prion infection dynamics- an analysis of conversion mechanisms to characterize propagation of prion...

H. Williams 4/28/14

Prion Infection Dynamics: An Analysis of Prion Conversion Mechanisms to Characterize the Propagation of Prion Disease

Henry W. Williams

Department of Biochemistry, Hobart and William Smith, Geneva, NY 14456 Submitted by: April 27, 2014

1.0 Introduction

Common cognitive diseases such as transmissible Spongiform Encephalopathy

(TSE), Scrapie, Creutzfeldt-Jacob disease, Gertmann-Straussler-Scheinker disease,

Familia Insomnia, Kuru, and Alzheimer’s Disease, have all been linked to fibrilization

caused by prion structural conversion. Proteinaceous infectious particles or prions (PrP),

are a distinguished family of pathogens that, unlike viruses or bacteria, do not use nucleic

acids for replication. Historically it was believed that infectious pathogenic agents require

nucleic acids to replicate and cause disease. Toxic prions however, are able to induce

conformational changes among other healthy prion proteins to adopt an infectious

structure causing the proliferation of amyloid fibers and progressive loss of structure and

function in nerve cells, resulting in neurodegeneration. The degeneration of neurons in

the brain, particularly in elderly populations, is a serious health concern resulting in wide

spread neurodegenerative diseases, severe brain damage, and even death.

Over the two past decades there has been substantial research on the

conformational change of healthy cellular prion (PrPC) to the toxic prion isoform (PrPSc).

Neurodegenerative diseases have been found to share a common prion conformational

transition that contributes to neural toxicity. This has stimulated further research to better

understand prion structural dynamics. A mechanistic understanding of prion conversion

could provide essential new information for future medical treatment for prion related

diseases. To fully appreciate how infectious prions aggregate, one needs to understand

H. Williams 4/28/14 prion interaction at the structural level and the evolution of prion conversion

mechanisms.

1.1 Prion Structure and Toxic Characteristics:

Prions are a gene family that consists of three members that comprise of Prnd

which encodes Doppel, a testis-specific protein involved in male reproduction, Sprn, a

prion protein expressed in the central nervous system, and Prnp which encodes for PrPC

the precursor to prion disease (Watts et al., 2007). Human Cellular Prion Protiens (PrPC)

are 253 amino acids peptides possessing one disulfide bond and three to four alpha

helical structures. PrPC is homologous protein, with similar prions found in bovine,

sheep, mice, and other mammals. Though highly expressed within the central nervous

system (CNS) of mammals, their function still remains unclear. The ability of PrPC to

bind to copper (II) ions with relatively high affinity is thought to play a role in

intracellular signaling within neurons (Steele et al., 2006). PrPC proteins are glycosylated

and integrated to the plasma membrane of neurons and can be readily digested by

proteinase K (Eigen, 1996). In fact, proteases function to regulate the concentration of

PrPC in the CNS via the selective cleavage of the glycophosphatidylinositol (GPI)

glycolipid anchor which attaches PrPC to the cellular surface of neurons (Eigen, 1996).

Cleavage results in the digestion of PrPC, allowing for the body to control the

concentration of prions from reaching toxic levels. However prions that have adopted the

toxic PrPSc conformation become particularly resistant to proteases digestion, disrupting

the normal concentration of prions. For a prion infection to occur, there needs to be

enough accumulation of the protease resistant PrPSc proteins to allow for development of

plaque in the brain.

H. Williams 4/28/14 While PrPSc and PrPC have very different effects on cell viability, structurally

prions only differ in secondary and tertiary structure. Edman sequencing and mass

spectrometry showed very similar amino acid sequences for both PrPC or PrPSc (Harrison

et al., 1997). In contrast fourier transform infrared (FTIR) and circular dichroism (CD)

spectroscopy has revealed that PrPC is comprised of primarily alpha helices, while PrPSc

has a characteristically high beta sheet content (Harrison et al., 1997). When the healthy

PrPC is converted to its toxic PrPSc form, the N terminus end of PrP proteins contains

approximately 90 residues that are resistant to proteinase-K activity. This inhibition of

proteinase activity is thought to be caused

by the conversion of four different alpha

helical regions of the prion protein to beta

sheets.

Spectroscopic studies have further

demonstrated that there are three predicted

alpha helical regions in healthy PrPC (figure

1) (Harrison et al., 1997). Each of these

regions can undergo conformational

transitions from alpha helices to beta sheet

structures. However, data has shown

that the PrP(109-122) region is the

essential for the conversion of PrPC to

PrPSc. The adoption of the beta sheet

Figure 1: Ribbon Representation of Healthy PrP. 3D diagram of structural composition of PrPC. The Binding surface is the shadowed region. The cavity is comprised of three alpha helices H1 in blue (F144, N146, E149), H2 in green (H180, V183, N184, V187, K188, T191, V192, T194, T195), and helix H3 in red (E203, I206, K207, E210, R211, E214, Q215). (B) Is the same structure except it is rotated by about 90°.

H. Williams 4/28/14 structure in this region would therefore be toxic. This conformation change to beta-

sheets is initiated by the interactions of PrP(109-122) with fragments PrP(104-122) and

PrP(129-141) (Harrison et. al., 1997).

This change in the structural conformation alters the way in which prion proteins

cooperate. These abnormal interactions result in the change in conformation of healthy

PrPC to the toxic isoform of PrPSc. Since toxic PrPSc is resistant to proteinase K, the build

up of PrPSc causes aggregation, forming amyloid fibers that build up and cause plaque in

brain tissue. The specific mechanism for this process has been highly controversial. A

progression of hypotheses has contributed to the evolutionary understanding of how

prions interact and whether prions undergo autocatalysis or enzymatic conversion.

1.2 “Prusiner Mechanism”-Linear Auto Catalysis In the 1990’s Stanley Benjamin

Prusiner proposed one of the first

mechanisms for prion conformation through

an autocatalytic approach (Eigen, 1996).

Prusiner hypothesis suggested that the toxic

PrPSc product of the catalytic conversion of

PrPC facilitates the conversion of more PrPC

to its infectious conformation. The

secondary, tertiary, and even quaternary

conformations are the determinants for a

prions’ pathological structure. Prusiner first

proposed a model that resembled an “induced fit process”, which later became known as

the Prusiner mechanism for linear autocatalysis. This process involves a PrPSc monomer

or dimer acting as a template to mediate the conformational change of PrPC to PrPSc.

