prp presentation
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The culmination of a lot of work during my postdoctoral fellowship.TRANSCRIPT
Human Prion Protein Amyloid:A Tale of Structure and Stability
Nathan J. Cobb
Department of Physiology and Biophysics
Case Western Reserve University, Cleveland, OH
The Prion Diseases
Disease Pathogenesis
Human Diseases• Creutzfeldt-Jakobs disease (CJD) Sporadic, iatrogenic
• variant CJD Infection from bovine prions?
• Familial CJDs Inherited
• Kuru Infection
• Gerstmann-Straussler-Sheinker (GSS) Inherited
• Fatal familial insomnia Sporadic, Inherited
Animal Diseases• Scrapie (sheep) Sporadic, Infection
• Bovine spongiform encephalopathy Sporadic, Infection
• Transmissible mink encephalopathy Sporadic, Infection
• Chronic wasting disease (cervids) Sporadic, Infection
• Feline spongiform encephalopathy Infection
• The infectious agent in the transmissible spongiform encephalopathies
(TSEs) is not a virus, but a misfolded form of the normal (cellular) prion
protein, PrPC
• This misfolded form, PrPSc (scrapie), is a β-sheet-rich aggregate which arises
from conformational conversion of the normally monomeric and primarily
α-helical PrPC
• Like other neurodegenerative diseases such as Alzheimer’s and Parkinson’s,
TSEs are often associated with neuronal accumulation of amyloid deposits
• PrPSc self-perpetuates by first binding PrPC and then inducing conformational
conversion of the latter protein to the PrPSc state
• Although the ‘Protein-only’ nature of the TSEs has yet to be conclusively
shown, studies in yeast and other fungi have provided the proof-of-principle
that proteins alone can act as self-perpetuating agents
• Challenge to the ‘Anfinsen Principle’?
The ‘Protein-Only’ Hypothesis of Prion Diseases
Leader Sequence: 1-22
Octapeptide Repeats: 51-91
-Helicies: 144-156,174-194, 200-228
-Sheets: 128-131, 161-164
Disulfide Bond: 179 and 214
N-linked Glycosylation: 181, 197
GPI Anchor: 231
GPI Anchor Signal: 232-254
The Structure of Cellular Prion Protein
S S
G
H1
1H2 H3
G
G
P
IPHGGGWGQ
PrPC PrPSc
23
S S
Proteinase K
S S
Proteinase K
S S
231
231~87-90
23 231
Digestion with Proteinase K
Surrogates for Structural Information
Congo Red
Thioflavin T
Binding of ‘Amyloid-Specific’ Dyes
Immunostaining for PrP
Immunostaining for Glial
Fibrillary Acidic Protein
Staining with
Hematoxylin-Eosin
Aguzzi et al. (2001) Nature
-39 kDa
-28 kDa
-14 kDa
-19 kDa
PrPC PrPSc
PK - + - +
FTIR spectra of brain-derived PrP
— PrPC
- - - PrPSc
••••• PrP 27-30
Pan et al. (1993) PNAS
Amyloid Fibrils
Nelson et al. (2005) Nature
X-ray diffraction structure of microcrystals
formed by the peptide GNNQQNY
Petkova et al. (2004) PNAS
Aβ1-40 structure as determined
by solid-state NMR
Like other neurodegenerative diseases such as Alzheimer’s and Parkinson’s,
TSEs are associated with neuronal accumulation of amyloid deposits
0 5 10
0.0
0.5
1.0
1.5
2.0
2.5
Th
T f
luo
res
ce
nc
e
Time, hrs
2% seed
No seed
Conversion of rPrP (residues 90-231) into Amyloid Fibrils: ‘Synthetic Prions’
Nucleation
Elongation
Fragmentation 2o Nucleation
What is the Relevance of rPrP Amyloid to PrPSc?
• It has been shown that in vitro converted amyloid fibrils induce a TSE-like
disease in mice overexpressing PrP89-231 (Legname et al. (2004) Science)
• The infectivity titer is very low, suggesting that only a small fraction of this
material is infectious
• rPrP amyloid allows for structural and biochemical characterization techniques
that are not amenable to brain-derived aggregates
Brain-Derived PrPSc ‘Synthetic Prions’
Glycosylated Unglycosylated
Membrane-associated NA
16 kDa PK-resistant core Shorter PK-resistant core
Site-Directed Spin Labeling
Nitroxide-labeled proteins yield three important pieces of information
Involves substitution of cysteine for
native residues followed by thiol-specific
modification with the nitroxide reagent
1) Distance estimates – dipolar broadening and spin exchange
2) Mobility information
3) Accessibility
EPR spectroscopy
Absorption of electromagnetic radiation by an unpaired electron
in an applied magnetic field
H
En
erg
y
MS = 1/2
MI = 1/2
Hres
signal
amplitude
modulation amplitude
MI = -1/2
MS = -1/2
H
Energ
y
H
Absorp
tion
Hres
H
Absorp
tion
H
D(A
bsorp
tion
)/dH
Hres
H
d(A
bsorp
tion
)/d
H
Nitroxide EPR spectra
H
+1/2
-1/2
En
erg
y
+1
0
-1
-1
0
+1
MI MS
Mobility
Margittai & Langen (2004) PNAS
Jayasinghe & Langen (2004) J. Biol. Chem
Spin Exchange
Distance Estimates
Dipolar Broadening
Initial Purification and Refolding of rPrP
Denaturing Wash (10 mM reduced glutathione, 6 M GdnHCl, pH 8.0)
Gradient from 6 M → 0 M GdnHCl
Wash
