Download Correlation of β-Amyloid Aggregate Size and Hydrophobicity

Alzheimer’s
Understanding The Role of
Membranes in Amyloid Aggregation
Presentation Based on
Correlation of β-Amyloid Aggregate Size and
Hydrophobicity with Decreased Bilayer Fluidity of Model
Membranes
John J. Kremer, Monica M. Pallitto, Daniel J. Sklansky, and Regina M. Murphy.
Biochemistry 2000, 39, 10309-10318.
Nadia J. Edwin
Macromolecular Seminar
December 5, 2003
OUTLINE
1. Background
- Amyloid
- Membranes
2.
Previous Studies
3. Goal of Reference Paper
4. Experimental Techniques Used
- DPH Anisotropy
- Dynamic Light Scattering
- Static Light Scattering
5. Conclusion
What is Amyloid?
1853 – Rudolf Virchow named cerebral deposits as amyloid
Amyloid –proteinaceous aggregates associated with diseases
(Alzheimer’s, Parkinson’s, spongiform encephalopathies)
Amyloid aggregates in brain cells are thought to play a causative
role in the onset of Alzheimer’s Disease
Dobson, C.M. Protein misfolding, evolution and disease. Trends Biochem.Sci. 1999, 24,
329-332.
Origins of Amyloid?
β-Amyloid peptide : (39- 42 amino acids) is a protein fragment cleaved
from a much larger protein
β-Amyloid Precursor Protein : (~ 695 amino acids)
β-APP – an inhibitory molecule that
regulates the activity of proteases
Amyloid Hypothesis
Neurodegeneration in Alzheimer’s disease (AD) may be
caused by deposition of amyloid β-peptide (Aβ) in plaques
in brain tissue
Current studies probe effects of physical conditions
(differing pH, temperature, salt concentration) on Aβ
aggregation
Hardy, J.; Selkoe, D.J. Science, 2002, 297, 353-356.
Plasma Membrane
Regulate transport of nutrients into and waste out of the cell
Provide a site for chemical reactions not likely to occur in an aqueous
environment
Alberts et al. Molecular Biology of the Cell, Garland Publishing, N.Y. Third edition, 1994.
Membrane Fluidity
Fluidity of a lipid bilayer depends on its composition and temperature
The greater the concentration of unsaturated fatty acid residues, the more fluid
the bilayer
At body temperature, the phospholipid bilayer has consistency of olive oil
Fluidity of the phospholipid bilayer allows cells to be pliable
Alberts et al. Molecular Biology of the Cell, Garland Publishing, N.Y. Third edition, 1994.
Membrane Composition
Saturated fats
no double bonds between carbons in the tail
saturated with hydrogen
solid at room temperature
most animal fats, bacon grease, lard, butter
Unsaturated fats
one or more double bonds in tail kinks the
tail so cannot pack closely enough to solidify
at room temperature
most plant fats
Effect of Temperature on membrane
fluidity
Phospholipids
POPC 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine
POPE
1-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine
POPG 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]
POPS
1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-1-serine]
Alberts et al. Molecular Biology of the Cell, Garland Publishing, N.Y. Third edition, 1994.
Cholesterol
Phospholipids, cholesterol, gangliosides
Alberts et al. Molecular Biology of the Cell, Garland Publishing, N.Y. Third edition, 1994.
Previous Studies: Side 1
Aβ associates with cells via membrane-bound receptors
1. Aβ binds to Serpin-enzyme Complex Receptor (SEC)
2. Aβ binds to class A scavenger receptor (SR)
3. Aβ binds to Receptor for advanced glycation end products
(RAGE)
4. Aβ binds to a hydroxysteroid dehydrogenase enzyme (ERAB)
Joslin, G.; et al; J.Biol.Chem.1991, 266, 21897-21902.
El Khoury, J.; et al.; Nature, 1996, 382, 716-719.
Yan, S.D.; et al.; Nature, 1996, 382, 685-691.
Yan, S.D.; et al.; Nature, 1997, 389, 689-695.
Side 2 of the Debate
Aβ aggregates are toxic via nonspecific association
with cell membranes
1. Membrane components promoted changes in Aβ secondary
structure and/or aggregation propensity.