Figure 2: Prusiner’s Linear Autocatalysis. Induced fit mechanism in causing prion conversion to toxic structure. (A) represents PrPC conformation, while (B) is indicative of the toxic PrPSc structure.

H. Williams 4/28/14 According to Prusiner, the binding of these two monomers lowers the free energy barrier

between PrPC and PrPSc structures (Harrison, 1997). Upon formation of the new PrPSc,

more PrPC monomers are recruited for further conversion, resulting from PrPC having a

high binding affinity for PrPSc.

Since beta-sheets are more stable than alpha helices the conversion of PrPC to

PrPSc is thermodynamically favored; however it is usually prevented due to a high kinetic

barrier. When PrPSc is present the activation energy for the conversion of healthy PrPC to

the toxic PrPSc is then lowered. Prusiner suggests that these conditions, provide enough

driving force to cause the conversion of PrPC to PrPSc (figure 2) (Eigen, 1996).

To better understand conformational kinetics of this mechanism, in regards to

prion infection, the propagation of prions at low concentrations must be examined over

time. At the initial stage of infection, the concentration of PrPSc is assumed to be zero.

Under the assumption that the original population of PrPC is on the order of ~1024, and

the PrPSc population was close to zero, then the non-catalytic rate kAB would have to be

between 10-22 or 10-23 s-1. This rate has to be small because if it was any higher,

spontaneous conversions could cause prion disease when an infection is not present.

However In order for an infection to occur the rate of turn over (kT = [A]/KM) (figure 2)

of the PrPSc-PrPSc dimer would have to be large enough to create an accumulation of

monomeric PrPSc. To cause disease, the linear autocatalysis mechanism would require kT

to be on the order of 10-8 to 10-7 s-1 to accumulate enough of the PrPSc. An examination of

the ratio of KT/KAB provides valuable information regarding the rate of enhancement of

the prion conversion. Here, Eigen et al. suggests that the ratio of KT/KAB is

approximately 1015, which they express to be unrealistic for a non-cooperative

autocatalytic mechanism. Eigen et al. states that there is no non-cooperative enzymatic

H. Williams 4/28/14 reactions that could have a turnover that high. Though the linear autocatalysis mechanism

was one of the first accepted models for prion conversion, it was unable accurately

express the true rate of toxic prion conversion. To correct this, Prusiner also suggest a

second hypothesis to provide a better representation of how toxic prions replicate. In this

second mechanism, prions undergo structural transition via cooperative autocatalysis.

1.3 “Prusiner Mechanism”-Cooperative Autocatalysis A second mechanism proposed by Prusiner is

the cooperative autocatalysis mechanism. The

cooperative autocatalysis model, suggests that PrPSc

is still thermodynamic favorable and causes the

decrease in the activation energy barrier in the

conversion of PrPC. However the two PrPSc

monomers remain bound after conversion is

complete. This accumulation of bound PrPSc results

in further recruitment of PrPC, lowering the activation

energy of conversion for each new PrPSc

that is converted (figure 3).

Each time a new PrPC binds to the

existing oligomer, the amount of energy

needed to convert to the toxic form decreases (Eigen et al., 1996). This method of

conversion is very similar to the process found in subunit cooperativitiy in hemoglobin

(Egen et al., 1996). As a result of this exponential decrease in activation energy, the rate

of conversion increases as PrPSc accumulates. However the cooperativity in binding

depends on the stability of the subunits.

Figure 3: Prusiner’s Cooperative Autocatalysis. Analyzing the catalyst affects of oligomeric Prion proteins. Cooperative Autocatalysis suggests PrPSc conformer is thermodynamically favored. Reaction is driven concentration of previous reaction step.

H. Williams 4/28/14 Figure 3 fails to account for how PrPC can affect the stability of other nearby

prions in the PrPSc and PrPC conformation (Laurent, 1997). According to Laurent et. al,

there are two hypothesis that have been formulated as a means of possibly determining

whether there are two different binding domains that bind PrPSc and PrPC or if PrPSc and

PrPC are competing for the same binding domain. In linear autocatalysis, there would

only be one binding domain. In this case the opposite isoform should have a stronger

binding affinity for that site. Meaning that PrPSc should have a higher binding affinity for

PrPC, while PrPC has a higher affinity for

PrPSc. While for cooperative autocatalysis

there could be heterogeneous and

homogenous binding domains.

Heterogenous binding domains would

allow for specific binding between PrPSc

and PrPC. While homogenous binding

domains would be a set of identical

domains binding, which could bind

without specificity. To understand the

difference between linear autocatalysis,

PrPSc-PrPC, and PrPSc-PrPSc coopertivity

autocatalysis, one has to compare between

the catalytic efficiency between each type

of aggregation.

Figure 4: Comparing Linear Autocatalysis to Cooperative Autocatalysis. Comparing the catalytic efficiency between three forms of autocatalysis to determine, catalytic activator causing increased PrPSc turnover. (A)Linear Autocatalysis is compared to autocatlysis with the formation of a PrP heterodimer (B) or PrP homodimer (C) .

H. Williams 4/28/14 In Prusiner’s cooperative autocatalysis hypothesis, either a heterodimer can form

from PrPSc and PrPC, or a homodimer can arise from two PrPSc with PrPSc. Each of these

dimers influences the catalytic efficiency of prion propagation differently. Luranet et al.

describes that understanding the differential activity of heterodimers and homodimers

reveals the key influences of prion propagation based on the relative binding affinities

that PrPSc has for either itself or PrPC. In figure 4, Luranet et al. compares the linear

autocatalytic efficiency (Figure 4.A) with the cooperative catalytic efficiency of when

PrPSc has a higher binding efficiency for PrPC ( Figure 4.B) or PrPSc ( Figure 4.C).

The formation of either a homo or a hetero-catalytic oligomer can have different

kinetic effects on prion propagation. Figure 4.B demonstrates that a heterodimer would

provide an excellent catalyst to initiate the infection. However this would mean that PrPSc

has a weak affinity for itself, which would, inhibits PrPSc monomers from assembling

into amyloid fibers. Assuming that monomeric PrPSc is incapable of catalyzing

aggregation, the cooperative autocatalysis of heterodimers would increase affinity for

binding of PrPC. Each PrPC conversion to PrPSc causes an increase in the affinity of PrPC

binding to PrPSc. In contrast, the homodimer illustrates that the PrPSc has a higher affinity

to PrPSc. Based on the information provided by Luranet et al., this hypothesis is less

likely to occur due to the fact that interactions between PrPSc would be too great,

decreasing PrPSc binding affinity for PrPC. Homodimers of PrPSc would dramatically

decreases the number of healthy PrPC converted to toxic PrPSc. Thus the heterodimer

hypothesis illustrates the most likely means by which PrPSc binds to PrPC, causing

elevated propagation of PrPSc in a cooperative way. However there is more scientific

evidence that illustrates that prions could propagate based on this homodimer hypothesis

through a Nucleation-dependent Mechanism.

