Week 2.3 Prion Flashcards
2.5 PrP trafficking in the cell
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- In vivo* Conversion of PrPC to PrPSc
- In vetro* Conversion Synthetic Prions
Prion folding intermediates and oligomers: Copper and the Prion protein
Objectives:
- Appreciate the trafficking and propagation of PrPSc at the cellular level
- Describe synthetic prions and their importance in the protein only hypothesis
- Describe possible folding pathways between PrPC and PrPSc and intermediates
- Appreciate PrP oligomers and cell toxicity
- Report the role for Copper in PrPC biology
Prion protein trafficking
What happens in the production of PrP-C in the ER?
What does the PrP-C travel through to get to the plasma membrane?
How is the PrP-C anchored to the lipid rafts?
What happens at the plasma membrane?
PrP-C is syntehsized in the ER:
- cleavage (removal) of cell signalling peptide
- N-linked oligosaccharides
- disulfide
- attachment of GPI
Travels through Golgi to the plasma membrane-anchored to Lipid rafts via GPI
Where it will be exocytosed onto the plasma membrane
Prion Protein-endocytosis
What can bind to make endocytosis more effective?
How can the prion protein be endocytosed back?
- Once it’s on the plasma membrane, it endocytosed, when it binds to copper ions it makes endocytosis more effective, forms a bud which buds off and you get a vesicle.
- Prion protein can come up from the golgi, get on the plasma membrane- but after a while of hanging on the Plasma Membrane, it can be endocytosed back into the cell in these vesicles.
In vivo: propagation of prions In vivo **Conversion of PrPC to PrPSc<br></br>Cell-to-Cell propagation of Scrapie-PrP **
Proppogation in the cell or cell surface?
Cell to cell transfer?
Where does PrP-C conversion (misfolding) occur initially?
What may also have a role after initial conversion?
PrP has a GPI anchoring to the membrane surface, where Initial prion conversion seems to occur first, perhaps in lipid rafts.
Intracellular compartments may also have a role in this; possibly by accelerating conversion after PrPSc has been endocytosed.
What about transfer of prion material from cell to another cell
**Cell to cell transmission of PrP-RES (Scrapie) **
**How does it get to another cell?
**
- Idea here is that little exosomes: where misfolding prion protein attaches to lipid raft that forms a part of the lipid bilayer.
- These can be budded off the lipid membrane and then released to extracellular medium. They can float around and then they come across another cell and because much of it is lipid, the lipid attaches itself to the next molecule in the membrane.
- Then you have infection (in terms of misfolding of the protein) spreading from one cell to its neighbor, which will spread throughout the brain.
Prion Protein misfolding at the cell surface
Where does initial prion conversion occur?
What possibly accelerates the conversion?
Is the toxic action on surface of plasma known?
What are the two ideas?
- Initial prion conversion seems to occur first at the plasma membrane, perhaps in lipid rafts.
- However, intracellular compartments may also have a role in this, possibly by accelerating conversion after PrPSc has been endocytosed.
No one knows if the toxic action is on the surface of plasma membrane –
- Very popular hypothesis is theses misfolded prion fibers grow on surface of membrane and as they grow they perhaps disrupt the membrane/ puncture membrane and all these processes perhaps disrupt integrality of plasma membrane and you get cell death as you disrupt homeostatic of cell.
- Another idea is that once endocytosis happens, the protein might enter cell and react with mitochondria and break up sub cellular membrane compartments rather than the plasma membrane on the surface.
Synthetic Prions confirms prion hypothesis
In vetro Conversion of PrPC to PrPSc
Synthetic prions produced. 2005 they managed to create fibers infectious enough to cause disease in mice.
How did they do this?
They took recombinant protein and added urea to destabilize the helical form –on unfolding
Destabilize the native form with 2M urea (disulphide remains)- so protein is verging on unfolding.
Promote self-association: by adding high levels of NaCl (help aggregation process, to assemble together)
Promote Amyloid growth: by shaking
They shook samples vigorously for a few weeks, generated fibres and those fibres were inoculated into the mice
Can infect mice via cerebral inoculated, mice didn’t get sick till 1.5 years…
Inoculating recombinant prion- a prion that has never seen an animal which is generated in the test tube using E.coli to over express the protein.
