Week 2.1.2 Prion

Question 1

Objectives

Compare and contrast the biophysical differences between the two isoforms of Prion protein
Describe and evaluate the structural and spectroscopic data available on Cellular and Scrapie Prion Protein.

How many isoforms of prions are there?

What are the characteristics of them?

Answer 1

1 PrP Cellular (found in all mammalian brains)
•Ubiquitous in neuronal cells
•High a-helical content (identified by solution NMR)
•Soluble
•Susceptible to proteolysis
Anchored by GPI protein on the lipid bilayer

2 PrP Scrapie
•Present in the infected cells
•High b-sheet content
•Insoluble
•Resistance to proteolysis

Unusual, because there were two structural forms of Prion protein-How can it encode two structures?

Question 2

Structural data of Scrapie isoform

Which two techniques identified this?

Why is each technique used?

What are the spectra results for PrPc? and the PrP Sc?

Answer 2

Rich in beta sheet (43%) 2 techniques identified this: circular dichromasm and infrared spectroscopy

1. Circular Dichromasm: which is very good at giving the percentrage of 2’ (secondary) structure
2. Infrared spectroscopy: which is good at looking at the 2’ (secondary) structure of insoluble proteins.

PrPC Helical, amide stretch 1660 cm-1

PrPSc b sheet, stretch at 1630 cm-1

Question 3

Structural data on Scrapie PrP

Why is there no NMR result of Scrapie PrP?

Are the molecular-level details of Scrapie confirmed?

Answer 3

• Insoluble therefore no solution NMR
• Molecular-level details of scrapie sructure not confirmed

Question 4

Scrapie:
Forms short amyloid fibres-Prion-Rods

How can we image them?

Answer 4

Transmition electron microscopy (T-EM), gives you nanometerscale images Prion Rods ~3-8 nm thick

Fiborous short prion rods are short and stumpy and have a slight twist in them

This is a halmark of misfolding disease (aggregated fibres)

Question 5

Scapie: Forms short amyloid fibres-Prion-Rods

What technique can be used to identify the secondary structure part that forms beta-sheet?

Why is this technique used?

What exchange does this technique exploit?
What does it tell you?

Answer 5

• Technique called **deuterium exchange **(H/D)
• The prion protein has a polypeptide backbone (with peptide bonds i.e amides)
• Each amide has a proton attached to it
• The proton (i.e H) will come off (layby) - for an instance and is replaced by a proton from water
• Exchanges with water surrounding the protein (this happens all the time and rapidly in solution)
• **We use this exchange property to identify the secondary structure **
• because if the aide is hydro bonded with carbonyl then the proton ceases to be layby

HOW DO WE MEASURE THIS EXCHANGE ABILITY

We put the protien into D2O rather hand H2O

Chemically D2O behaves the same but will not be picked up by NMR as proton NMR is used

When it exchanges with D (assuming there is no hydrobond there), it becomes a deutrated amide, it becomes silent and not visible in NMR spectrum.
So you can see which amide regularly exchanges and which parts are protected and which ones form beta sheets (2’ structure). From that data, we know that from residue 169-225 are in beta sheets, the rest is not amide protected – has little secondary structure.

Question 6

Another experiment using Spin labelling- gives similar results to the H/D

Gives the same core beta-sheet residue

What is the beta sheet structure fibre orientation?
Are the sheets formed within a single molecule?

Answer 6

Models of the structure of these fibres.
160-220 stretch and form beta sheets and each stack with its neighbour- described as cross beta structure because the fibre orientation is into the board and beta sheets are at 90 degrees to that.
Thus, if you have a fibre, and the beta sheets form at 90degrees.
The sheets are not formed within a single molecule- beta sheets normally form in the same protein and we say they are intra-molecular.
However, with fibres the beta strands form H bonding between adjacent molecules- intermolecular.

Question 7

Cellular form —- structure identified

Scrapie form —— models based on experimental data not structure

Question 8

Epitope mapping: to try to understand the structure of the Scrapie isoform

What do the antibodies recognise?

Answer 8

Some antibodies that recognize the normal form of protein and the Scrapie form of protein. Antibodies bind to protein in cellular and same bond in Scrapie- argument that those regions are the same and where its different you can argue that it’s a different structure.
•In particular helix B and C have same epitope mapping in PrPSc and PrPC. While the rest of the molecule gives very different mapping patterns

•Molecular-level details of scrapie structure are still not confirmed

Question 9

Glossary:

Defining and the meaning of words, defining terms that are important

Define the following terms:

**Prion
Scrapie
PrP-Sc
PrP-C
Amyloid/fibre
Mutated prion protein
Misfolded prion protein **

Answer 9

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• <!--[endif]-->Prion = proteinous infectious particle (usually found in mammalian species but also associated with TSE (yeast) but can apply to any protein that has replication/ transmissible properties, often oligomeric protein aggregate)