H. Williams 4/28/14 1.4 Nucleation-Dependent Mechanism One of the most widely excepted hypotheses within “protein-only” aggregation

mechanism is the nucleation-dependent model. According to this model, PrPSc and PrPC

differ in their quaternary structures. Lansbury suggests that nucleation of four PrPSc

monomers must occur to accelerate the fibrilization and progression of disease.. Prusiner

and Luranet originally suggested that accumulation of PrPSc was based on the formation

of PrPC-PrPSc heterodimers subunits. According to Lansbury, PrPSc has a higher affinity

for PrPSc than PrPC, as a result of its monomeric instability. Lansbury assumes that PrPSc

is stabilized by the intermolecular (allosteric) interactions between other PrPSc proteins.

Unlike Prusiner, where the concentration of monomeric PrPSc concentration determines

the speed of the conversion and development of disease, Lansbury suggests that the

formation of PrPSc oliogmers determines rate of fibrilization. Lansbury suggest that the

accelerated conformational change and fibrilization are due to the development of nuclei,

which then polymerize together to form long fibril like structures. In order for there to be

rapid aggregation, a nucleation step is needed to stabilize PrPSc and facilitate the

polymerization of oligomers into fibril structures. This oligomerization step has been

seen numerous recent studies involving how prevent the conversion of prions (Cavaliere

et al., 2013).

In short, there are three fundamental modifications from cooperative autocatlysis

that make up the nucleation-dependent mechanism (NDM) (Eigen et al., 1997). The first

requires NDM to produce quaternary structures to catalyze the formation of fibrils.

Second, in the NDM, unlike Prusiner model, PrPC is the thermodynamically preferred

state relative to PrPSc.. Finally, according to NDM PrPSc monomers are less stable than

PrPC, however in their dimeric state, bound PrPSc is more stable than PrPC. Eigen et al.,

H. Williams 4/28/14 explains that free PrPSc particles can be stable, however their stability is increased upon

binding to other PrPSc molecules (forming a nucleus) (Eigen et al, 1996).

Eventually, according to NDM, these PrPSc homodimers would form a sextet

(seed), which causes a shift in the equilibrium to form fibrils (figure 5). The nucleation

step of these seeds limits the development of disease. PrPC is more stable than

monomeric PrPSc, preventing PrPC from spontaneously converting to PrPSc. However

PrPSc gains greater stability when oligomerized. The nucleation of PrPSc into seeds then

causes a shift in the activation energy barrier, resulting in rapid polymerization into

fibrils. This rapid fibirlization of seeds accounts for the rapid growth in the disease phase.

While the nucleation-dependent mechanism provides a possible alternative to

accumulation of PrPSc in an autocatalytic fashion, this hypothesis still does not take into

account whether there are other proteins involved that could stimulate the conformation

of these prion proteins. It is known that there are chaperon proteins within the cell that

facilitate the aggregation of proteins when under various stresses.

Figure 5: The Nucleation-dependent mechanism as a means to convert PrPC to PrPSc. This mechanism results from the idea that prion oliogmers must form first before the accelerated aggregation. According to Lansbury, prion aggregation is dependent on two steps. A oliogmerization step and secondly a accelerated fibrilization step.

H. Williams 4/28/14 1.5 Which mechanism is the correct one? Eigen et al. demonstrates that there is a logical progression from prusiner’s

original linear autocatalysis to Lansbury’s nucleation mediated mechanism. Prusiner’s

autocatalysis mechanisms are capable of causing exponential growth of toxic prions.

However mathematical analysis of each mechanism revealed that the method of

conversion is unrealistic to conditions found in a cell. The issues with Prusiner’s

autocatalysis method is that the rate of production of PrPSc is either slower than the rate

of degradation, meaning disease can never occur, or spontaneous conversion of PrPC to

PrPSc occurs far too readily resulting in the development of disease every time (Eigen et

al., 1997). Both of these inaccuracies illustrate that the prusiner models fail to accurate

represent the true method of prion conversion. Both the cooperative autocatalysis and

linear autocatalysis method do not have the ability to control spontaneous growth of PrPSc

and don’t provide realistic conditions for PrPSc to accumulate non-spontaneously.

Adapting from Prusiner’s cooperative autocatalysis mechanism, the NDM asserts

that there needs to be a nucleation step that develops seeds which can then activate the

accelerated formation of fibrils. Based on the fact that seed formation decreases the

activation energy barrier in order to cause development of the disease, the nucleation step

acts as a threshold preventing or allowing accelerated fibrilization. Where either

accumulation of PrPSc is to low to form seeds, preventing disease from spontaneously

occurring, or the concentration is high enough allowing forward progression of

fibrilization due to the presence of PrPSc oliogmers. Though research demonstrates that

the NDM can occur under cellular conditions, Eigen et al. questions whether the

mechanism can occur autocatalylitcally. Though nucleation provides a barrier to prevent

the onset of spontaneous disease, Egien et al. questions how the cellular environment is

H. Williams 4/28/14 able to provide the conditions needed to cause the formation of seeds when PrPC is the

thermodynamically favored state. According to Eigen et al., there has be skepticism on

whether the accumulation of PrPSc is enough to overcome the kinetic barrier needed to

convert PrPC to PrPSc or if there is a separate agent that could be catalyzing prion

aggregation (Eigen et al., 1996).

1.6 Chaperons Proteins: In more recent studies, researchers have adopted an alternative theory that

diverges from “protein-only” hypotheses, previously described by Prusiner and Lansbury.

It has been proposed that there are molecular chaperones that can regulate the

conformational transitions between the two PrP structural states. This is a radically

different proposal compared to the previous propagation models, which demonstrate that

prions facilitate their own conformational change through autocataylsis.

