein molecules that partially

resemble the secondary structures of natively-folded proteins, yet lack a cohesive and

defined tertiary structure. Intermediates such as the molten-globule state often are

detected on the pathway to refolding the protein into the native state and allow refolding

to occur on the microseconds to seconds time scale. Folding intermediates may be

populated by destabilizing the native state through low/high pH, increasing temperatures

and denaturants. In the partially folded state, specific moieties that are conducive to

forming cross-β structures may be exposed or specifically-oriented to initiate aggregation

or fibrillation. For example, X-ray crystallography of microcrystals of the SNQNNF

peptide of the prion protein (residues 169 to 175) reveal that these segments can self-

associate into a parallel, cross-β, steric zippers [112]. Whether or not these motifs

provide a contact site that enables partially-folded PrPC to multimerize has yet to be

determined.

Researchers were unsuccessful on their first attempt to characterize kinetic

folding intermediates using the C-terminal domain of mouse PrP (MoPrP 121-231), since

the refolding and folding kinetics were within the dead-time of the measurement [113].

However, equipped with a better, state-of-the-art stop-flow, fluorimeter, Surewicz and

colleagues characterized kinetic intermediates of recombinant human PrP as an on-

pathway intermediate [114]. Furthermore, human PrP sequences containing single,

point-mutations that lead to inherited human prion diseases such as CJD and GSS

populated the intermediate state more readily than the wild-type sequence [115, 116].

- 17 -

These results lead the authors to conclude that kinetic folding intermediates of PrP play a

role in the spontaneous formation of PrPSc.

Equilibrium folding intermediates have also been characterized for both human

and mouse recombinant PrP [117, 118]. By monitoring PrP unfolding or refolding with

circular dichroism (CD), equilibrium folding intermediates were populated at low pH and

in moderate concentrations of Gdn-HCl or urea, and yielded biphasic, unfolding/refolding

curves. On the other hand, the same experiments performed at neutral pH yield a

sigmoidal shaped curve, which suggests that only natively-folded or unfolded PrP were

populated. Since acidic endosomes and lysosomes represent a potential site for

conversion of PrPC to PrPSc, the authors suggested that perhaps equilibrium, folding

intermediates of PrP are on pathway or play a role in the formation of PrPSc [118].

The equilibrium, folding intermediates have been extensively characterized by

Baskakov and colleagues. Refolding recombinant PrP under acid conditions and mild

amounts of denaturant yielded round spherical particles observable by electron

microscopy (EM) [119, 120]. The authors labeled these particles as β-oligomers, and

further characterization of β-oligomers revealed that they were soluble, yielded a β-sheet

CD spectra, were partially resistant to proteinase K and were octameric. Alternatively,

refolding PrP under near neutral pHs, in mild amounts of denaturant and with aggitation

caused recombinant PrP to form amyloid [121]. Amyloid was distinguished from β-

oligomers in its ability to bind Thioflavin T and a fibril morphology when viewed with

EM. Whereas β-oligomers show no seeding capabilities, the amyloid reaction can be

seeded in an autocatalytic fashion [122]. Using an auto-catalytic approach to seed for

amyloid formation, recombinant MoPrP89-231 was found to be infectious when injected

- 18 -

into Tg20 mice expressing a mutant form of PrP 89-231 [123, 124]. The authors

indicated that the β-oligomers were kinetically, off-pathway to amyloid formation.

However, the two ultra-structures were shown to be linked through a monomeric PrP

species. When β-oligomers were placed in near-neutral conditions with aggitation, they

disassembled into a monomeric PrP species, and after a lag phase, monomeric PrP

reassembled into amyloid [120]. Finally, both β-oligomers and amyloid were shown to

be toxic to primary culture neuron cells at micromolar concentrations [125].

Rationale, Hypothesis and Objectives

The term species barrier reflects the long incubation times observed when

infectious prions that have been adapted in one animal (the donor animal) are transmitted

to another of a different species (the recipient animal). Experimentally, there can be two

outcomes; the recipient animals may display neurological symptoms and succumb to the

disease, thus rendering a measurable incubation time, or the recipient animals may not

show any symptoms of the disease and survive beyond the time limits of the experiment.

In the first case, the recipient animal can be classified as a species that is susceptible to

the transmitted prions. In the second case, the animal is considered to have a low

susceptibility to the prions. We use the term ‘low susceptibility’ and not ‘resistance’

because there could be cases where subclinical infection could occur in animals where

the infectious agent that does accumulate is below the threshold amount required to yield

outward disease symptoms [126]. Thus, both species barrier and prion susceptibility are

intertwined concepts in that if the species barrier is overcome in the recipient animal,

then the animal is considered susceptible to that particular prion.

- 19 -

By reviewing the literature on accidental or experimental transmission of

infectious prions to animals, one can generally classify species as susceptible or less

susceptible to certain prion strains. The BSE crisis in the United Kingdom is a case of

accidental transmission of BSE (bovine spongiform encephalopathy) – or prions adapted

in cows, to animals through the consumption of contaminated meat. As mentioned

previously, humans and many members of the family Felidae including household cats,

cheetahs, and pumas were susceptible to the BSE agent, while no cases of prion disease

were reported in canines. Studies on experimental transmission of infectious prions to

laboratory and rodent animals reveal varying susceptibility to prions. Golden Syrian

hamsters are susceptible to a variety of prions adapted in hosts such as humans, sheep,

cows, mice, and minks. Like hamsters, mice also share similar susceptibility to the

animal-adapted prions listed above; however, they have low susceptibility to hamster and

mink-adapted prions. On the other hand, rabbits are considered a species that is less

susceptible to prion diseases, as human, sheep and mouse-adapted prions did not cause

noticeable neurological symptoms, nor produced measurable incubation times when

rabbits were challenged with these particular prion strains.

The molecular basis of conversion of host-encoded PrPC to PrPSc in the presence

of exogenous PrPSc, prions from the donor animal, has not been fully resolved. Early

theories suggested that primary sequence similarity between the prions from the donor

animal and the PrPC from recipient animal plays a critical role in the success of prion

transmission. For example, it was mentioned that wild-type mice show low susceptibility

to hamster-adapted prions, yet transgenic mice expressing hamster PrPC are susceptible.

However, there have been cases where primary sequence similarity does not determine an

- 20 -

animal’s susceptibility to prion strains. Bank voles render lower incubation times and are

more susceptible to human-adapted prions versus hamster or mice-adapted prions, despite

sharing greater PrPC sequence similarity to hamsters and mice [127]. Also, vCJD isolates

are transmitted more readily to wild-type mice rather than transgenic mice expressing

human PrPC [128]. And finally, the fact that many prion strains exist for a single

sequence of PrPC, and exhibit varying biochemical, histopathological, and

neuropathological characteristics when introduced in the same species of animal [129],

supports the notion that species and transmission barriers involve more than just primary

sequence similarity between donor prions and host PrPC.

A recent theory that may help to explain species and transmission barriers

involves the conformational selection model [130]. This model states that mammalian

PrPC in general is capable of forming a multitude of PrPSc conformations. The PrPC from

individual animals are capable of forming a subset of these PrPSc conformations. The

species barrier is overcome when the subsets of PrPSc conformers between two

mammalian PrPC sequences overlap. Conversely, if the subsets do not overlap, then a

species barrier exists and disease transmission will not occur. If we apply this model to

the animals that display varying susceptibility to prions, we could say that the PrPC from

animals that show susceptibility to various prion strains like hamsters, mice, humans and

cats are capable of forming a greater variety of PrPSc conformations than rabbit or canine

PrPC, which in turn limit their subset of PrPSc conformers. Thus, the primary sequence of

PrP from a given animal may conformationally favour or limit it’s propensity to adopt

PrPSc conformer(s); thus, rendering it either susceptible or less susceptible when

challenged with prion strains, or in other words, other PrPSc conformers.

- 21 -

The transition from PrPC to PrPSc is accompanied by a secondary structural change

from a protein that is primarily α-helix to an isoform or an oligomeric state that contains

higher β-sheet content. A method for observing an equilibrium, β-sheet rich

conformation of recombinant PrP has been established by Hornemann and Glockshuber

[118], who monitored the unfolding and refolding of mouse PrP by using CD, in varying

conditions of urea denaturant and pH. If we apply this method to PrP from species that

vary in their degree of susceptibility to prion diseases, our hypothesis states -

Hypothesis:

The PrP from susceptible species populates the misfolded, β-structured conformation

more readily than the PrP from less susceptible species.

Objectives:

1) To compare the relative populations of β-sheet, equilibrium folding intermediate(s),

or 'β-state PrP', between recombinantly-expressed, hamster, mouse and rabbit PrP 90-

231.