2. Aβ or its fragments caused:
- formation of large ion channels in phospholipid bilayers
- leakage of encapsulated dyes from phospholipid vesicles
- fusion of small unilamellar vesicles
3. Loss of impermeability in lysosomal and endosomal
membranes
Goal of Paper
• Determine whether changes in membrane physical properties
were correlated with Aβ aggregates
• Relations of any such effects with biological membrane with
specific membrane components
• Does changes depend on Aβ aggregation state
ANISOTROPY
r=
I par  gI per
I par  2 gI per
PTI Spectrofluorometer with
manual polarizers
Excitation wavelength – 360 nm
Emission wavelength – 430 nm
1,6-Diphenyl-1,3,5-hexatriene (DPH)
CH
CH
CH
CH
CH
CH
Absorption – 350nm
Emission – 452nm
Absorption and emission spectra of DPH in
hexane at 25°, polarization spectrum of DPH
in frozen (propylene glycol at -50°)
Partition itself in the hydrophobic region of the bilayer at or
near the ends of the acyl chains
Detect changes in the order of the acyl chains
Lentz, B.R.; Barenholz, Y.; Thompson, T.E. Biochemistry 1976, 15, 4529-4537.
Shinitzky, M.; Barenholzz, Y. J.of Biological Chemistry 1974, 25, 2652-2657.
Dynamic Light Scattering
Lens
Sample Cell
LASER
θ
Photomultiplier
Detector
Compute
r
Correlator
Amplifier
DLS Results
Figure 1: Growth kinetics of Aβ aggregates at physiological pH. Aβ was dissolved
in DMSO and then diluted 20-fold into PBS, pH 7.4, to a final concentration of 0.5
mg/mL. The average apparent hydrodynamic diameter, dsph, was determined from
cumulants analysis of dynamic light-scattering data taken at 90º scattering angle.
Static Light Scattering
Figure 2: Change in Aβ aggregate molecular weight and size with time. Static light-scattering data taken at 24
(), 30 (), 44 (), 52 (), 69 (), and 93 h () after initiation of aggregation are shown as Kratky plots. Lines indicate
nonlinear regression fit of semiflexible chain (24-52 h) or semiflexible star (69-93 h) models to the data. The
increase in the y-axis intercept is indicative of an increase in average molecular weight, whereas the
appearance of a maximum in the curves at intermediate values of q is characteristic of branched structures.
SLS Results Cont’d
Figure 3: Growth of Aβ aggregates at physiological pH. Weightaveraged molecular weight <M>w ( ) and average fibril length Lc
( ) were determined by nonlinear regression fit of model
equations to the light-scattering data of Figure 2, as described in
more detail in the text. Error bars represent 95% confidence
intervals for fitted parameters.
Microscopy Shows
Figure 4: Electron micrographs of Aβ aggregated for 2
days at neutral and acidic pH. (A) pH 7 fibrillar aggregates,
scale bar = 50 nm; (B) pH 6 agglomerated aggregates;
scale bar = 200 nm.
Fluorescence Results
Figure 5: Effect of Aβ aggregation on bis-ANS fluorescence. PBS ( , n = 18), freshly diluted Aβ ( , n = 4),
and Aβ aggregated for 2 days in PBS at pH 7 ( , n = 6) or pH 6 ( , n = 6) were added to PBS containing
the dye bis-ANS. Fluorescence spectra were collected from 450 to 550 nm, with excitation at 360 nm.
Results shown are averaged scans from 4-18 samples; the error bars signify one standard deviation. Two
other data sets were taken with samples prepared on different days with similar results (data not shown).
Binding of bis-ANS to exposed hydrophobic sites is signaled by an increase in fluorescence intensity and
blue-shifting of the peak. Fluorescence intensity of Aβ aggregated at pH 6 and 7 was statistically different
(p < 0.01).