Molecular chaperons are proteins that assist the non-covalent folding or unfolding

of other proteins. Though their primary function is to prevent polypeptide chain or

protein subunits from aggregating, chaperones have been found to mediate the misfolding

of secondary and tertiary structures. Many of these chaperons are heat-shock proteins

(Hsp) because proteins have a tendency to aggregate more readily when under heat-

related stress. Heat-shock proteins attempt to prevent aggregation by facilitating protein

interactions. Chaperone proteins are typically activated due to infections, inflammation,

hypoxia, or other related stresses to a cell.

There is evidence to suggest that heat shock proteins facilitate the conversion of

PrPC to PrPSc conformation, specifically the chaperons, GroEL and Hsp104 (DeBurman

et al., 1997). This chaperone mediated conversion hypothesis of prions provides a new

understanding of the nature of PrP conversion. If chaperones help to elevate

conformational change of PrPC to PrPSc then this would change our understanding of the

H. Williams 4/28/14 nature of PrP intermediates. In doing so, researchers have shifted how prions propagation

is viewed as a disease, suggesting that the conversion of prions is more complex than we

originally thought.

Hsp104 is a protein-remolding factor that belongs to the AAA+ (Adenosine

triphosphatases Associative with diverse Activities) family. This family of proteins is

able to hydrolyze ATP to cause conformational changes. AAA+ proteins are found in all

organisms. These proteins are essential for many cellular functions such as DNA

replication, protein degradation, membrane fusion, signal transduction, and the regulation

of gene expression (Grimminger-Marquardt et al., 2010). As a molecular chaperone

HsP104 has a central role in the digestion of aggregates after heat shock (Grimminger-

Marquardt et al., 2010). However Hsp 104 has the capacity to catalyze protein

aggregation as disaggregase causing the misfolding of proteins. This disaggregation

activity has been linked to prion propagation. In yeast Hsp104 is able to cutting prionic

fibrils creating prion seeds (propagons). These seeds are then inherited to the next

generation of cells, when the parent cell divides its cytoplasm into two daughter cells.

This phenomenon is known as inherited prionic disease.

GroEL belongs to a family of chaperons that is found in a number of bacteria

including Escherichia coli. To function properly GroEL requires GroES as a cochaperone

protein to prevent the undesired aggregation of proteins (Martin et al., 1993). GroEL is

both structurally functionally similar to Hso60 and Hsp10 found in eukaryotes. Within

the cell the GroEl/ES complex accelerates the binding, conformation, and release of

various protein reactions. In order for cause a change in the conformation, GroEL needs

to bind to the substrate protein and then bind to ATP. The assembly of this complex then

recruits GroES, which causes the substrate protein to be fold. Hydrolysis of ATP and the

H. Williams 4/28/14 binding of a new substrate cause the protein to be released into the cytosol. However the

ATP-dependent release of GroEL in the absence of GroES results in aggregation rather

than favorable folding (Martin et al., 1993). Both GroEL and Hsp104 have significantly

influenced prion propagation and assembly of fibril aggregates in Prion disease.

2.0 Analysis

2.1 The Involvement of Chaperon Proteins in Prion Conformation. To asses the role chaperone proteins play in prion conversion, DebBurman et al.

examined seven major cellular chaperones GroES (Hsp10), Hsp26, Hsp40,

GroEL(Hsp60), Hsp70, Hsp90, and Hsp104 for their ability to activate the change in

conformation and physical states of other proteins (DebBurman et al,. 1997). First, they

Figure 6: Effects of chaperones of in vitro conversion of PrPC to PrPSc as a means of increasing Prion disease. (A) Conversion obtained after 24hrs either PrPSc or without PrPSc, with indicated chaperon proitens. (B) Same conversion preformed as (A), however contained preexisting PrPSc. (C) concentration dependent effects of chaperons in promitng conversion with untreated PrPSc. (D) SDS-PAGE of each reaction treated or untreated with Proteinase K (PK). (E)ATP dependence of GroEL-mediated conversion.

H. Williams 4/28/14 confirmed that none of the chaperon proteins could induce the conformational change of

PrPC into PrPSc in the absence of PrPSc (figure 6.A). In contrast chaperones can induce the

conversion of PrP proteins, in the presence of PrPSc (figure 6.B). This finding illustrates

that preexisting PrPSc is needed within the system to causes an infection (DebBurman

eal., 1997).

Currently it has been found that in order for PrPC to convert PrPSc, preexisting

PrPSc need to be slightly denatured in order to facilitate the transition between the two

conformations. Looking at the SDS-Page gel in figure 6.D, the denaturation of PrPSc (6M

Gdn-HCl pretreatment) caused a large accumulation of PrPSc. When this occurs,

proteinase K cannot degrade toxic prions. This is unlike PrPC, which can easily be

degraded by proteinase K. Knowing this DebBurman et al., wanted to test whether

chaperones were capable of converting undenatured PrPSc. It was found that many of the

chaperons were incapable of reproducing PrPSc (figure 6.B) in the presence of preexisting

untreated PrPSc monomers except for GroEL. GroEL was able to convert around 25-30%

and occasionally 50-100% of PrPC to PrPSc(Figure 6.B). Looking back at figure 6.D, they

found that GroEL was able to yield the similar PrPSc accumulations as the completely

denatured PrPSc (6M Gdn-HCl pretreatment). This demonstrated that GroEL was capable

of reducing the quantity of preexisting PrPSc required for detectable conversion and it

raised maximal number of conversions by ten fold (DebBurman et al., 1997). By

comparing different doses of GroEL and other Hsps, researchers found similar results

(figure 6.C), illustrating that GroEL has the ability to promote the conversion of untreated

PrPSc to a statistically significant higher rate of conversion. Interestingly, the activity of

GroEL was stunted by either the presence of GroES or lack of ATP.

H. Williams 4/28/14 PrP conversion was not observed in the absence of ATP or in the presence of

GroES. Looking at figure 6.E the SDS PAGE gel shows that in the presence of ATP and

the wild type (WT) GroEL, PrPSc was able to accumulate in the presence of Proteinase K.

However when GroEL contains a mutation that blocks the release of the substrate protein

(D87K) or when ATP was not present, a reduction was seen in ability of GroEL to

convert PrPC to PrPSc (figure 6.E). It is known that GroEL hydrolyzes ATP to release the

old substrate protein to bind to a new substrate. Thus if ATP or the binding domain is

unable to release the substrate protein, there is an inhibition in the function of GroEL.

