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Prion Diseases: Protein Agent & Isoform Structures (TWO ISOFORMS OF…
Prion Diseases: Protein Agent & Isoform Structures
EVIDENCE
Initial thought:
Virus?
"slow virus" b/c years-long incubation period
infection initiates production of millions of similar infectious particles
species barrier
HOWEVER (1) no immune response (2) strong resistance to heat treatment (3) unaffected by UV & ionizing radiation unlike DNA and RNA
Protein Only Hypothesis
Stan Prusiner, 1982
lab isolated refined fractions of infected brain material
Injected each fraction into hamsters to identify infectious agent
Infectious fraction almost exclusively contained
protein
infectious material called
Prion (proteinaceous infectious) particle
Viral modification procedures have no effect on infectivity of Prion particle
BUT protein-digestion caused reduction in scrapie infectivity
Prion particles derived from single Prion protein
Confirmation
Knockout mice that don't express prion protein at all -> unaffected by infectious material
Wild-type mice (control) that did express protein -> easily infected by cerebral inoculation
inheritable human prion diseases shown to contain mutations in primary sequence of prion protein
WHY CONTROVERSIAL?
1st infectious disease reported to be caused by protein
previous belief: a disease must have genetic info (DNA or RNA) to infect host and replicate
YET no DNA/RNA longer than 50bp detected in prion partials
MAMMALIAN PRION PROTEIN (PrP)
254 amino acids
before
post-translation modifications
209 amino acids (PrP 23-231)
Components for Cell Location
GPI anchor
(residues 231-254)
Hydrophobic signal peptide
Inserts into cell membrane to anchor protein to outside cell surface
Remove N-terminal secretory signal
Key Features
Disulfide bridge Cys179-Cys214
Two O-linked carbohydrate chains @ Asn181 and Asn197
TWO ISOFORMS OF PRION PROTEIN
Cellular PrP-C
Ubiquitous on neuronal cells
Monomeric
High alpha-helical content
Soluble
Susceptible to proteolysis
Structural Details
Circular Dichroism (CD) Spectroscopy
: strong negative CD bands indicate predominantly helical protein
42% alpha helix 3% beta sheet
NMR Chem Shifts
: data from alpha carbons
2 structurally distinct domains
residues 23-126 (1st half) UNSTRUCTURED
residues 126-231 (2nd half) ALPHA HELICAL
80-90% homology b/w all mammalian PrP-C's
Highly flexible, unstructured N-terminal half can bind Cu2+ and induce endocytosis of protein
OVERVIEW: 3 helices packed together with long flexible N-terminal tail
Scrapie PrP-Sc
Oligomeric (aggregative, self-associating)
Present on infected cells
High Beta-sheet content
Insoluble
Resistant to proteolysis
Structural Details
insoluble -> no solution NMR
Molecular-level details unconfirmed
FT-IR
on residues 23-231
43% beta sheet 30% alpha helix
Forms Prion Rods: short flat amyloid fibrils that twist, w/gaps in protein density that resemble a ladder
H/D Exchange Maps
Hydrogen bonding along protein using deuteron (neutron-heavy Hydrogen w/in H2O -> D2O) exchange
Any amide not H-bonded already will bond w/ Deuteron of heavy water b/c amide is labile (water's electronegative Oxygen pulls off amide's original Hydrogen and replaces w/ Deuteron
Deuteron-bonded amides can be mapped along protein, indicate lack of Beta sheet structure
RESULTS: residues 169-225 likely form stable Beta sheet
Beta strands stack together parallel into fibers
inter-Beta-strand H-bonding is b/w diff. polypeptide chains/protein molecules
No H-bonding within same molecule at all
, even if fiber folds back on itself
Fibers grow by shorter monomer fibers stacking end to end, either via monomeric or dimeric interactions
Same primary sequence
Diff. tertiary structure b/c environmental diffs
Prion disease = Misfolding disease
Epitope Mapping
antibodies that bind to specific regions of PrP-C bind to some regions of PrP-Sc
ppl infer that some of the original alpha helices of cellular form are maintained in the misfolded isoform