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• <!--[endif]-->Scrapie = A TSE(prion disease) found in sheep and goats

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• <!--[endif]-->PrP-Sc = Misfolded mammalian prion protein (not just sheep but in ANY mammal)

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• <!--[endif]-->PrP-C = Cellular mammalian prion protein

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• <!--[endif]-->Amyloid/fibre =Long fibrilar protein aggregates with ordered cross-beta-sheet structure found in plaques

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• <!--[endif]-->Mutated prion protein =Prion protein with an altered primary sequence

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• <!--[endif]-->Misfolded prion protein = Prion protein with a non-native possible pathogenic structure

Question 10

2. 4: Mechanisms of prion propagation; for transmitted and inherited forms
   - Mechanisms of how prion can enter a host and copy themselves

Objectives

• Discuss the seeded polymerisation reaction and the conversion of PrPC to PrPSc
• Describe amyloid fibre growth – how do these fibres form
• Explain how a protein with the same primary sequence can have two structures
• Illustrate how mutations in the primary sequence of PrP might cause the production of PrPSc
• Relate prion disease strains to fibre structure
• <!--[if !supportLists]-->§ <!--[endif]-->Relate prion disease strains to fibre structure

Question 11

Prion Propagation and fibril growth

Explain the method of template assisted conformation switch?

Answer 11

Concept of template assisted conformational switch; proteins copy themselves.

• Scrapie form passes the blood brain barrier and acts as a seed.

§Cellular form of protein recognizes the misfolded form binds together and the act of binding the misfolded (scapie form) induces normal (cellula) form (not to mutate) but to misfold.

§Scrapie plaques present and large fibrous collections of aggregated protein, toxic to neurons

Question 12

What is the template assisted conformational switch of PrP-C –> PrP-Sc analogous too?

Answer 12

Sometimes described as the misfolded form acting as seed- same as in crystallography

• Beaker of copper sulphate, clear blue solution
• Add one crystallised piece of solid copper sulphate
• ~24 hours, beaker full of crystals
• **Process is analogous to structural switch from alpha helica to beta sheet rich protein in prions **

Question 13

Mechanism of Prion Replication

In the infectious disease?
In the inherited disease?

Answer 13

One infectious particle gets to your brain and replicates in your brain by inducing normal prion protein to change its structure- how protein can infect and propagate in an animal or human.

• *Infectious disease:** template assisted conformational switch
• *Inherited disease:** mutations in the primary sequence promote misfolding

Question 14

Amyloid Fibres grow from ends recruiting more monomeric PrP

Answer 14

At a strucutural level: Amyloid Fibres grow from ends recruiting more monomeric PrP

End of fibre has hydrophobic residues exposed, it becomes unstable. These act to induce the misfolding of the normally folded protein.

Question 15

What structure is amyloid fibre?

What does it recruit?

How does the misfolded spread throughout the brain?

Answer 15

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§ <!--[endif]-->Amyloid fibre with cross beta structure, end of the fibre, its exposed and the side chains are hydrophobic,

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§ <!--[endif]-->Because its exposed to hydrophobic environment- it becomes unstable (because its hydrophilic on its surface) and thus unfolds and turns inside out and makes contact with hydrophobic residues.

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§ <!--[endif]-->Now attached itself to fibre and now new end of fibre and now another correctly folded PrP molecule will come up and as a consequence, at the other end the fibre is now growing.

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§ <!--[endif]-->Over time they extend and get to a certain length and then snaps in two and now you have 2 fibres.

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§ <!--[endif]-->So twice as much seeds ready to grow fibre and this is a constant cycle. Process of fibre growth, fragmentation and growth again. This is how the misfolded (scrapie isoform) spreads throughout the brain

Question 16

How can you monitor fibril growth?

What does the graph look like?

Answer 16

In a test tube:

§You can monitor this fibre growth using a dye that binds to fibres and fluorescence- more dye fluoresces the more fibres you have. (ThT, thioflavin)

§Can take your solution of monomeric prion protein where you have no fibres, depending on concentration of prion solution, eventually sample will start to progress.

§Lag phase-slow ^ but once you have the seeds, rate increases- rapid elongation and then eventually you get no more increase in fibres because all the cellular protein in test tube is converted.

§No more correctly formed prion protein is left. This happens in test tube but similar thing happens in vivo.

Question 17

**Fibril growth **

How can you demonstrate the seeding process?

Answer 17

In a test tube you can demonstrate the seeding process by taking a 1% extract from a previous experiment with fibres in it - adding it to a fresh solution

This shows immediate formation of fibres without any lag phase

Question 18

Prion Propagation

Two models proposed for nucleation propagation:

Probably a mixture of the two is a reality, but you can conceive two scenarios

What are the two models?