You can get that synthetic protein to mimic and then use to infect animals with it.
Synthetic Prions confirms prion hypothesis
Better ways to do it, what is it called?
How do they do it?
Why do pepole doubt?
But what was done so that there couldnt of been any left in the solutoin?
**PMCA: Protein misfolding cyclic amplification **
A Cell-free method for formation of misfolded prion protein with prion infectivity
They take recombinant fibers, but found that if they took fibers from a hamster brain and added it to recombinant protein (from test tube) then shake it and weeks later turned all the recombinant alpha helical monomeric cellular protein into Scrapie form- causing the whole test tube to misfold.
Mouse injected and got sicker quicker.
Doubters will say that ‘yes of course because you took the recombinant from an animal’
But this is what they did to it…
Took 10% added to 90% recombinant and then took 10% of that and added to monomericà
Here at start you have 5% animal derived prion protein–à .0>05%…0.0005….
Did this 15 times and diluted the small amount of animal fibers so much that there could not have been one single molecule left in solution.
Of their 1L there was maybe 1 molecule and only took a fraction of a microliter.
Chances of animal derived protein were zero.
Yet, fresh fibrous sample was highly infectious.
What is the infectious form?
Oligomers not Amyloids are most infectious and toxic
Amyloids my be inert reservoir while small oligomers are neuro-toxic
The most infectious prion particles: Silveira et al Nature 2005
Partials as small as 5 PrP molecules can be infectious
Most efficient initiators of TSE disease Non-fibrillar particles 14-28 molecules in size
How is PrP-Sc Neuro-Toxic ?
•Accumulation of misfolded PrP on membrane surface may compromise the integrity of the membrane and is thus cytotoxic
Protein misfolding pathway and folding intermediates
What is used to identify stability of unfolded protein?
Pathway where cellular helical form –> fibrous form
There must be a folding intermediate between the transitions
Protein Folding (funnel) Energy landscape: unfolded protein unstable on top and fold to stable folded state – more stable
Folding pathway
What are the three pathways? *Viles didnt go through it you can read through it
•Pathway 1: PrPb is a folding intermediary to a more stable PrPC.
–Nucleated Polymerisation
•Pathway 2: Formation of PrPb is an off pathway and may contain no structural elements from PrPC
•Pathway 3 : PrPC is a kinetically trapped intermediate in the folding pathway of PrPb. The infectious prion “seed” lowers the barrier of the conversion of PrPC to PrPb
–Template Assisted
Conversion of PrPC to PrPSc:
Changing the conditions to see what change this has on the structure
CD Folding studies on PrPC with pH
If you drop the pH to pH 4 what happens? how can we monitor this?
At pH 7, the prion protein is largely helical
Strong negative CD band at 222 nm
At pH 4, the protein converts
to a b-sheet rich conformation.
Negative CD band at 217 nm, will form oligomers and then amyloid
Change pH and convert to helical protein rich in beta sheet.
Monitor by circular dichromasm.
Stable at pH4- folding intermediate and fully unfolds at lower pH and that folding intermediate, has strong beta sheet character.
Conversion of PrPC to PrPSc
CD folding studies: conversion of PrP-B
What is used to characterize on a per residue basis?
Able to monitor structural change?
Characterize on a per residue basis: solution NMR. Each one of the peaks represents amino acid in polypeptide sequence.
e.g – 150 amino acids in prion protein, there is 150 peaks.
Monitor structural change for amino acid at each pH.
- *Flexibility of the backbone:
- by NMR relaxation measurements **
What does it recognize?
Key residues in loop region associated with familiar mutation that have additional flexibility.
So already an unstable part of the protein.
Those mutations on the prion protein house area of flex
Oxidation of prion protein:
What is oxidized?
What triggers fiber formation?
Oxidative markers are a hallmark of prion diseases
PrP-Sc scrapie isolates show most of the Methionines are oxidized to sulphoxide
- prion diseases linked with oxidative stress: is it the protein becoming oxidized and that is triggering diseases?