Low Imidazole (50 mM imidazole, pH 8.0)
Collect High Imidazole (350 - 500 mM imidazole, pH 5.8 - 6.4)
Standard Buffer: 100 mM phosphate, 10 mM Tris, pH 7.0
Final Purification and Labeling of rPrP
• Dialyze collected fractions into 50 mM actetate, pH 4.0
• Pool and concentrate, storage at -80 oC
• Incubate with 5x molar equivalents of TCEP at 4 oC, dilute with 50 mM phosphate,
pH ~7.0, and add 10x molar equivalents of nitroxide spin-label and thrombin
• Verify nitroxide-labeling and 6xHis-tag cleavage by MALDI
• Remove excess spin label, uncleaved rPrP, rPrP aggregates, and remaining
impurities by cation exchange chromatography
• Measure monomeric EPR signal, concentrate, aliquot and store protein at -80 oC
Characterization of rPrP Cysteine Variants
1) Verify native-like α-helical
content by CD spectroscopy
3) Verify native disulfide formation by HPLC/MS of tryptic digests
2) Verify the proper formation
of amyloid fibrils by AFM
Monomer
FibrilUndiluted Fibril
1:4 Spin-Diluted
Fibril
Spin Exchange in Labeled rPrP Amyloid Fibrils
EPR Signals for Nitroxide-Labeled rPrP Fibrils
Cobb et al. (2007) PNAS
Monomer
Fibril
4 M GdnHCl Fibril (no denaturant)
Fibril 4 M GdnHCl
Cobb et al. (2007) PNAS
Denaturation of Nitroxide-Labeled rPrP Fibrils
no denaturant
1 M GdnHCl
2 M GdnHCl
-helix -sheet1:4 dilution
undiluted
1:1 dilution
191 undiluted
190/191, 191/192
191 1:1 dilution
Only a Parallel In-Register -Structure can Describe EPR Data
Modeling of Prion Protein Amyloid
• Residues ~160-220 are in-register and parallel with one another
• The native disulfide is maintained in the amyloid structure
• Asn 181 and 197 must point toward the outside of the amyloid structure
PrP monomer
Two-bend modelOne-bend model
N181
N197
N181
N197
Molecular Details of the Two-Bend Model
N181
N197
Cobb et al. (2007) PNAS
Correlation with H/D Exchange Data
Lu et al. (2007) PNAS
23190
EPR data (160-220)
H/D exchange (170-220)
Pathogenic Mutations of the Prion Protein
2531 51 91
P102L
P105L
P105T
A117V
Y145stop
Q160stop
D178N
V180I
T183A
H187R
T188K
T188R
E196K
F198S
E200K
D202N
V203I
R208H
V210I
E211Q
Q212P
Q217R
M232R
P238S
23
Insertion of
1,2, or 4-9 repeats
EPR data (160-220)
H/D exchange (170-220)
Spiral model (116-164)
Left-handed helical model (89-175)
Left-handed
β-helical modelGovaerts et al.
(2004) PNAS
Spiral modelDeMarco and
Daggett (2004)
PNAS
Parallel an in-register
β-structure of rPrP amyloidCobb et al. (2007) PNAS
Structural Models of Prion Protein Aggregates
Sim and Caughey
(2008)
Neurobiol. Aging
Wille et al.
(2002) PNAS
90 231
Similar Amyloid Fibrils are Formed under Native Conditions
Native conditions 50 mM acetate
pH 4.0
Denaturing Conditions 50 mM phosphate
2M GdnHCl, pH 7.0
Cobb & Surewicz (2008) J. Biol. Chem.
pH-Induced Structural Differences at the N-Terminus are Reversible
Cobb & Surewicz (2008) J. Biol. Chem.
• Fibrils formed under
native (pH 4.0) conditions
• Equilibrated to pH 7.0
• Fibrils formed under denaturing
(2 M GdnHCl, pH 7.0)conditions
• Equilibrated to pH 4.0
193
103
132
193
103
132
113
113
103
192
Native and Denaturing Conditions Form rPrPFibrils of Similar Stability
Cobb & Surewicz (2008) J. Biol. Chem.
Monomer
Fibril
4 M GdnHCl
● Denaturing
○ Native
● EPR
Against GdnHCl Against pH
● Denaturing
○ Native
● EPR
Highly Acidic Conditions Stabilize rPrPAmyloid Fibrils
179
214
pH 4.0-10.0
179
214
pH 2.0
Fewer Stacked Charges
at Very Low pH
rPrP Amyloid is more Resistant
to GdnHCl at Low pH
Replacement of Hydrophobic with Charged Residues Yields Intriguing Results
Prion Strains and the Species Barrier
Cobb & Surewicz (2009) Biochemistry
23
S S
Proteinase K
S S
231
231~87-90
Structurally Distinct rPrP Amyloid Fibrils
4 M GdnHCl
2 M GdnHCl
500 nm
Conclusions
• As deduced by SDSL/EPR, the amyloid core of rPrP amyloid fibrils is comprised
of residues ~160/170-220 - there is no evidence of stable secondary structure
outside this β-core region
• The molecular architecture of rPrP amyloid fibrils is a parallel and in-register
stacking motif, where same residues are in direct contact with their counterparts
on neighboring molecules
• An identical amyloid core is formed under both denaturing and native conditions,
indicating that this region is strongly predisposed to forming amyloid fibrils
• Stacking of same charges appears to be the main destabilizing force in parallel
and in-register amyloid fibrils
• Substitution at only a single residue can result in structurally distinct amyloid
fibrils
• Varying buffer conditions can result in structurally distinct amyloid fibrils – the N-
terminal residues 23-90 are important for amyloid formation at high denaturant
concentrations