Answer 18

1. Autocatalytic nucleated polymerixation:

**2. Non-catalytic nucleated polymerization: **

Question 19

Mechanism of inherited prion disease

What is the cause of inherited prion disease?

Answer 19

One of the key first observations between prion protein and the diseases is that the inherited form we looked at the primary sequence and found mutations in that sequence – a particular mutation for insomnia, GSS.

Question 20

Prion protein structure and familial diseases

Mutations in more detail

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• <!--[endif]-->Familial prion diseases exhibit mutations in prion protein primary sequence.

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• <!--[endif]-->A number of these mutations destabilise the packing of the b-sheet on to helix B and C

Question 21

Familial prion diseases exhibit mutations in prion protein primary sequence

What are the mutations in FFI? what does this result in?

What are the mutations in fCJD? whaat does this result in?

Thr183-to-Ala mutation what does this result in?

Answer 21

FFI has a Asp178-to-Asn mutation
•Loss of Salt bridge Asp178 -Arg164
•178 (Helix B) to 164 (Sheet 2)

**fCJD has a Met129-to-Val and Asp178-to-Asn mutation **
•Loss of H bonding network between 129 and 164
• 129 (Sheet 1) to 164 (Sheet 2)

•Thr183-to-Ala mutation
•Loss of a hydrogen bond between 183 (Helix B) to 162 (Sheet 2)

Question 22

Familial Mutations

Although the PrP-C structure suggest some mutation destabilize PrPC. Folding stability measurements suggest no difference.

What may be the affect of mutation?

Are all the mutations found within the structured doman of PrP-C?

Answer 22

•rather than destabilize PrP-C these mutation may stabilize the intermediate or/and the Scrapie isoform

• In addition- Not all mutations are found in the structured domain of PrP-C not between 126-231
• eg A117V, P105L, P102L

Question 23

Familial mutations effect
on Free Energy

Is PrP-C –> PrP-Sc energetically favourable?

Give example of mutations in
C-terminal half
N-terminal half

Answer 23

Exothermic reaction

Energentically favourable to form PrP-Sc form

Examlples

• *Mutations in C-terminal half: **
  e. g Asp178-to-Asn

**Mutations in N-terminal half: **

eg P105L or P102L or A117V

Question 24

Strains of Prion diseases: Prion species barrier

What is different between prion diseases?

Give an example of infective and familial forms that are different?

Answer 24

§Different mutations in PrP produce different pathologies in inherited (and infectcious) prion diseases.

§Idea of strains came up due to different forms

For example:

§BSE transmitted to sheep results in a prion disease different from Scrapie

§Kuru and vCJD both of which are transmissible in humans have different pathologies

§ Differences in strains are explained as differences in the structure of the misfolded aggregates

§Have different symptoms , pathology is different, appearance of plaques differ.

§KURU and CJD transmissible in humans but again ^^

Question 25

Synthetic prion strains

Such as those grown in mice

What were the different conditions?

Answer 25

Work with mice that they injected cerebral inoculation- inject misfolded prion onto brain, mice gets sick. Prion protein generated recombinately under diff. conditions form diff. fibres.

Different Synthetic prion strains with different fibre structure

Fibres were grown under different conditions e.g. NaCl, temperature, pH

have different incubation periods and pathology in mice

mice incubation times relates to stabilities of fibre.

Question 26

Parent-todaughter seeding and prion inheritance: seed will influence morphology

Is the structure retained from parent to progeny?

Answer 26

Concept of structure of the fibre remaining and also being replicated in the mouse your injected, structure is retained in brain and spreads that form of misfolded protein.

Key experiment showed the morphology of parent seed dictated morphology of daughter fiber- (Tycho)

Analogues to strains of prion disease in CJD

Question 27

**The Species Barrier: **

What was believed initially regarding transmissibility of BSE to humans?

How long has Scrapie been around - has it been transmissble?

Answer 27

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§ <!--[endif]-->It was initially believe that BSE in cows could not be transmitted to Humans

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§ <!--[endif]-->Scrapie in sheep had been around for hundreds of years without evidence of transfer to humans

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§ <!--[endif]-->Studies show that initial infection from one species to another results in a delay in the incubation time for the disease

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§ <!--[endif]-->Species Barrier believed to be caused by differences in primary sequence between mammals

Question 28

Summary:

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§ <!--[endif]-->PrP^sc can act as a seed or template in the conversion of PrP^c to PrP^Sc

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§ <!--[endif]-->Formation of amyloid involves a nucleation followed by elongation

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§ <!--[endif]-->Mutations in the primary sequence may may alter kinetics of misfolding destabilises PrP^c or stabilize the beta form

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§ <!--[endif]-->Phenomena of Prion strains in TSE’s have a structural basis