2) To determine the molecular weight(s) of species that form β-state PrP

3) To determine the toxicity of β-state PrP

4) To solve the X-ray crystal structure of rabbit PrP 121-231 (in collaboration with

Braden Sweeting from the Pai Laboratory, University of Toronto)

- 22 -

- 23 -

Hamster 90 GQGGGTHNQW NKPNKPKTSM KHMAGAAAAG AVVGGLGGYM LGSAMSRPML Mouse GQGGGTHNQW NKPSKPKTNL KHVAGAAAAG AVVGGLGGYM LGSAMSRPMIRabbit GQGG.THNQW GKPSKPKTSM KHVAGAAAAG AVVGGLGGYM LGSAMSRPLI --β1-

Hamster 140 HFGNDWEDRY YRENMNRYPN QVYYRPVDQYN NQNNFVHDCV NITIKQHTVTMouse HFGNDWEDRY YRENMYRYPN QVYYRPVDQYS NQNNFVHDCV NITIKQHTVTRabbit HFGNDYEDRY YRENMYRYPN QVYYRPVDQYS NQNSFVHDCV NITVKQHTVT ------α1---- -β2- -----------α2------

Hamster 190 TTTKGENFTE TDVKMMERVV EQMCVTQYQK ESQAYYDGRR S Mouse TTTKGENFTE TDVKMMERVV EQMCVTQYQK ESQAYYDGRR SRabbit TTTKGENFTE TDIKIMERVV EQMCITQYQQ ESQAAYQRAA G ---- ------------α3-----------------

Figure 1.1. Sequence alignment of golden hamster, mouse and rabbit PrP (90-231) using ClustalW2 (www.ebi.ac.uk/clustalw/ ). The accension numbers for hamster, mouse and rabbit PrP are AAA37093, AAA39997 and AAD01554, respectively. Secondary structural elements and their positions are indicated with dashes below the sequences. Residues highlighted in green represent important residues that define the species barriers between hamsters and mice. Residues highlighed in red may represent important residues that contribute to lower susceptibility of rabbits and higher susceptibility of hamsters and mice to various strains of prions.

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Prion disease susceptibility tracks with β-structure folding of the prion

protein in golden hamsters, mice, and rabbits

Short Title

Prion susceptibility and β-structure folding

Keywords

Prion protein, prion disease, prion susceptibility, protein misfolding, β-structure, circular

dichroism, analytical ultracentrifugation, X-ray crystallography

Authors and Affiliations

M. Qasim Khan1, Braden Sweeting2, Vikram Khipple Mulligan1, Pharhad Eli Arslan1,2,

Neil R. Cashman3, Emil Pai1,2 and Avijit Chakrabartty1,2*

1. Ontario Cancer Institute, Department of Biochemistry, University of Toronto, TMDT

4-307, 101 College Street, Toronto, Ontario, Canada, M5G 1L7

2. Ontario Cancer Institute, Dept. of Medical Biophysics, University of Toronto, TMDT

4-305, 101 College Street, Toronto, Ontario, Canada, M5G 1L7

3. Brain Research Centre, Division of Neurology, Department of Medicine, University of

British Columbia and Vancouver Coastal Health, UBC Hospital, 2211 Wesbrook Mall,

Vancouver, BC, Canada V6T 2B5

* Corresponding author (416) 581-7553 Fax: (416) 581-7555

chakrab@uhnres.utoronto.ca

Abbreviations

BSE, bovine spongiform encephalopathy; CD, circular dichroism; CJD, Creutzfeld Jacob

disease; FFI, fatal familial insomnia; GHaPrP 90-231, golden hamster PrP residues 90-

231; GSS, Gerstmann-Straussler-Scheinker; HEPES, 4-(2-Hydroxyethyl)-1-

- 36 -

piperazineethanesulfonic acid; MoPrP 90-231, mouse PrP residues 90-231; NGF, nerve

growth factor; NMR, nuclear magnetic resonance; PMCA, protein-misfolding cyclic

amplification; PrP, prion protein; PrPC, normal cellular isoform of PrP; PrPSc,

pathological isoform of PrP; RaPrP 90-231, rabbit PrP residues 90-231; RMSD, root

mean squared deviation; SEC, size exclusion chromatography; Tris,

tris(hydroxymethyl)aminomethane.

Abstract

Prion diseases occur in animals when their normally α-helical prion protein (PrPC)

converts to a pathological state that possesses β-structure and prion infectivity (PrPSc).

This conversion can be catalyzed by exposure to PrPSc from other animals. Different

animal species are known to vary in their susceptibility to prion diseases: hamsters are

highly susceptible, mice show moderate susceptibility, and rabbits have low prion

susceptibility. We find that exposure of prion proteins from hamsters, mice, and rabbits to

acid and urea denaturation cause conversion from their normal α-helical state to a β-

structured state. This β-state comprises a β-structured monomer-octamer equilibrium,

and is highly neurotoxic. Using a model-free approach for analyzing protein denaturation,

we find that the propensity to form this β-structured state correlates with prion

susceptibility. We have examined the X-ray crystal structure of rabbit prion protein and

have identified a key helix capping motif implicated in the low prion susceptibility

phenomenon of rabbits. These results support a role for β-state PrP as a marker for prion

disease pathology, and yield new insight into susceptibility mechanisms.

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Introduction

Prion diseases are a group of fatal, neurodegenerative diseases that include

Creutzfeldt-Jakob Disease (CJD), Gerstmann-Straussler-Scheinker (GSS) Syndrome,

fatal familial insomnia (FFI), and Kuru in humans, scrapie in goats and sheep, bovine

spongiform encephalopathy (BSE) in cattle, and chronic wasting disease (CWD) in elk

and deer1. According to the “protein-only” hypothesis, prion diseases are caused by the

misfolding of the prion protein (PrP) into a conformation that is pathogenic and

infectious2,3. PrP exists normally in a monomeric, mostly α-helical state (PrPC) and upon

prion infection refolds into an aggregated, β-sheet structured infectious form (PrPSc).

Experiments from the 1960s on the transmission of the infectious prion agent

from brain extracts of Kuru patients to chimpanzees demonstrated that when infectious

prions are transmitted between species, long incubation periods can result4; this

phenomenon has since been designated the “species barrier”5. To develop more efficient

animal models for studying prion disease, efforts were made to passage these diseases to

laboratory animals. These experiments demonstrated that different species have varying

susceptibility to prion disease6.

The prion disease susceptibility of an organism is defined here as the propensity

of its PrPC molecules to convert to the pathogenic and infectious PrPSc isoform upon

exposure to a prion agent. Prion disease susceptibility can be modulated both by kinetic

effects (i.e. the length of the incubation time) and equilibrium effects (the general

tendency of PrPC to convert to the pathologic isoform). The species barrier is one aspect

of prion susceptibility, and can be described thus: the PrPC molecules of species A may

be less susceptible to pathologic conversion when exposed to prions originating from

- 38 -

species B compared to the conversion event elicited by prions from species C. Prion

adaptation is a modifier of prion susceptibility. With prion adaptation, the long incubation

time experienced when species A is infected by prions from species B becomes

progressively shorter with increasing rounds of passaging the prions from one member of

species A to another. Recent studies using PMCA (protein-misfolding cyclic

amplification) demonstrate that the species barrier effect can be abrogated by in vitro

generation and passaging of prions7,8 a process that can be termed in vitro prion

adaptation. Mouse PrPC when exposed to hamster prions can cause the de novo

generation of prions via the PMCA process. Increasing rounds of PMCA induces in vitro

prion adaptation, in that mice become more susceptible (i.e. lower incubation times and

better attack rates) to hamster prions with increasing rounds of PMCA7.

Different animal species vary in their degree of prion susceptibility. A survey of

various prion transmission studies reveal that golden Syrian hamsters are susceptible to a

variety of mammalian prions, which include those from humans, cows, sheep, mice, and

minks6, 9, 10, 11. Mice show susceptibility to human, cow, sheep and mice prions, yet show

low susceptibility to hamster and mink prions6, 9, 12-15. Rabbits show low susceptibility to

prions from humans, sheep, mice and minks, and can survive after prion exposure for the

remainder of their natural lifespan6, 16. The term low susceptibility is used instead of

resistance because animals may display kinetics of PrPSc formation too slow to result in

clinical symptoms during these animals’ lifetime despite successful initiation of the

infection process15.

It has been shown that prion susceptibility can be affected through changes in the

primary structure of PrP 17-19. Using a cell culture model of prion propagation, mouse PrP

- 39 -

was shown to resist conversion to the pathological isoform when it was mutated at

several amino acid positions to corresponding residues of PrP from rabbits, a species that

demonstrates low susceptibility20. It had been suggested that these single amino acid

changes caused differences in the structure of PrPC between species, which accounted for

prion susceptibility; however, structures of PrPC show little difference in overall native-

state folds among many mammalian species21-23. A second possibility is that these amino

acid differences lead to formation of uniquely structured PrPSc that are specific for a

particular organism and have limited capacity for interspecies conversion. A third

possibility is that the amino acid differences modulate the formation of an alternate

conformational state that is critical for the conversion mechanism.