The same issue occurs in the presence of GroES. The SDS-PAGE gel in Figure 6.D,

illustrates that when proteinase K is active and both GroES and GroEL are present, there

is a decrease in the accumulation of PrPSc. Comparing this result to GroEL is alone, there

is greater aggregation when GroES is not present. This is because in the absence of

GroES, GroEL is able to make errors, resulting in aggregation rather than favorable

folding (Martin et al., 1993).

To gain insight about the chaperone-mediated processes of GroEL, researchers

analyzed kinetics of the conversion of PrPC to PrPSc by evaluating protease resistance and

insolubility of PrPSc. It was found that in the presence of GroEL and monomeric PrPSc,

the rate of nucleation didn’t change from reactions where GroEL was absent(DebBurman

et al., 1997). However when PrPSc has been nucleated into oligomers and GroEL is

present, GroEL is able to increase the rate of the PrPSc forming a polymer. GroEL is able

to facilitate the conversion of PrPC by bringing PrPSc oligomers and PrPC in close

proximity. DebBurman et al.’s protease resistance analysis provided evidence that the

conversion of PrPC is a two-step process. the initial formation of nucleus which then

leads to a chaperone driven reaction which promotes PrPSc propagation. This data that

H. Williams 4/28/14 DebBurman et al. presents demonstrates chaperones have a significant effect on prion

propagation. Based on SDS-PAGE and percent conversion analysis, DebBurman et al.

had shown that GroEL has the ability to faciltate rate of the conversion of PrPC to PrPSc.

Although there were no other proteins that facilitated the conversion in the presence of

untreated PrPSc, Hsp104 demonstrated that it could convert PrPC when preexisting PrPSc

was slightly denatured.

For this reaction PrPSc was exposed to urea which is a milder denaturant because

Gdn-HCl could inhibit the function of certain chaperones. When PrPSc is partially

denatured chaperones only stimulate conversion to a small degree. However Hsp104 was

found to strongly stimulated conversion (Figure 7.A), contributing to 20-30% (and

occasionally more than 50%) of the total PrPC converted (DebBurman et al., 1997).

DebBurman et. al, then compared the activity of GroEL and Hsp104 to one another in the

presence of protienase K and a pretreatment of Urea. It was found from the SDS-PAGE

gel in figure 7.B that GroEL and Hsp104 could accumulate PrPSc under the same

conditions to a significantly higher level then the control (which did not contain any

chaperones). DebBurman et al. then tested the ATP dependence of Hsp104. Figure 7. C,

demonstrates Hsp104 does not depend on ATP. Even with ATPase-deficient Hsp104

mutants (KT218 and KT620), the conversion of PrPSc was the same as the wild type

(DebBurman et al., 1997).

The data that DebBurman presents not only provides significant evidence that

GroEL and Hsp104 affect prion conversion but they also illustrate that the state of which

prions are denatured can improve the ability of chaperones to facilitate conversion. This

data provides noteworthy insight about how the state of prions affects how chaperone

function. This demonstrates that many chaperones can have a primary role in prion

H. Williams 4/28/14 propagation. This give further insight into how prion diseases could be inhibited.

Targeting prion interactions within the conversion mechanism provides an answer in how

to prevent prion diseases, .

2.2 Potential Inhibition of Prion Structural Conformation Current research on prions has focused more on the medical application

preventing prion disease. . To prevent the growth of disease and the formation of fibrils,

the most logical step is to prevent the conversion of PrPC to its toxic PrPSc form. This can

be achieved by stabilizing PrPC, causing it to reduce it binding affinity for PrPSc. Some

antiprion conformational compounds that are currently known include sulfated glycans

(pentosan polysufate dextran sulfate), azo dyes (i.e. Congo red), dendritic polyamines

(i.e. polyamidoamine, polypropyleneimine, and poly-ethyleneimine), nitrogen

heterocycles (i.e. quinacrine and chloropromzine), and certain tetrapyrole molecules

(Mays et. al, 2012). These chemical inhibitors were first characterized in NMR. They

Figure 7: Combined effects of chaperones and partially denatured PrPSc conversions. (A) conversion obtained with partially denature PrPSc. (B) SDS-PAGE of representative conversion reactions obtained with GroEL (with and without Protienase K (PK) treatment). (C) SDS-PAGE presenting conversion reactions involving Hsp104.

H. Williams 4/28/14 were found to stabilize the PrPC conformation, and prevent the structural change of PrPC

to PrPSc (Hosokawa-Muto et. al, 2009). In order to determine a viable drug for prion

disease, researchers need to look at current compounds that have similar characteristics to

antiprion compounds.

In a recent study Methylene blue (MB), which is commonly used for treatment of

several medical conditions concerning memory consolidation and neuro-protective

effects, has been under investigation for its potential anti-prion characteristics (Cavaliere

et al., 2013). This FDA approved oral and I.V. administered compound is a heterocylic

aromatic phenothianzine compound that is water-soluble. It is able to pass the blood-

brain barrier, making it suitable to reach neuronal targets such as prions (Cavaliere et al.,

2013). This particular compounds has been shown to have anti-aggregating properties in

relation to Alzehimers Disease (AD), and has been shown to restrict protein chaperons

such as hsp70/hsp90 (Cavaliere et al., 2013). Knowing that this compound has potential

to provide treatment of neurodegenerative disease and prevent protein aggregation,

Cavaliere et al. tested whether this compound had an affect on the PrP aggregation

process.

In order to explore whether MB has the capability of interacting with the PrPC

ground state, interactions with monomeric PrPC were analyzed through NMR

experiments at ph 4.6 and 7.0. This allowed for researchers to map out the region of

where MB could interact with PrPC. From this NMR study they found that there were 20

residues located around the helix H1, H2, and the N-terminal region helix H3 that have

the potential to interact with MB. Out of the nine residues that showed chemical shifts

N246, K188, 1T191, T194, T195, and Q215, all fell in the active binding domain of PrPC

(figure 1). This high number of ionizable residues (1 Arg, 2 Lys, 1 His, and 5 Glu), could

H. Williams 4/28/14 potentially affect the attraction of the positively charged MB molecule within a pH of

4.6-7.0 (Cavaliere et al., 2013).

