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Prion Diseases: Cellular Trafficking & Copper (OXIDATION OF PRION…
Prion Diseases: Cellular Trafficking & Copper
SYNTHETIC PRIONS: confirms prion hypothesis
destabilize native form w/urea, promote self-association w/NaCl making protein less soluble, use agitation to promote amyloid growth -> can slowly but significantly infect mice via cerebral inoculation
PMCA: Protein Misfolding Cyclic Amplification
original 1%-soln fiber seed from infected animal converts test tube to all insoluble infectious fibers
continue successive 1% seeds into new tubes, completely diluting out the original animal-derived misfolded fibers
statistically, final inoculation of healthy animal contains only fibers produced in the lab and none from an animal
ability to cause infection solely from synthetic fibers
supports protein-only prion hypothesis
CELLULAR TRAFFICKING
From ER to Cell Surface
Synthesized in ER
Modifications: cleavage of signal peptide, N-linked oligosaccharides, disulfide bonds, attachment of GPI
Travel thru Golgi to plasma membrane -> anchored to cholesterol-rich lipid rafts via GPI
Cycling
protein's half-life is 7 hours, doesn't last long in cell
Endocytic cycling via formation of endosome, then lysosome
Cell-Cell Transmission/Propagation
Budding
: exosomes containing misfolded PrP-Sc transferred to another cell and endocytosed
Accumulation in Brain
PrP-Sc with GPI anchor
(wild-type): accumulation is granular/diffuse in membrane-few amyloid fibers
Anchorless PrP-Sc: accumulation into amyloid plaques
In vivo
CONVERSION OF PrP-C TO PrP-Sc
experiments show that cells that don't even express PrP-C on surface can still become infected and produce misfolded protein
unknown how or where in the cell the misfolded protein causes damage
Model:
PrP-C misfolding on membrane surface
initial conversion seem to occur here from Scrapie interaction, perhaps in lipid rafts
intracellular compartments may be involved by possibly accelerating conversion after endocytosis of PrP-Sc
OLIGOMERS MOST TOXIC
oligomers (a few monomers) proving more infectious/toxic than amyloid fibers themselves
amyloids may be inert reservoirs of prion protein while small oligomers are neuro-toxic
separated diff sizes of prion aggregates (dimers, trimers, etc) into fractions/partials and tested infectivity
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?
Model: accumulation of PrP-Sc on membrane surface may compromise membrane integrity -> cytotoxic
some experiments showing PrP-Sc forming unregulated ion channels in membrane -> disrupts bilayer integrity, thus cytotoxic
MISFOLDING PATHWAY & INTERMEDIATES
must be folding intermediates during conversion of PrP from helical-form to beta-form
Protein Folding Funnel Energy Landscape
Double folding funnel shows possibilities of both native state folding and aggregate misfolding
Jump in energy (energy barrier) required from intermediate to misfolding funnel
Energy of misfolded aggregate likely lower than that of native state b/c more stable
Interest in intermediate form b/c could be vital to development of therapeutics for prion diseases
several folding pathways proposed
CD folding studies on PrP-C w/ pH
pH 7: protein is largely helical
pH 4: protein converts to Beta-sheet rich conformation
Unique NMR Fingerprint of PrP
NMR peaks disappear after dropping to pH 4, then reappear when returning solution to pH 7
Disappearance can't be b/c of formation of fibers at pH 4, b/c fiber formation is NOT reversible
Protein form must be
molten globule
--transient, changing structure rich in Beta-sheets w/ N-terminal tail remaining flexible
Molten globule is probably pentamer, could be intermediate form
NMR Relaxation Measurements
show particularly flexible residues--involved in milli-sec conformational fluctuations
OXIDATION OF PRION PROTEIN
oxidative markers are hallmark of prion disease
PrP-Sc isolates show most methionine are oxidized
Oxidation: adding double-bond Oxygen to the Sulfur atom in Methionine
Main Q
: Does oxidation cause misfolding or result from it?
Oxidation of Met perturbs core amino acids
thus destabilizes native fold of PrP-C
Regular PrP-C unfolds @ 5.5M urea, but oxidized PrP unfolds @ 2M urea
COPPER AND PRION PROTEIN
Normal Function
function of prion protein is unclear
Ideas
: PrP involved in endocytosis of metal ions into cells, or functions as antioxidant for metal ions
Prion knockout mice don't develop disease and are healthy, so disease isn't loss of prion protein normal function
Copper Binds to PrP-C
Cu2+ has
nano-molar affinity
for PrP-C
octarepeat section of PrP contains histidine residues and is highly conserved
histidine residues
in unstructured half of protein (region vital for prion propagation) actually bind Copper ions
transgenic mice deficient in PrP-C show: 10-fold reduction in Copper conc. in brain,
increased sensitivity to copper toxicity
presence of Cu promotes endocytosis of PrP-C
protein (concentrated at synapses) can bind up to 6 Cu ions
protein may be involved in releasing Copper to de-ionize neuronal membrane (change its charge)
Prion Function?
could be sacrificial quencher of hydroxyl radicals (sacrificial anti-oxidant) by binding Copper in order to mitigate the ions' toxicity when released at synapse
hydroxyl radicals (damaging to DNA, membranes, proteins) formed due to free-moving Copper ions
NEXT UP....
prion replication in other proteins:
prionoids
Ex. yeast prions exhibit "prion" inheritance but no sequence homology
other misfolding diseases can have some prion properties, e.g.
Alzheimer's Disease
(AD)