We have investigated the tendency of PrP from three species that differ in prion

disease susceptibility to undergo structural transitions between native, non-native, and

unfolded conformational states. The species selected are golden hamsters, mice, and

rabbits, which respectively display high, moderate, and low susceptibility to prion

infection. Using urea and acid denaturation to induce structural transitions, we find that

PrP from these three species can form four distinct conformational states: native

monomers, β-structured monomers, β-structured octamers, and unfolded monomers. We

have developed a novel two-wavelength, model-free method of circular dichroism (CD)

for quantifying the fractional population of β-state PrP that exists in a monomer-octamer

equilibrium. We find that the propensity to form this β-state PrP from golden hamster,

mouse and rabbit correlates with the prion disease susceptibility of the host animals.

Furthermore, we show that β-state PrP is highly toxic to cultured nerve growth factor

(NGF)-differentiated PC12 cells. We have also identified unique intramolecular contacts

- 40 -

within the X-ray crystal structure of rabbit PrP which may contribute to its lower

propensity to populate the β-state and thereby reduce its susceptibility to prion disease.

Taken together, these results strongly support involvement of β-state PrP in prion disease

pathology, and provide new insight into the origin of varying levels of prion disease

susceptibility from species to species.

Results

Urea denaturation GHaPrP 90-231 displays a biphasic transition from α -helical to β -

structured to unfolded states

Purified, recombinant, golden hamster PrP encompassing amino acid residues 90

to 231 (GHaPrP 90-231) was diluted into solutions buffered at pH 4 containing

increasing concentrations of urea denaturant from 0 M to 8.0 M. The final concentration

of GHaPrP 90-231 was 9.5 μM. After incubating samples at room temperature for 4-5

days, CD measurements were made of the samples at 220 nm. When plotted as a

function of urea concentration, the mean residue ellipticity of GHaPrP 90 -231 shows a

biphasic transition (Figure 1a). The CD spectra of the protein were obtained under

varying concentrations of urea in order to delineate the secondary structural changes in

the protein as a function of urea. At pH 4.0 in the absence of urea GHaPrP 90-231

exhibited a typical α-helical CD spectrum. Increasing the urea concentration to 2.5 M

caused the protein to adopt a β-sheet rich structure, and at 7.5 M urea GHaPrP 90-231

exhibited an unfolded CD spectrum (Figure 1b). By relating the three unique CD spectra

of GHaPrP 90-231 in Figure 1b to the biphasic unfolding curve in Figure 1a, it is

apparent that the denaturant urea does not cause unfolding of the native state to a partially

folded intermediate state, which then completely unfolds. Instead, moderate amounts of

- 41 -

urea at pH 4.0 cause the native α-helical state of GHaPrP 90-231 to convert to a very

different β-sheet rich conformation, and addition of higher amounts of urea results in

complete unfolding of this unique β-sheet rich state. We denote the β-sheet rich

conformation formed at moderate urea concentrations, between 2.5 and 4.5 M, “β-state

PrP”.

Figure 1: (A) Urea-unfolding curve of GHaPrP 90-231 at pH 4 monitored by circular dichroism at 220 nm. (B) CD spectra of GHaPrP 90-231 in the native (○),β-State (■) and unfolded (□) conformations at pH 4.

A monomer-octamer equilibrium best describes the oligomerization state of GHaPrP 90-

231 in the β-state

In usual practice, denaturant-induced unfolding or refolding curves that display a

characteristic biphasic shape are fit to a 3-state transition model to determine the

thermodynamic stabilities of the 3 stably-populated species. However, these models are

based on the assumption that the protein remains monomeric at all denaturant

concentrations. Therefore, before fitting our CD data to any particular model, we sought

to establish the oligomerization states of native, β-state and unfolded PrP.

- 42 -

GHaPrP 90-231 samples were analyzed by sedimentation equilibrium to

determine the molecular weights of native, β-state and unfolded PrP. Sedimentation data

indicated that while native (in 0 M urea, pH 4.0) and unfolded PrP (in 7.5 M urea, pH

4.0) were monomeric with a molecular weight of ~ 16.0 kDa (data not shown), β-State

PrP (in either 2.5 M or 3.2 M urea, pH 4.0) had a significantly higher number average

molecular weight. This finding suggested that GHaPrP 90-231, under conditions that

promote β-state PrP formation (from 2.5 M to 4.5 M urea, pH 4.0), self-associates into

oligomeric species that account for the high number average molecular weights. In order

to determine the size, and thus the molecular weight of the oligomer, various self-

association models were applied to the sedimentation data of β-state PrP and the

goodness-of-fit as assessed by the sample variance was plotted as a function of oligomer

size (Figure 2a). Our analysis indicated that a monomer-octamer association model was

the best fit to our sedimentation data (Figure 2a) and residuals to the fit displayed the

least magnitude and a random pattern (Figure 2b). In order to confirm that only 2 species

(i.e. monomer and octamer) exist under conditions that promote β-state PrP formation,

size exclusion chromatography (SEC) was performed on β-state PrP (Figure 2c), which

showed that only 2 species exist under these conditions: an octameric species eluting at

10.1 mL, and a monomeric species eluting at 15.4 mL. Taken together, our results from

sedimentation equilibrium and SEC analysis on β-state PrP indicate that unlike native or

unfolded PrP, β-State PrP exists as an equilibrium mixture of two species: monomers and

octamers.

- 43 -

Figure 2: (A) Goodness-of-fit (variance) of sedimentation equilibrium data of GHaPrP 90-231 analyzed by various self-association models in 2.5M (▲) and 3.2M (○) urea at pH 4. (B) Sedimentation equilibrium data of GHaPrP90-231 in 3.2M, pH 4 at 15 000 rpm, fit to a monomer-octamer equilibrium model (lower panel). Residuals to the fit of the data to a monomer-octamer model (upper panel) (C) Size exclusion chromatography of GHaPrP 90-231 in 3.6M urea at pH 4.

Quantification of the fractional concentrations of β-state PrP by a model-free method

The finding that β-state PrP exists as monomers and octamers indicates that a

simple 3-state model is inadequate to fit the biphasic, urea-unfolding curves of PrP.

Analysis of the data using a 4-state equilibrium model is also not straightforward because

various sequential or branched models are equally probable and cannot be discerned from

the urea-unfolding curves. Furthermore, the greater complexity of the four-state model,

with its higher number of parameters, increases the difficulty in differentiating local

quality-of-fit maxima in the parameter landscape from the global maximum. For these

reasons, we decided to develop a model-free approach. Our model-free method takes

advantage of the fact that β-state PrP possesses a β-sheet CD spectrum that is distinct

from both the α-helical native and random coil-like unfolded PrP spectrum (Figure 1b).

Because the CD spectrum is a sum of the CD contributions from all protein molecules

present in their particular conformations, our observed CD signal measured in degrees

(θ220, obs) in a sample can be expressed as follows:

- 44 -

θ220, obs = [θ]220, Native[Native] + [θ]220, β-state [β-state] + [θ]220,Unfolded[Unfolded] [1]

In the above, [θ]220, Native, [θ]220, β-state, and [θ]220,Unfolded represent the molar ellipticity

of native, β-state and unfolded conformations of PrP at 220 nm, respectively. The above

expression can be simplified by normalizing the CD data to fraction apparent values

where the native state and unfolded state have values of 1 and 0, respectively:

Fapp 220 = 1(FNative) + z220(Fβ-state) [2]

Here, F app 220 is the observed normalized CD signal, z220 is the normalized CD

signal for β-state PrP at 220 nm, and FNative and Fβ-state are fractional concentrations of the

native and β-state PrP, respectively.

To solve for Fβ-state, a system of two equations can be generated by monitoring the

unfolding of PrP at a second wavelength, allowing generation of a second equation of the

same form as Eq. 2. In order to solve for Fβ-state, the normalized CD signal zλ for the β-

state at a particular wavelength λ would need to be different than z220, and distinct from

the native and the unfolded CD signals. Based on the spectra in Figure 1b, a wavelength

of 229 nm was selected as the second wavelength for monitoring GHaPrP 90-231

unfolding as a function of urea, yielding the following second equation:

Fapp 229 = (FNative) + z229 (Fβ-state) [3]

- 45 -

Combining Eqs. 2 and 3 and solving for Fβ-state yields:

Fβ-state = (F app 220 – F app 229) / (z220 – z229) [4]

Thus, by monitoring urea-unfolding of PrP at both 220 and 229 nm, the relative

fraction of β-state PrP can be calculated at any given urea concentration and pH value.

Note that this model free method treats the CD spectrum of the native, β-state, and

unfolded PrP as a unique signature of that particular state, and that procedures to

deconvolute the CD spectra into percent helix, percent β-sheet, and percent random coil

were not used.