To understand the kinetic affects that MB has on the polymerization of prions, a

static light scattering analysis (SLS) was ran on three different prion species. Depending

on the PrP species, mutants, and experimental conditions used, prions could aggregate to

form different types of oligomers. In this experiment Cavaliere et al., demonstrated that

the MB disrupts the oligomerization of the prion species wt-OvPrP, wt-MoPrP,

PvPrP_H190A, and the mutant OvPrP_H190K1208M.

The influence of MB on the

process of oligomerization of these

pathways is indicative of the relative

decrease in SLS intensity. Static light

scattering is a technique in which the

intensity of the light scattered obtained

relates to the average molecular weight of

a macromolecule. The scattering intensity

can be used to calculate the root mean

square radius, which relates to the size of

the particle in the suspension. Thus a

decrease in light intensity would indicate

that MB would decrease the size of the oligomers able to form, restricting polymerization

of prion aggregates under thermal treatment (Cavaliere et al., 2013) . Here Cavaliere et

al. used SLS to measure the rate of oliogmerization in the presence of MB. When

compared to the control (no MB present), the oligomers showed a 40% decrease in SLS

Figure 8: SLS intensity indicating the amount of oligomerization overtime of Prion aggregates. The following black kinetic curve indicates an absence of MB. While the red curve illustrates a molar ration of PrP:MB equaling 1:1. Lastly, the blue curve indicates a molar ration of 1:3.

H. Williams 4/28/14 intensity when the ratio of PrP to MB is 1:1 and 1:3 (figure 8). Figure 8 demonstrates that

the ability of PrP to oligomerize in the presence or absence of MB. By comparing the

SLS intensities of PrP oligomerization without MB (black line) with the intensities of

different ratios (red and blue lines) of PrP to MB, it can be seen that MB is able to reduce

the amount of oligomeization. By restricting the number of oligomers forming prevents

or slows the process of fibrillation.

The last experiment preformed by Cavaliere et al. studied the affects of MB on

fibril formation. As a control, two prion fibril species, wt-HuPrp and wt-OvPrP, were

generated at pH 6.0 in the absence and presence of MB. Prion fibril formation was

monitored using Thiolavin T (ThT) spectroscopic assay. Figure 9.A and 9.B illustrates

the differences in binding curves of wt-HuPrP and wt-OvPrP at different molar ratios of

PrP:MB. In both species, in the absence of MB, the ThT signal indicated that there was a

sigmodial in protein aggregation and fibrilization (black line). In both cases, there was a

significant decreases in fibrilization when MB was present in any concentration (colored

lines) (Figure 9 A and B). This suggests that MB is able to suppress fibril formation. This

conclusion can be supported by TEM images, showing that both wtHPrP and wt-OPrP in

the presence and absence of MB (figures 9.C,D,E, and F). Figure 9.C illustrates the

fibrilization of wt-HuPRp in the absence of MB, while figure D illustrates the fibrilzation

in the presence of MB. The same was done for wt-OvPrP, where figure 9.E illustrated

fibrilization with MB and Figure 9.F illustrated with fibrillation. In both Cases (figures

9.D and 9.E), MB was able to inhibit the formation of fibrils. This alone demonstrates

that MB could prevent the onset of prion disease.

H. Williams 4/28/14

This article shows that although there are newly discovered prion inhibiting

compounds, there is much to be learned about their medical capabilities. This article

provides one FDA approved drug that has the potential of reducing the oligmerization

and fibrilization of prion aggregates. From this in vitro analysis, Cavaliere et al.

concludes that MB has highly valuable features that prevent prion disease and that their

results pave the way for further evaluation of MB in vivo studies and preclinical testing

(Cavaliere, et al., 2013) What also is important these findings is that the

pharmacokinetics and toxiocological properties of MB are known (Cavaliere et al.,

2013). MB has shown to overpass the blood-brain barrier and thus can target proteins on

neurons. By binding to residues located on the c-terminal tip of H2 helix, MB is able to

Figure 9: Inhibition of wt-HuPrP and Wt-OvPrP fibrilization mediated by MB. (A) Illustrates the affects of MB on the fibrilization of HuPrP at different ratios of HuPrP:MB. (B) illustrates the amount of fibrilization of OvPrP mediated by MB (1:5 ratio). Fibrilization was analyzed through a ThT-binding assay. (C) shows the fibrilization of wt-HuPrP in the absence of MB, while (D) illustrates fibrilization in the presence of MB. While (E) shows the fibriization of wt-OvPrP in the absence of MB and the presence with MB (F).

H. Williams 4/28/14 stabilize this structurally unstable region, leading to the prevention of unfolding and

aggregation of health PrPC proteins. MB was also found to significantly affects the rate of

oligomerization for each of the prion forms tested. This limits the amount of oligomers

formed during heat-induced unfolding processes, and completely suppress fibril

formation (Cavaliere et al., 2013). The following experiment provides strong evidence

for future studies in vivo to advance research in furthering clinical trials to develop a drug

or treatment to combat prion diseases.

3.0 Summary Prions are an infectious pathogen that is responsible for numerous fatal

neurodegenerative diseases. These PrP proteins are transmissible particles that lack

nucleic acids and seem to be composed of exclusively modified proteins. PrPC and PrPSc

are identical with respect to all chemical features however compared to the PrPC form,

PrPSc has a higher proportion of beta sheets rather than alpha helices. Each of theses

structural conformations have an effect on the way in which prion proteins cooperate.

These abnormal interactions result in the change in conformation of healthy PrPC to the

toxic isoform of PrPSc. Since toxic PrPSc isoforms are resistant to proteinase K, the build

up of PrPSc causes aggregation, forming amyloid fibers accumulate and create plaque in

brain tissue. The cause of the initial infection is still undetermined. It is believed to occur

through a spontaneous conformation or through the interaction of prions from different

species.

Though characterization of prion structure is important for the initial infection.