Differences in the urea-induced unfolding of PrP at various pH values monitored by CD

at two wavelengths

Figure 3 represents the normalized, urea-unfolding curves of GHaPrP90-231 at

pH values of 4.0, 4.5, 5.0 and 7.0. The CD signals were monitored at wavelengths of 220

and 229 nm and the data was normalized to Fapp. A relationship is observed between the

shape of the urea-unfolding curves and the difference between the normalized CD signals

at the two wavelengths of 220 and 229 nm. At pH values of 4.0 and 4.5, the urea-

unfolding curves display a biphasic shape and the difference between the normalized CD

signals at both wavelengths are strikingly different (Figure 3a and 3b). At pH 5, the

biphasic shape and the differences between the curves monitored at two wavelengths are

both diminished (Figure 3c). At pH 7, the urea-unfolding curves are monophasic in shape

and the curves at the two different wavelengths are superimposable (Figure 3d). In sum,

at pH 4.0, 4.5, and 5.0, the unfolding curves at two wavelengths do not overlay at

- 46 -

intermediate urea concentrations, thus giving a qualitative indication that β-state PrP is

populated. Quantification of the amount of β-state PrP is possible with our model-free

method.

Figure 3: Urea-unfolding curves of GHaPrP 90-231 monitored by circular dichroism at 220 nm (▲), and 229 nm (○) at pH (A) 4.0, (B) 4.5, (C) 5.0, and (D) 7.0. The lines are intended as a guide to the eye.

- 47 -

Interspecies comparison of the β-State PrP fraction from GHa, Mo and RaPrP 90-231

measured using the model-free method

Recombinant PrP encompassing residues 90-231 from mouse (MoPrP 90-231)

and rabbit (RaPrP 90-231) displayed α-helical, β-sheet, and unfolded CD spectra very

similar to those of GHaPrP 90-231 at pH 4 in 0, 2.5-4.5, and 7.5 M urea, respectively

(data not shown). Both Mo and RaPrP 90-231 formed monomer-octamer equilibrium

mixtures at pH 4, 4-5 M urea that were similar to GHaPrP 90-231 (Figure 4).

Figure 4: (A) Goodness-of-fit (variance) of equilibrium sedimentation data at pH 4 of MoPrP 90-231 in 5M urea (▲) and RaPrP 90-231 in 4M urea (○) to various self-association models. Sedimentation data of (B) MoPrP 90-231 and (C) RaPrP 90-231 at 15 000 rpm fitted to a monomer-octamer equilibrium model.

Urea-induced unfolding of Mo and RaPrP 90-231 at pH values of 4.0, 4.5, 5.0 and

7.0 was monitored by CD measurements at 220 and 229 nm (Figures 5 and 6). At acidic

pH values both Mo and RaPrP 90-231 produce biphasic unfolding curves and populate

three, distinct conformations of PrP (native, β-state, and unfolded) much like GHaPrP 90-

231; however, a clear gradation is observed when the differences in CD measurements at

220 and 229 nm are compared between the three species. Differences between the two

wavelength curves are greatest with GHaPrP 90-231, intermediate with MoPrP 90-231

- 48 -

and least with RaPrP 90-231; these differences provide a qualitative indication of the

fractional concentrations of β-state PrP in the three species.

Figure 5: Urea-unfolding curves of MoPrP 90-231 monitored by circular dichroism at 220 nm (▲), and 229 nm (○) at pH (A) 4.0, (B) 4.5, (C) 5.0, and (D) 7.0. The lines are intended as a guide to the eye.

- 49 -

Figure 6: Urea-unfolding curves of RaPrP 90-231 monitored by circular dichroism at 220 nm (▲), and 229 nm (○) at pH (A) 4.0, (B) 4.5, (C) 5.0, and (D) 7.0. The lines are intended as a guide to the eye.

Importantly, refolding experiments of all three PrP proteins demonstrate that at

pH values of 7.0 and 4.0, the unfolding and refolding of the proteins are completely

reversible, indicating that equilibrium has been achieved (Figure 7).

- 50 -

Figure 7: Urea-unfolding (○) and refolding (●) at pH 7, and urea-unfolding (□) and refolding (■) at pH 4 for (A) GHa, (B) Mo and (C) RaPrP 90-231. Circular dichroism data was collected at 220 nm.

The normalized difference in CD signal of the β-state PrP at 220 nm and 229 nm

(defined by the expression (z220 – z229)) was calculated at the pH values that rendered a

biphasic curve for GHa, Mo and RaPrP 90-231. For GHaPrP at pH 4.0 and 4.5, for

MoPrP at pH 4.0, and for RaPrP at pH 4.0, the (z220 – z229) expression yielded values of

0.144 ± 0.020. This similarity in (z220 – z229) indicates that the normalized differences in

CD signal of β-State PrP are essentially the same for GHa, Mo and RaPrP 90-231.

Using a value of 0.144 for (z220 – z229) and the model-free method outlined above,

the fractional concentration of β-state PrP was calculated for each of the three species at

various urea concentrations and pH values (Figure 8). At pH 4.0, the peak of the β-state

PrP fraction reaches ~1.0 for all three PrP proteins (Figure 8a). However, GHa and

MoPrP 90-231 populate β-state PrP over a greater range of urea concentrations in

comparison to RaPrP 90-231. A similar trend is noticeable at pH 4.5, where GHa and

MoPrP 90-231 exhibit high β-state contents over extended ranges of urea concentrations

relative to RaPrP 90-231, which only reaches a maximum β-State fraction of ~0.2 over a

short urea concentration range (Figure 8b). At pH 5.0, only GHaPrP 90-231 significantly

- 51 -

populates the β-State fraction to a maximum of ~0.8 at 4.5M urea (Figure 8c). Both Mo

and RaPrP 90-231 show no significant fraction of β-state PrP at pH 5.0. At pH 7.0, GHa,

Mo and RaPrP 90-231 show no significant fraction of β-State PrP (Figure 8d). Thus, at

acidic pH values of 4.0 and 4.5, both GHa and MoPrP 90-231 sustain higher β-state PrP

fractions over greater urea concentration ranges relative to RaPrP 90-231.

Figure 8: Comparison of the fractional populations of β-State PrP of GHaPrP (red), MoPrP (green) and RaPrP (blue) 90-231 as a function of urea concentration at pH (A) 4.0, (B) 4.5, (C) 5.0, and (D) 7.0. The lines are intended as a guide to the eye.

- 52 -

Comparing the fraction octamer from sedimentation equilibrium analysis with the β-state

fraction from CD analysis leads to the detection of a monomeric, β-state PrP species

The fractional concentrations of octamer in the various samples can be

determined from sedimentation equilibrium data from GHa, Mo and RaPrP 90-231 by

fitting the sedimentation data to a double-exponential equation (Figure 9).

Figure 9: Determining fractional concentrations of octamer: (A) Sedimentation data of GHaPrP 90-231 (2.5M Urea, pH 4) collected at 10 000 rpm (○) fitted to a monomer-octamer equilibrium model (solid curve). The dashed line represents the radius at which point the data collected at 3 000 rpm (■) intersects with the fitted data at 10 000 rpm. (B) The monomer (□) and octamer (▲) absorbance fractions can be plotted as a function of radius from the solid line that represents the fitted data in (A). The dashed line represents the radius at which the octamer fraction is calculated.

The fraction octamer was calculated at urea concentrations of 2.5, 3.2, 4.0 and 4.5

M for GHaPrP, at 3.7, 4.3 and 5.0 M urea for MoPrP and at 3.0, 3.7 and 4.4 M urea for

RaPrP 90-231, all at pH 4.0. In comparing the fraction octamer with the fraction of β-

state PrP, it is clear that the fractional concentration of β-state PrP is significantly higher

than that of the octamer. Specifically, the fraction octamer only accounts for ~60-80 %

of the total β-state PrP fraction for GHaPrP 90-231 (Figure 10a), for ~50-75% of the total

β-state PrP fraction for MoPrP 90-231 (Figure 10b) and ~25-75% of the total β-state PrP

- 53 -

fraction for RaPrP 90-231 (Figure 10c). Since sedimentation equilibrium and SEC

analysis indicated that only monomers and octamers exist in solution, we conclude that

the β-state PrP fraction is a mixture of β-structured monomers and octamers for GHa, Mo

and RaPrP 90-231.

Figure 10: Comparison of the fractional populations of β-State PrP (●) with the octamer fractions (black bars) as a function of urea at pH 4 for (A) GHaPrP, (B) MoPrP and (C) RaPrP. The lines are intended as a guide to the eye.

Cytotoxicity of β-state PrP from MoPrP 90-231

We have observed that once formed, β-state PrP retains its secondary structure

and remains soluble, when the solution conditions are changed from 4.0 M urea, pH 4.0

to phosphate buffered saline, pH 7.4 (data not shown). This stability of β-state PrP

allowed investigation of its cytotoxic properties using differentiated PC12 cells, a

common cell culture model of neurons. Native-state and β-state MoPrP 90-231 were

added to differentiated PC12 cells and their effect on cell survival was monitored (Figure

11). Bee venom mellitin, a known cytotoxin, was used as a positive control. While native

state PrP did not display any toxic properties, β-state PrP was as cytotoxic as bee venom

mellitin on a molar basis. These data indicate that β-state PrP is stable under

physiological conditions and possesses potent neurotoxic activity.

- 54 -

Figure 11: Toxicity of β-State PrP when exposed to differentiated PC-12 cells. Absorbance readings were taken at 550 nm after performing the SRB assay to measure cell viability of cells exposed to no protein (*), β-State PrP of MoPrP 90-231 (▲), Native PrP (■), and melittin (○). The SRB assay was performed after 4 days incubation of cells at 37 °C.

Three-dimensional structure of RaPrP 121-230

Our circular dichroism experiments indicate that the propensity to form β-state

PrP is highest with hamster PrP, intermediate with mouse PrP, and least with rabbit PrP.

Sidechain-sidechain or sidechain-mainchain interactions that affect the stability of either

the native state or the β-state PrP likely determine the propensity of these proteins to

convert to the β-state. To determine whether the structure of RaPrP contains any unique

structural elements that might contribute to its resistance to conversion into the β-state,

we solved the X-ray crystal structure of rabbit RaPrP 121-230 to 1.6 Å resolution (see

Table 1 for data collection and refinement statistics).

- 55 -

Table 1: Summary of crystallographic data collection and model refinement statistics.

Data StatisticsSpace group P212121

Cell constants a (Å) 29.5b (Å) 86.2c (Å) 87.1

Resolution (Å) 30 – 1.6 (1.7 - 1.6)Overall reflections 214 933 (35 458)Unique reflections 30 148 (4 929)Redundancy 7.1 (7.2)Completeness (%) 99.6 ( 99.6)R merge 7.0 (24.6)<I>/σI 18.35 (7.71)

Refinement StatisticsFinal R Cryst (%) 20.3R-free (%) 23.5Solvent (%) 40.1No. of molecules 2No. of all atoms 1892No. of water molecules 182No. of sodium ions 1No. of Chloride ions 2Average B-factor (Å2) 7.5Ramachandran plot

Favored 99.5%Allowed 0.5%

R.M.S from ideal valuesLengths 0.005Angles 0.8

While numerous structures of PrP from various species have been solved by

nuclear magnetic resonance spectroscopy, to date there are only X-ray crystal structures

of sheep PrP26, human PrP24, and human PrP bound to an antibody27. The X-ray structure

of the human PrP is a nonbiological domain-swapped dimer.

The asymmetric unit of the RaPrP 121-231 crystal structure contains two

molecules. The two molecules are not domain swapped as in the previous dimeric

- 56 -

structure of human PrP24 but rather RaPrP 121-231 associates closely along a 2-fold

symmetry axis that is near parallel with helix-2 (Figure 12).

Figure 12: Observed dimeric arrangement in the asymmetric unit of wild-type RaPrP 121-230 crystal lattice. The two molecules are not domain swapped and closely associate between the helix-2 – loop – helix-3 (arrows) region of the C-terminal half of the ordered domain.

In the first molecule, electron density was observed for residues 126-230 and for

residues 127-222 in the second. The root mean squared deviation (RMSD) between the

two molecules, considering all backbone atoms, was 0.38 Å indicating that they are

nearly identical. The interface of the observed dimeric arrangement buries >1600 Å2 of

surface area, and contains several hydrophobic, hydrogen bonding, and ionic interactions

that potentially could occur in solution25. Additional lattice contacts between β-strand 1

of symmetry mates, form a 4-stranded anti-parallel β-sheet as has been observed in

previous crystal structures of sheep PrP26 and antibody-bound human PrP27.

- 57 -

The overall fold of RaPrP 121-230 is similar to other reported PrP structures; the

backbone RMSD was 0.80Å when comparing the structure of RaPrP 121-230 to the

crystal structure of sheep PrPC 28. As with previous structures, RaPrP 121-230 contains 3

α-helices: α1 (144-152), α2 (172-193) and α3 (200-222), and a small, two stranded, anti-

parallel β-sheet: β1 (129-131), β2 (161-163). There is also a disulfide bond which links

C179 of helix-2 with C214 of helix-3.

The loop between β-strand 2 and helix-2 (residues 166-174), sometimes referred

to as the β2-α2 loop or rigid-loop29, has been implicated as a possible amyloidogenic

motif that forms a steric zipper35. In the RaPrP 121-230 structure, electron density for

resides 165-175 is fairly well defined (Figure 13) indicating that the region is well

ordered. The main-chain carbonyls of P165 and V166 form hydrogen bonds with the

amides of Q168 and Y169 respectively, forming a small 310-helical loop. Similar loops

have been observed in previous crystal structures and NMR structure in which the β2-α2

loop was well ordered30-33. The side chains of D167 and Q168 are surface exposed but

that of V166 forms hydrophobic interactions with Y218 of helix-3 possibly contributing

to the stability of this loop (Figure 14). The 310-helix produces a small kink in the loop

before the 171-NNQS-174 sequence at the N-terminus of helix-2.

- 58 -

Figure 13: Omit FO-FC electron density (at 2.5σ) for resides V166-N168 (left) and Y169-S174 (right) of the β2-α2 loop. Residues 166-168 are well ordered forming a 310-helical loop and show good electron density over the main and side chains. Residues 169-174 also show good electron density except for S170 for which no side chain electron density was observed. Electron density of S174 shows that the side-chain hydroxyl points inward toward the backbone, making a hydrogen bond with backbone amide.

Figure 14: Long rang contacts between the β2-α2 loop and Helix-3. V166 and F175 make hydrophobic contacts with Y218. Polar contacts are also formed between the backbone of Helix-3 and N172.

- 59 -

The amino acid at position 174 is of particular interest because it is known to

affect prion susceptibility. The amino acid residue at position 174 is a serine in rabbit PrP

and an asparagine in mouse PrP. While mouse neuroblastoma cells expressing wild-type

PrP show susceptibility to scrapie infection, an N174S mutant of mouse PrP (the

analogous residue in rabbit PrP) is not susceptible to infection20. In the crystal structure

of RaPrP 121-231, the electron density for residues N171 and S174 (Figure 13) shows

that side chain carbonyl of N171 forms a hydrogen bond with the backbone amide of

S174 and the side chain hydroxyl of S174 forms a hydrogen bond with the backbone

carbonyl of N171 (Figure 15). The reciprocal side-chain to backbone hydrogen bonds

and flanking hydrophobic residues, Y169 and F175, form a helix capping motif similar to

the “hydrophobic staple”35, 36 that may contribute additional stability to the N-terminus of

helix-2.

- 60 -

Figure 15: The crystal structure of RaPrP 121-230. (Left) The observed monomeric fold is similar to previously observed structures of the ordered domain of PrPC. The β2-α2 loop is highlighted in the inset box. (Right) Comparison of residues 170-174 of the rigid loop from RaPrP 121-230 structures and the lowest energy structures from the hamster and mouse PrPC

NMR structure ensembles. The differing polar contacts observed within this region of the loop may result in the varying propensities to populate β-state PrP.

- 61 -

Discussion

Correlating prion susceptibility with the propensity of an animal’s PrP C to adopt the β-

state conformation

Data from accidental and experimental transmission of infectious prions to

animals can be used to gauge the prion susceptibility of those particular animals. The

BSE crisis in the United Kingdom exemplifies accidental transmission of BSE to other

animals through the consumption of contaminated feed materials. Humans and many

members of the family Felidae, including cheetahs, pumas, and domestic cats were

susceptible to the BSE agent, while no cases of prion disease were reported in canines37,

38. Studies on experimental transmission of infectious prions to laboratory animals reveal

varying susceptibility to prions. As discussed above, golden hamsters display greater

susceptibility than mice, which in turn are more susceptible than rabbits.

The molecular basis of susceptibility of host-encoded PrPC to convert to PrPSc

after exposure to exogenous PrPSc from the donor animal is still not fully understood.

Early hypotheses suggested that primary sequence similarity between the prions from the

donor animal and the PrPC from recipient animal plays an important role in prion

transmission39. For example, wild-type mice show low susceptibility to hamster prions,

yet transgenic mice expressing hamster PrPC are much more susceptible40. However,

there have been cases where primary sequence similarity does not determine an animal’s

susceptibility to prion strains. Bank voles show very low incubation times for prion

disease and are more susceptible to human prions than hamster or mice prions, despite

sharing greater PrPC sequence similarity to hamsters and mice41. Also, variant CJD

isolates are transmitted more readily to wild-type mice compared to transgenic mice

- 62 -

expressing human PrPC 42. Furthermore, the fact that many prion strains exist for a single

sequence of PrPC, and exhibit varying biochemical, histopathological, and

neuropathological characteristics when introduced in the same species of animal43,

suggests that prion susceptibility involves more than just primary sequence similarity

between donor prions and host PrPC.