Even more important for disease prevention is the further characterization of the

conformational transition from PrPC to PrPSc toxic aggregates essential for the prevention

of the propagation of disease. The mechanism by which the PrPC protein converts to its

toxic conformation has evolved from many suggested hypotheses. The linear

H. Williams 4/28/14 autocatalysis model (heterodimer mechanism) stated that a single PrPSc protein is able to

change the conformation of another PrPC molecule (Eigen et al., 1996). The cooperative

autocatalysis, implied conformational change through symmetry conversion (Eigen et

al., 1996). Here, conformation of PrPC to PrPSc is compared to the cooperitivity of

hemoglobin through allosteric interactions, causing each subunit to aggregate to the

stable PrPSc form. Leading to the nucleation-dependent aggregation mechanism (NDM

mechanism), which illustrates how monomeric PrPSc aggregates into oliogmeric PrPSc

and then is polymerized to cause the formation of fibril structures(Eigen et al., 1996). By

comparing the nucleation model of growth to elements of the cell, current research has

suggested a the chaperone-assisted model, which illustrates that chaperone proteins

facilitate the nucleation or the polymerization of fibril formation. Unlike the previous

models the chaperone-mediated aggregation provides a more realistic method for how

prions can aggregate. Each of the autocatalysis methods seams to be lacking the

understanding that in cellular environment, where there are high incidences for

interaction and protein interaction. Knowing that chaperone proteins are able to facilitate

the prion interactions, questions arise concerning how can one inhibit prion conformation

and prevent prion disease.

Methlyene blue is a FDA approved drug that has been shown to support cognitive

function. Where it is able to pass the blood-brain barrier, making it suitable to reach

neuronal targets such as prions (Cavaliere et al., 2013). This particular compounds has

been shown to have anti-aggregating properties in relation to Alzehimers Disease (AD)

and restricts protein chaperons such as GroEL and Hsp104 (Cavaliere et al., 2013).

Knowing that this compound has significant nuerological applications in terms of treating

neurodegenerative disease and protein aggregation, Cavaliere et al. wanted to test

H. Williams 4/28/14 whether this compound had an affect on the PrP aggregation process. Based on the results

found from Cavaliere et al., MB is able to cause inhibtion of prion conformation by

stabilizing the PrPC conformation in vitro. However if these compound is going to be

used as a drug to treat prion disease, there needs to be in vivo testing that provides

evidence that MB is able to first maintain its function within the microenvironment.

Secondly it must demonstrates that prions have a higher affinity for MB then they do for

chaperon proteins. If MB is able to simultaneously stop prion interactions and chaperone

function, MB and other similar compounds, could inhibit the propagation of prion

diseases.

H. Williams 4/28/14 4.0 The Next Experiment: Analyzing the effect of Chaperon Proteins on Antiprion Inhibition Compounds PrPC is involved in the neuroprotective response of the brain. In cases such as the

cellular oxidative stress response, prions protect against cerebral ischaemia and traumatic

brain injury. Prions can’t be knocked out as a therapeutic treatment due to their role in

regulating presynaptic copper concentrations, calcium homeostasis, as well as activation

and proliferation of lymphocytes, astroytes, and signal transduction (Roettger et al.,

2013). PrPC is essential in interacting with synaptic proteins, cell adhesion molecules, and

apoptosis regulator Bcl-2 proteins. PrPC is able to activate these signally pathways by

activating protein kinases such as cAMP-protein kinase A to ensure neural survival

(Roettger et al, 2013). In order to prevent disease while maintaining PrPC function,

conversion of PrPC to PrPSc must be inhibited.

Current research has been explored concerning prion inhibition by targeting the

prion binding domains that facilitates conversion of PrPC to PrPSc. From what was

previously discussed, Methelyene Blue (MB) has shown promising results that suggest

MB has the potential to inhibit both prion oliogmerization and fibrilization. By inhibiting

the ability of prions to form oligomers and prevent the formation of fibril structures, MB

allows for the suppression of prion related diseases. As previously discussed, prion

disease is related to the formation of fibril structures that are incapable of being broken

down by protienase K. This resistance to proteases causes an accumulation of plaque in

the brain causes the death of neurons. By preventing this process from occurring,

compounds able to occupy the prion binding domains of PrPC could provide therapeutic

value to prevent the growth of protease-resistant aggregates. Though MB has been shown

to have antiprion capabilities, tests have only been done in vitro. There has yet to be an in

vivo study that measures the affects of how the microenvironment affects MB’s ability to

H. Williams 4/28/14 prevent prion aggregation. Prions may have a higher binding affinity for chaperones

rather than MB. It has been found that a majority of chaperone proteins bind to the same

binding site as MB (Moran et al., 2013). The relative binding affinity of prions to

chaperones, in contrast to other proteins within the cellular environment, could result in

the repression of MB as an inhibitor. In vitro studies have demonstrated that MB has the

capabilities to prevent accumulation of prion aggregates. In vivo experimentation could

further validate that MB has the characteristic need to become a preventative treatment

for prion diseases.

4.1 Hypothesis: It is hypothesized that MB would be able to inhibit prion aggregation even in the

presence of chaperones in vivo. If MB was analyzed through an in vivo Thiofalvin T

assay (ThT-binding assay) then the results should indicate no fibrilization within the cell

because MB is able to target prion proteins directly by blocking the PrPC binding site for

PrPSc. This would prevent the nucleation step from occurring, which would restrict the

rate at which chaperones could facilitate oliogmerization and fibrilization.

4.2 Methods: This experiment would be carried out in a similar manor as the in vitro study

conducted by Cavaliere et al. In order for the results to be comparable with Cavaliere et

al., the same prion species will be tested from the Cavaliere et al experiment. Full length

PrP from sheep OvPrP (A136 R154 Q171 variant) , mouse PrP (MoPrP), and human PrP

(HuPrP) would be amplified through PCR using cDNA or genomic DNA. The PCR

product will be placed into a pET22bC vector and the plasmids would then be transfected

into mice using an i.v. or direct injection into the brian. To test for successful

transfection, an immunofluorescence assay will use fluorescently tagged antibodies with

specific binding to each prion species. A mouse from each prion plasmid transfection will

H. Williams 4/28/14 be sacrificed and brain tissue will be harvested. Subjecting brain tissue to these specific

antibodies will mark the eah type of PrPC, because healthy PrPC is attached on the surface

of neurons thorugh the GPI anchor. If the microphotography indicates fluorescence on

the surface of cells this would indicate that successful transfection of prions had occurred.

DAPI dye will indicate the location of the nucleus to reference where the prions are

located in relation to the cell.