A recent hypothesis that addresses species barriers and prion susceptibility

involves the conformational selection model44. This model posits that while mammalian

PrPC in general is capable of forming a multitude of PrPSc conformations, the PrPC from a

particular species is capable of forming only a subset of these PrPSc conformations. The

species barrier is overcome when the subsets of PrPSc conformers between two

mammalian PrPC sequences overlap. Conversely, if the subsets have little overlap, then a

species barrier exists and disease transmission is impeded. Applying this model to the

animals that display varying susceptibility to prions, one can postulate that the PrPC from

animals that show high prion susceptibility like hamsters, mice, humans and cats are

capable of forming a greater subset of PrPSc conformations than rabbits or dog PrPC,

which have low prion susceptibility and a very limited subset of PrPSc conformers.

The conformational transition from PrPC to unfolded PrP is accompanied by a side

reaction involving structural changes from a protein that is primarily α-helix to an

isoform that contains higher β-sheet content, the β-state. Using CD spectroscopy, we

have assessed the relative propensity of α-helical, recombinant PrP 90-231 from

hamsters, mice, and rabbits to adopt the β-state. We find that the propensity to adopt the

β-state correlates with prion susceptibility in these three species. Thus, in the context of

the conformational selection model, we find that relative to PrP from hamsters and mice,

- 63 -

rabbit PrP conformationally restricts its ability to adopt the β-State, and this observation

correlates with experimental transmission studies that classify rabbits, mice, and hamsters

as a species with low, moderate, and high susceptibility to prion diseases, respectively.

Structural features of native RaPrP that may impede conformational transition into the

β-State

Studies have shown that by exposing scrapie-infected, mouse neuroblastoma cells

(ScN2a) to molecules that stabilize PrPC, such as antibodies27 or chemical chaperones like

trimethylamine N-oxide and dimethylsulfoxide45, enables improved survival when

cultured cells are exposed to infectious scrapie material. Thus, stabilizing the native

structure of PrPC hinders the conformational transition into the PrPSc isoform when

exposed to the scrapie agent. From our studies it appears that the difference in the

propensity of GHa, Mo and RaPrP 90-231 to enter the conformational transition and

adopt the β-state resides within the covalent structure of PrP. Thus, certain structural

features of RaPrP may cause stabilization of the native state and reduce conversion to the

β-state.

Structural studies on PrPC from a variety of mammalian species indicate that

single amino acids changes do not have drastic structural effects on the overall fold of the

ordered domain. Rather, variation in local interactions in the PrPC monomer may affect

the mechanism of conversion to the infectious form. For example, in our high-resolution

crystal structure of rabbit PrPC 121-231, N171 and S174 form a hydrophobic staple-like

helix capping interaction at the N-terminus of α-helix-2. Such an arrangement is the first

one described for any PrPC structure and it may suggest how structural features of the

folded domain of PrP could influence prion infectivity and species barrier variations by

- 64 -

stabilizing the native state and impeding conversion to the infectious state. The

importance of residue 174 to prion infectivity has been highlighted in a cell culture model

of prion propogation20.

Relating PrP native state stability with kinetic and equilibrium folding intermediates

Inherited forms of CJD account for ~10 to 15% of all CJD cases; thus, carriers of

these PrP variants have a higher susceptibility to spontaneously develop prion diseases.

The relationship between the stability of on-pathway kinetic intermediates of human PrP

variants and inherited forms of prion disease has been detected48. Using stopped-flow

protein folding experiments, it was discovered that variants of human PrP which result in

inherited human prion diseases populate greater amounts of partially-folded, kinetic

intermediates than the wild-type protein. Further investigations into α-helix 2 and 3, a

region of the primary sequence that houses 20 of 33 known point mutations that lead to

human prion diseases, reveal that changes in hydrophobic core residues within this region

lead to kinetically trapping partially-folded states of PrP49.

Whether the transiently populated kinetic intermediates of PrP unfolding are on

the pathway to the formation of the β-state conformation is unknown. However, the

observation that both the β-state and the kinetic intermediates correlate with prion

susceptibility suggests that there may be a relationship between these two states, and thus

the β-state propensity of CJD and GSS mutants of human PrP should be examined.

Monomeric β-state PrP is populated and in equilibrium with the octamer

Analysis of the equilibrium sedimentation data of the β-state of GHa, Mo and

RaPrP 90-231indicated that the model which most accurately represented the data was

that of a monomer-octamer equilibrium, and the fraction octamer at different urea

- 65 -

concentrations and pH was calculated from the raw data by a method that is independent

of the relative abundances of native, β-state, and unfolded PrP. Comparison of the

fraction octamer with the fraction β-state for GHa, Mo and RaPrP 90-231 at pH 4.0

indicated that the fraction octamer was substantially lower than the β-state fraction.

According to our sedimentation and SEC data, only octamers and monomers exist in

solution; therefore, the remainder of the β-state population that is not octameric must be

monomeric.

Isolating monomeric, β-sheet PrP has proven to be difficult due to aggregation.

Previous studies indicated that diluting unfolded MoPrP 90-231 under acidic conditions

in 1M urea denaturant causes a time-dependent association of the protein into larger

oligomers, and that the relative ratio of oligomers to monomers increased over time51. In

one study, monomeric PrP with reduced disulfides was detected at low pH and it yielded

β-sheet spectra; however, addition of chaotropes or agitation caused the monomers to

form oligomeric and fibrillar structures52. It should be noted that the transition from PrPC

to PrPSc occurs without reduction of the disulphide bond53, thus the relevance to prion

disease pathology of the reduced, monomeric, β-sheet-rich, PrP species reported by these

previous studies is uncertain.

Using CD, SEC, and sedimentation equilibrium analysis we have determined that

under denaturational stress, conformational transitions exist between a minimum of four

states of PrP 90-231. These states are the monomeric, α-helical native state, β-sheet rich

monomers, β-sheet rich octamers, and the monomeric, unfolded state. We find that less

denaturational stress is required to populate both monomeric and octameric, β-sheet

conformational states of GHa and MoPrP compared to RaPrP 90-231.

- 66 -

Toxicity of β-state PrP

When the β-state of MoPrP 90-231 was added to NGF-differentiated PC12 cells,

the LD50 concentration of β-state PrP was ~ 1 μM after a 4 day incubation of the cells at

37 ºC. Other investigators have found a similar result with the β-structured

conformations of PrP, where concentrations of 1 μM were sufficient enough to cause a

substantial toxic effect in primary culture neurons after 24 hours55. Interestingly, β-state

PrP had to be in the soluble form in order to produce the toxic effect when exposed to

PC12 cells. Rapid-dilution of β-state PrP from low pH, urea solutions into physiological

conditions produced large, noticeable aggregates, which did not produce any toxic effects

in PC12 cells (data not shown). The cytolytic polypeptide melittin was used as a positive

control and it also yielded a similar LD50 concentration. Melittin is a major component of

bee venom and in aqueous solution the polypeptide remains monomeric and mostly

unstructured. Cytolytic properties of melittin are manifested in the presence of lipid

bilayers, where the monomers assemble into tetramers with pronounced α-helical content

and insert to form transmembrane pores56, 57. The mode of toxicity of β-structured forms

of PrP has been a contentious issue in the field of research. In general, amyloid

aggregates can cause cell toxicity by permeabilizing the cell membrane, either through

creating pores in the membrane, or by indirectly affecting membrane conductance58, 59.

The ability of low molecular weight PrP oligomers to cause toxicity in cells by disrupting

the 26S proteosome machinery has also been reported60. Although oligomers of β-sheet-

rich PrP may be toxic, there is no consensus on how toxicity comes about in an in vivo

context. As regards the relationship between β-state PrP and PrPSc, β-state PrP has not yet

demonstrated infectivity in animals, but contains many of the characteristics of PrPSc

- 67 -

including: β-structured conformation, PK resistance and cytotoxic properties. For now,

our findings demonstrate that the propensity to form β-state PrP is a valid surrogate

marker of prion disease susceptibility in golden hamsters, mice and rabbits; this

correlation may apply to other species.

Materials and Methods

Protein Expression and Purification

DNA constructs of golden Syrian hamster (GHa), mouse (Mo), and rabbit (Ra)

PrP 23-231 were generous gifts from M. Coulthart (Health Canada, Winnipeg, Canada),

D. Westaway and J. Watts (University of Toronto, Toronto, Canada), and S. Priola

(Rocky Mountain Laboratories, Hamilton, Montana, USA), respectively. The DNA

sequences for GHa, Mo, and Ra PrP 90-231, were cloned and expressed using the

pProEX-Htb plasmid (Invitrogen, Burlington, Canada) with an N-terminal 6xHis-tag and

a Tobacco Etch Virus (TEV) protease cleavage site. RaPrP 121-231 was cloned and

expressed using pET28a (Novagen,Gibbstown NJ, USA) with an N-terminal 6xHis-tag

and a thrombin protease cleavage site.

All PrP proteins were expressed as inclusion bodies using E. coli BL21 AI cells

(Invitrogen, Burlington, Canada) and refolded and purified according to Zahn et al. (ref.