Since there are four different types of prions being tested, 4 different groups of

mice would be used, each injected with a different plasmid. Each of these groups would

then be split into two different groups, a control group where no MB was administered,

and an experimental group where MB was injected into body. Once MB and the plasmids

were injected, mice will killed and their brain tissue will be subjected to an in vivo ThT-

assay to measure the amount of prion protein aggregation occurring in the presence and

in the absence of MB over a 30 day period. Mice from each group of transfections would

be sacrificed every 100hrs to perform a ThT-assay to determine the amount of

fibrilization present. Typically a ThT-assay is meant to visualize and quantify the

presence of fibrilization of misfolded protein aggregates both in vitro and in vivo. A

comparison between the ThT-assays of the control and experimental groups, would allow

one to compare how fibrilization was occurring between each strain of prion with or

without MB present. The results of this experiment could lead to confirmation that MB

has the ability inhibit prion propagation in the cellular environment, or it would illustrate

that something in the cellular environment restricts MB’s ability to inhibit prion

conversion.

4.3 Outcomes and Interpretations: If MB was able to prevent fibrilization the results of the ThT-assay could

resemble what is found in figure 10.A. If MB was able to prevent fibrilization in vivo this

H. Williams 4/28/14 result could provide grounds for future clinical and human testing. However MB were

unable to restrict the conversion of prions, a second experiment would be conducted

using knockouts of either GroEL or Hsp104 chaperones to determine whether chaperones

were causing MB to become inhibited. Figure 10.B illustrates the possible results that

would be characteristic of this conclusion. A large sigmodal increase in ThT fluorescence

illustrates fibrilization is present (Cavaliere et al., 2013). As previously described,

chaperone proteins have been found to facilitate oligomerization and fibrilization of

prions and bind to the same active sites as MB (DebBurman et al., 1997). Chaperones

could compete with MB for prion binding sites. Prions may have a stronger binding

affinity for chaperones than MB. By knocking out potential prion chaperones, one could

learn whether prions have a higher binding affinity to chaperones or another cellular

component.

A second ThT-assay would have to be done using chaperone deficient mice, to

see if prion fibrilization is affected. If no fibrilization is present then this would suggest

MB was able to inhibit prion-prion interactions in the absence of chaperones. Figure 11.A

illustrates evidence that would characteristic of this result. When MB treatment in the

absence of chaperones, the amount of fibrilization should be significantly lower than

when mice are treated with MB. Figure 11.A indicates that each prion species shows

lower ThT-flouresence when MB is present compared to the control (when MB is absent

(purple line)). This would demonstrate that the decreased fibrilization is due to prions

having a higher binding affinity for MB. If fibrilization does occur then there is likely

something in the cellular environment that MB has a higher affinity for. Thus inhibiting

MB’s ability to interact with PrPC. This would indicate that further research is needed to

understand how MB is interacting with the cellular environment and whether changes in

H. Williams 4/28/14 conditions (in vitro to in vivo) affect the prion binding affinities for MB. However

knocking out chaperone proteins could be fatal for mice and may cause death due GroEL

and Hsp104 can mediate other protein interactions. If this was the case an

immunflouresence assay could be a useful substitute experiment to illustrate what protein

interactions are occurring. MB is a fluorescent dye, therefore by fluorescently tagging

GroEL, Hsp104, and PrPC one could determine protein interactions based on

microphotograph. (A concept map of the future experiment is included in the appendix).

Figure 10: Hypothesized results for in vivo ThT-assay with treatment of MB. (A) Illustrates possible result where MB is able to reduce the fibrilization in vivo. In this case the all three-prion species should show low levels of ThT fluorescence, indicating little fibrilzation was occurring. A positive control where no MB was administered was added to contrast the reduction in fibrilization. (B) Illustrates the possible result of MB not being able to reduce PrPSc fibrilization. In this case the amount of fibrilization would be equal or slightly less to the amount of fibrilization when MB is not administered (purple line).

H. Williams 4/28/14

B

Figure 11: Hypothesized results for in vivo ThT-assay with treatment of MB and knockouts of GroEl and HsP104. (A) Illustrates possible result where MB is able to reduce the fibrilization in vivo. In this case the all three-prion species should show low levels of ThT fluorescence, indicating little fibrilzation was occurring. A positive control where no MB was administered was added to contrast the reduction in fibrilization. (B) Illustrates the possible result of MB not being able to reduce PrPSc fibrilization. In this case the amount of fibrilization would be equal or slightly less to the amount of fibrilization when MB is not administered (purple line).

A

B

H. Williams 4/28/14 Bibliography: Cavaliere, Paola, et al. "Binding of methylene blue to a surface cleft inhibits the oligomerization and fibrillization of prion protein." Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1832.1 (2013): 20-28. Eigen, Manfred. "Prionics or the kinetic basis of prion diseases." Biophysical chemistry 63.1 (1996): A1-A18. Harrison, Paul M., et al. "The prion folding problem." Current opinion in structural biology 7.1 (1997): 53-59. Grimminger‐Marquardt, Valerie, and Hilal A. Lashuel. "Structure and function of the molecular chaperone Hsp104 from yeast." Biopolymers 93.3 (2010): 252-276. Hosokawa-Muto, Junji, et al. "Variety of antiprion compounds discovered through an in silico screen based on cellular-form prion protein structure: Correlation between antiprion activity and binding affinity." Antimicrobial agents and chemotherapy 53.2 (2009): 765-771. Laurent, Michel. "Autocatalytic processes in cooperative mechanisms of prion diseases." FEBS letters 407.1 (1997): 1-6. Martin, Jörg, et al. "The reaction cycle of GroEL and GroES in chaperonin-assisted protein folding." Nature 366.6452 (1993): 228-233. Mays, Charles E., et al. "Prion inhibition with multivalent PrP< sup> Sc</sup> binding compounds." Biomaterials 33.28 (2012): 6808-6822. Roettger, Yvonne, et al. "Immunotherapy in prion disease." Nature Reviews Neurology 9.2 (2013): 98-105. Taguchi, Yuzuru, and Hermann M. Schätzl. "Identifying critical sites of PrPc-PrPSc interaction in prion-infected cells by dominant-negative inhibition." Prion 7.6 (2013): e1003466-1. Steele, Andrew D., et al. "Prion protein (PrPc) positively regulates neural precursor proliferation during developmental and adult mammalian neurogenesis." Proceedings of the National Academy of Sciences of the United States of America 103.9 (2006): 3416-3421. Watts, Joel C., and David Westaway. "The prion protein family: diversity, rivalry, and dysfunction." Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 1772.6 (2007): 654-672. Wills, Peter R. "Autocatalysis, information and coding." Biosystems 60.1 (2001): 49-57.

H. Williams 4/28/14 5.0 Appendix:

H. Williams 4/28/14

prion infection dynamics- an analysis of conversion mechanisms to characterize propagation of prion...

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