61) with slight modifications. For PrP 90-231, the His-tag was cleaved with His-tagged

TEV protease (Invitrogen, Burlington, Canada) and both the His-tag and TEV were

removed with Ni-NTA (Qiagen, Mississauga, Canada). For PrP 121-231, anion

exchange was performed using HiTrap Q-sepharose 5/5 anion exchange column on an

AKTA FPLC system (GE Bioscience, Mississauga, Canada).

CD Spectroscopy

- 68 -

CD spectroscopy was performed on an Aviv circular dichroism spectrometer

model 62DS. All CD measurements were taken using a 1 mm quartz cuvette and a

spectral bandwidth of 1 nm at 25 °C. Far-UV CD spectra were averaged from three

wavelength scans from 250 nm to 190 nm with a 5 second averaging time, and blanked

with corresponding buffers.

Urea-unfolding of PrP 90-231 monitored by CD at 220 nm and 229 nm.

Stocks (95 μM) of folded PrP 90-231 were dialyzed into the following buffers

that were of identical ionic strength; pH 7 (50 mM sodium phosphate), pH 5.0 (50 mM

sodium acetate, 67 mM NaCl), pH 4.5 (50 mM sodium acetate, 70 mM NaCl), pH 4.0 (50

mM sodium acetate, 74 mM NaCl). All buffer solutions contained 5 mM EDTA.

Equilibrium samples were prepared by diluting the PrP stock to final concentration of 9.5

μM into buffered solutions containing urea at concentrations ranging from 0 M to 9 M.

Samples were incubated for 4-5 days at room temperature before making CD

measurements.

CD measurements were obtained at wavelengths of both 220 nm and 229 nm; 100

measurements at a rate of 1 measurement/s were averaged. For each pH value,

equilibrium curves were normalized according to Santoro et al. (ref. 62). The fraction β-

state PrP at each urea concentration was determined with Eq. 4. The z parameters in Eq.

4 represent the normalized CD signal of β-state PrP at wavelengths 220 and 229 nm; the

values of these critical points were determined graphically63.

Urea-refolding of PrP 90-231 monitored by CD

Unfolded PrP stocks (95μM) were prepared by denaturing folded PrP in 9.9M urea in pH

7 and pH 4 buffers using 3500 MWCO dialysis cups (Pierce, Rockford, Illinois). Urea-

- 69 -

refolding samples were prepared by diluting the PrP stock 10-fold (to final concentration

of 9.5 μM) into pH-buffered solutions containing urea to final concentrations ranging

from 1 M to 9 M urea. Samples were incubated for 5 days at room temperature. The CD

signal for each sample was the mean of 100 separate 1 s readings at a wavelength of 220

nm.

Size Exclusion Chromatography

GHaPrP 90-231 at 9.5 uM was incubated at room temperature in 3.6 M urea with

50 mM sodium acetate (pH 4.0) and 80 mM NaCl for a minimum of 72 hours. An

aliquot of 300 μL was then injected onto a Superdex 200 10/30 column (GE Bioscience)

equilibrated in the same buffer as above. The flow rate was 0.25 mL/min, and elution

was monitored by absorbance at 280 nm using an AKTA FPLC system at room

temperature.

Analytical Ultracentrifugation

Samples that contained high fractional concentrations of β-State PrP (based on

CD analysis) were subjected to equilibrium sedimentation experiments to determine the

oligomerization state. Experiments were performed on GHa, Mo and RaPrP 90-231 at

pH 4 in urea concentrations ranging from 2.5M to 5.0M, at protein concentrations of 9.5

µM. Experiments were performed in a six-sectored cell with quartz windows using rotor

speeds of 3000, 10000, 15000, 20000, 25000, 30000, 35000 and 40000 rpm at 25 °C. For

each speed, 5 replicates of data were collected every 0.001 cm at a wavelength of 280 nm

after 18 to 34 hours. Molecular weight determinations involved global analysis of data

acquired at 5 different speeds using Beckman XL-I software, where absorbance versus

radial position data were fitted to the sedimentation equilibrium equation using non-linear

- 70 -

least squares techniques. The partial specific volume and density of the sample were

calculated using the program SEDNTERP from the amino acid sequence and buffer

composition, respectively.

Determination of the Fraction Octamer

Sedimentation data collected from GHa, Mo and RaPrP 90-231 at pH 4 in urea

concentrations ranging from 2.5 to 5.0 M were fitted to a double-exponential model,

where the absorbance of the PrP sample equals the summation of the absorbance of the

monomer and the absorbance of the octamer, as a function of radius:

Absorbance (PrP sample) = Absorbance (Monomer) + Absorbance (Octamer) [5]

Absorbance (PrP sample) = A*exp(k*r2) + B*exp(8*k*r2), [6]

where A and B represent proportionality constants, r is the radius from the centre of the

rotor (in cm), and k equals the term (ω2/2RT)*(M*(1- νρ)). Here, ω represents the

angular velocity of the rotor (in radians per second), R represents the gas constant

(8.314*107 erg/(mol*K)), T represents the temperature in Kelvin, M represents the gram

molecular weight of the protein, ν represents the partial specific volume of the protein

and ρ represents the density of the solvent. For each sample, the fraction octamer was

determined at the radius value where the sedimentation absorbance readings at speeds

ranging from 10,000 to 15,000 rpm intersects with the absorbance readings at 3000 rpm;

that is, the radius at which the protein concentration equals the concentration in the

cuvette prior to sedimentation. The fraction octamer was calculated using 3-6 separate

data sets.

- 71 -

Sulforhodamine B Cytotoxicity Assay

Pheochromocytoma (PC-12) cells were plated at 1,000 cells per well in a 96-well

plate and incubated in 60 ng/mL NGF diluted in N2 supplement/Dulbecco's Modified

Eagle’s Medium (Gibco BRL). Cells were differentiated for 6 days. Samples (0 to 9 µM)

of β-state MoPrP 90-231, native state MoPrP 90-231, and bee venom mellitin were

prepared by diluting concentrated stock solutions into cell culture media and then

dialyzing the samples against large volumes of cell culture media in a 3,500 Da MWCO

dialysis cup to remove urea from the samples. After differentiation, the media was

exchanged with the PrP/mellitin samples and the cells were incubated for 4 days at 37 ºC.

Toxicity was assayed using the sulfhydryl rhodamine B (SRB) assay. Briefly, cells were

fixed with 8.5% trichloroacetic acid for 30 minutes. The plates were washed with

distilled H20 and air-dried. Protein was stained with 0.4% SRB (Molecular Probes Inc.) in

1% acetic acid for 30 minutes. Plates were washed with 1% acetic acid and air-dried. The

dye was extracted in 10 mM Tris (pH 9.0) and absorbance was assayed at 550 nm on a

plate reader.

Crystallization of RaPrP 121-231 and Data Collection

Initial screens using Crystal Screens I & II (Hampton Research, Aliso Viejo, CA)

yielded showers of crystalline plates in 100 mM HEPES, pH 7.5, 4.3M NaCl. Further

optimization and microseeding gave single, plate-like crystals (300 μm × 300 μm × 30

μm) that grew overnight in 100 mM Tris, pH 7.0, with 2.5 M NaCl and diffracted to 1.6

Å. Data was collected from a single crystal at 100 K with 100 mM Tris, pH 6.5, 2.5 M

NaCl, and 30% v/v glycerol as cryoprotectant. Data was collected using a Marmosaic

- 72 -

CCD300 at the CMCF beamline at the Canadian Light Source Synchrotron (Saskatoon,

Canada).

Data Processing, Refinement and Model Building

Data were processed with XDS64. Statistics for data collection and unit cell

parameters are summarized in Table 1. Structure was solved using molecular

replacement with PHASER65 using sheep PrP (1UW3) as a search model. The model was

refined without NCS with ML-method using REFMAC66 and TLS refinement67 assisted

by TLSMD68. Model building was done using Coot69. Summary of refinement statistics

are given in Table 1. Atomic coordinates have been deposited in the Brookhaven Protein

Data Bank (PDB ID: XXXX).

Acknowledgements

This study was supported by a grant from PrioNet Canada. B. Sweeting was the

recipient of Ontario Graduate Scholarships. Braden Sweeting contributed significantly to

the crystallization and analysis of RaPrP 121-231. V.K. Mulligan was the recipient of a

CIHR Frederick Banting and Charles Best Canada Graduate Scholarship Doctoral

Award.

- 73 -

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Conclusion and Future Experiments

We have developed a model-free method that can be used as a tool to rank the

propensities of particular PrP 90-231 sequences to convert from the α-helical state to the

β-state. This conversion propensity of an animal’s PrP90-231 is correlated with its

susceptibility to prion disease. We find that the β-state encompasses both monomeric and

octameric forms of PrP 90-231, and is achieved when denaturational stress (urea, pH) is

added to the folded protein under equilibrium conditions. Our method and results have

potential predictive value. For example, if a pH and urea dependent unfolding/refolding

profile of a particular PrP 90-231 is similar to that of GHaPrP 90-231, we would predict

that this sequence when expressed in animals could cause them to have prion disease

susceptibility similar to hamsters. Similarly, if the denaturation profile matches that of

Mo or RaPrP 90-231, prion disease susceptibility similar to mice and rabbits,

respectively, would be predicted. While this method is a potential predictor of general

prion susceptibility, it cannot predict which animal species will be susceptible prions

originating in a different animal species.

Conservation of primary and secondary structures of the β-state amoung mammalian

PrP90-231

The fact that GHa, Mo and RaPrP 90-231 convert into both monomeric and

octameric, β-sheet states suggests that the transition from the α-helical, native state to the

β-state occurs through a misfolding and oligomerization pathway that is common to PrP

from these animals. Experimentally, we have concluded that the circular dichroism (CD)

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signal of the β-state is nearly identical for GHa, Mo and RaPrP 90-231. From a primary

sequence perspective, the common β-state PrP structures are most likely encoded by key

stretches of amino acid sequences that are not only conserved by these three proteins, but

also by other mammalian PrPs. There are also regions of the mammalian PrP primary

sequence where variability occurs. The residues that exist in the variable regions for a

particular PrP sequence can perform one of the three functions: they can either serve to

stabilize the β-state, they can serve to destabilize the β-state, or they have no effect on the

β-state. Since GHa, Mo and RaPrP can all populate the β-state suggests that the primary

sequence that promotes the β-state is conserved, and, relative to RaPrP, both GHaPrP and

MoPrP 90-231 have residues within the variable regions of the PrP sequence that either

act together or independently to stabilize the β-state.

Theoretically, there could be cases where a sequence of PrP would contain

residues within the variable or conserved regions of the sequence that would completely

destabilize the β-state, essentially causing the protein to unfold/refold between the native

and the unfolded states at neutral and acidic pH values. Once expressed in animals, in

theory, these sequences will prevent the host PrPC from interacting and/or converting into

the scrapie isoform. There are two sequences which show such promise. During the

BSE crisis in the United Kingdom, household cats fell ill to the disease; however, there

were no reports of dogs being susceptible to BSE [1, 2]. Cell culture studies revealed that

Madin Darby canine kidney (MDCK) cells that harbor the canine PrP sequence did not

convert to PrPSc once challenged with CJD and mouse-adapted prions [3]. Furthermore,

dog and horse PrPC isolated from brain have not been able to be amplified by PMCA

technique - the same technique that is able to amplify PrPC from rabbit brains (J. Castillo,

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personal communication). Thus, our hypothesis is that certain key amino acid differences

within the sequence of the dog and horse PrP play a key role in destabilizing β-state PrP.

Interestingly, there are key polymorphisms present in these sequences of PrP 90-231 that

are not present in other species of mammals, and also reside near the N174 residue of the

SNQNNF motif of hamster PrP. When compared with 46 mammalian PrP sequences, the

dog family contains two unique mutations: D160N, and R178H, which flank the

SNQNNF motif. Horses contain a very unique K173N mutation, which replaces the third

asparagine of the SNQNNF motif [4].

I plan to perform equilibrium, unfolding and refolding of dog and horse PrP90-

231 sequences using the model-free method for calculating β-state PrP at various pH and

urea concentrations. If these unique polymorphism destabilize monomeric and/or

octameric β-sheet conformations, then I will introduction these mutations in GHa, Mo

and RaPrP which I hypothesize would similar effects on their β-states. Conversely, by

replacing the unique residues present in the dog and horse PrP with those present in GHa,

Mo and RaPrP, I predict these changes would promote β-state formation in both dog and

horse PrP.

Other Future Experiments: Assessing infectivity of β-state PrP and X-ray crystal

structure determination of the octamer

We have defined β-state PrP as the β-sheet rich conformation of PrP, either

monomeric or octameric, that is induced by low pH and moderate concentrations of urea.

However, β-state PrP, once taken out of these denaturing conditions and into

physiological conditions, can retain its oligomerization status and we have demonstrated

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its cytotoxicity on neuronal-differentiated PC12 cells. Stabilizing β-state PrP under

physiological conditions offers the opportunity to perform other key experiments.

In the field of prion diseases, the ‘truly infectious’ unit of PrPSc, and mode of

toxicity in vivo have not yet been identified. The β-sheet rich form of PrP in the

oligomeric or amyloid form can be generated in the laboratory [5]. We and other

researchers have demonstrated that β-sheet rich, oligomeric PrP from recombinant

sources is cytotoxic to cell cultures [6, 7]. In vitro preparation of amyloid from

recombinant PrP not only exhibits cytotoxicity to cell cultures, but has been shown to be

infectious using animal bioassays [6, 8]. Transgenic mice (Tg) expressing MoPrP 89-231

eventually succumbed to a neurological diseases when injected with amyloid fibrils

prepared from recombinant MoPrP 89-231. What has not been assessed is whether β-

sheet rich, PrP oligomers are infectious.

I intend to directly inject β-sheet rich, oligomeric PrP from recombinant GHaPrP

90-231, stabilized under physiological conditions, into the brains of hamsters and test the

effectiveness of β-state PrP to induce a prion-like, neurodegenerative disease. This

experiment, if successful, will not only support the notion that smaller oligomers of PrPSc

possess toxic and infectious properties [9], it will also provide additional support of the

protein-only hypothesis in that misfolded PrP prepared in the laboratory can induce prion

infection in animals.

The second experiment includes solving the X-ray crystal structure of β-sheet

octamers. There exist models on how PrPC may adopt a β-sheet conformation (see

Introduction section); however, to date, there has been no high-resolution structures

published of PrP in a β-sheet conformation. Due to its tendency to aggregate in the β-

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sheet conformation, achieving a highly-concentrated sample of octamers under

physiological conditions is unlikely. Thus, we intend to stabilize the octamers with PrPSc-

specific antibodies that have been generated in our laboratory to the sequence which

includes the YYR epitope located within β-strand 2 of the PrPC structure. This region of

the protein is buried when PrP is folded in its native state, and is exposed when PrP

adopts an acid-induced, β-sheet conformation [10].

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References:

[1] Aldhous, P. BSE: spongiform encephalopathy found in cat. Nature 345, 194. (1990).

[2] Kirkwood, J. K. & Cunningham, A.A. Epidemiological observations on spongiform encephalopathies in captive wild animals in the British Isles. Vet. Records 135, 296-303. (1994).

[3] Polymenidou, M., Trusheim, H., Stallmach, L., Moos, R., Julius, C., Miele, G., Lenz-Bauer, C. & Aguzzi, A. Canine MDCK cell lines are refractory to infection with human and mouse prions. Vaccine 26, 2601-2614. (2008).

[4] Wopfner, F., Weidenhofer, G., Schneider, R., von Brunn, A., Gilch, S., Schwarz, T. F., Werner, T., & Schatzl, H. M. Analysis of 27 mammalian and 9 avian PrPs reveals high conservation of flexible regions of the prion protein. J. Mol. Biol. 289, 1163-1178. (1999).

[5] Baskakov, I. V., Legname, G., Baldwin, M. A., Prusiner, S. B. & Cohen, F. E. Pathway complexity of prion protein assembly into amyloid. J. Biol. Chem. 277, 21140-21148. (2002).

[6] Novitskaya, V., Bocharova, O. V., Bronstein, I. & Baskakov, I. V. Amyloid fibrils of mammalian prion protein are highly toxic to cultured cells and primary neurons. J. Biol. Chem. 281, 13828-13836. (2006).

[7] Kristiansen, M., Deriziotis, P., Dimcheff, D. E., Jackson, G. S., Ovaa, H., Naumann, H., Clarke, A. R., van Leeuwen, F. W., Menendez-Benito, V., Dantuma, N. P., Portis, J. L., Collinge, J., & Tabrizi, S. J. Disease-associated prion protein oligomers inhibit the 26S proteasome. Mol. Cell 26, 175-188. (2007).

[8] Legname, G., Baskakov, I. V., Nguyen, H. O., Riesner, D., Cohen, F. E., DeArmond, S. J. & Prusiner, S. B. Synthetic mammalian prions. Science 305, 673-676. (2004).

[9] Collinge, J. & A. R. Clarke. A general model of prion strains and their pathogenicity. Science 318, 930-936. (2007).

[10] Paramithiotis, E., Pinard, M., Lawton, T., LaBoissiere, S., Leathers, V. L., Zou, W. Q., Estey, L. A., Lamontagne, J., Lehto, M. T., Kondejewski, L. H., Francoeur, G. P., Papadopoulos, M., Haghighat, A., Spatz, S. J., Head, M., Will, R., Ironside, J., O'Rourke, K., Tonelli, Q., Ledebur, H. C., Chakrabartty, A. & Cashman, N. R. A prion protein epitope selective for the pathologically misfolded conformation. Nat. Med. 9, 893-899. (2003).

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