Aggregation Results
Figure 6: Sketch of Aβ aggregation at (A) neutral and (B) acidic pH. (A) At pH 7, Aβ steadily increases
from an average fibril length of ~960 nm at 1 day to ~4000 nm at 4 days. These fibrils possess hydrophobic
patches as shown by bis-ANS binding (Figure 5). Fibril-fibril entanglement is detectable as "branching" at
3 days and increases with time. Precipitation occurs around 5 days, accompanied by a loss of bis-ANS
binding when tested at 7 days. Together these results suggest that at the later stages of Aβ aggregation at
neutral pH, fibril-fibril association mediated by hydrophobic interaction occurs, reducing solvent-exposed
hydrophobic patches but generating macroscopic fibril bundles. (B) At pH 6, Aβ instantaneously forms
large, amorphous aggregates that precipitate in less than 24 h. These aggregates contain many highly
hydrophobic solvent-exposed patches, which are present even at 7 days. This suggests that Aβ aggregation
at pH 6 does not occur through orderly self-association via burial of hydrophobic interactions and that
precipitation occurs due to poor aggregate solubility near the isoelectric point.
DPH Anisotropy Results
Figure 7: Effect of Aβ Aggregation at pH 7 on DPH anisotropy. Freshly diluted ( )
and 2 day-aged ( ) Aβ samples were added to (A) POPC and (B) POPG liposomes
with embedded DPH. Data are compilation of 2-4 replicate experiments at each
condition.
DPH Anisotropy
Figure 8: Effect of Aβ Aggregation at pH 6 on DPH anisotropy. Freshly
diluted ( ) and 2 day-aged ( ) Aβ samples were added to (A) POPC and
(B) POPG liposomes with embedded DPH. Data are compilation of 2-4
replicate experiments at each condition.
DPH Anisotropy
Figure 9: DPH anisotropy with (A) type I and (B) type 2 vesicles at pH 7 (
,
)
and pH 6 (
,
) upon addition of freshly diluted (
,
) and 2 day-aged
(
,
) Aβ. Aβ induces a significantly larger anisotropy increase in vesicles
containing gangliosides at both pH 6 and pH 7.
Conclusion
Observed decreases in membrane fluidity, detected as an increase
in DPH anisotropy.
Changes in membrane fluidity are not solely dependent on binding
of Aβ to the bilayer surface.
Acknowledgements
• NSF-IGERT
• Dr. Paul Russo
• Russo Group Members
Fluidizing action of Aβ
Mason, R.P.; Jacob, R.F.; Walter, M.F.; Mason, P.E.; Avdulov, N.A.; Chochina, S.V.; Igbavboa, U.; Wood,
W.G. J.Biol.Chem. 1999, 274, 18801-18807.
Figure 5.10 The synthesis and structure of a fat, or triacylglycerol
Carboxyl group has acid properties
Hydrocarbon chain, 16-18 carbons
Nonpolar C-H bonds, hydrophobic
(Condensation Reaction)
(bond between hydroxyl group and a carboxyl group)
Fats:
hydrophobic, not water soluble
variation due to fatty acid composition
fatty acids can be the same or different
fatty acids can vary in length
fatty acids can vary in the number and location
of double bonds (saturation)
A triglyceride
Lipids:
Diverse Hydrophobic
Molecules
Lipids = Diverse group o f organic compoun ds th at are insolub le in w at er,
but wi ll dissolv e i n nonp olar s olvents ( e.g., eth er chlorof orm, benz ene) .
Importa nt gro ups are fat s, phospholipids, and st eroids.
Fat s st ore large amount s of energ y
Fat s = Macromolecules are c onst ruct ed f rom:
Glycerol, a th ree-carbon alcoho l
Fat t y acid ( carboxylic acid) = Compos ed of a carboxyl gro up at
one e nd a nd an a t ta ched hyd rocarbon ch ain (“ t ail” )
PALMITIC ACID: Palmitate. Fatty Acids. From fats, oils (see Fatty Acids) mixed with
stearic acid (see). Occurs in many animal fats and plant oils. In shampoos, shaving
soaps, creams. Alternatives: palm oil and other vegetable sources.
OLEIC ACID: Oleth-2, -3, -20, etc. Oleyl Alcohol. Oleamine. Oleyl Betaine. Obtained
from various animal and vegetable fats and oils. Is usually obtained commercially from
inedible tallow (see). In foods, soft soaps, bar soaps, permanent wave solutions,
shampoos, creams, nail polish, lips ticks, liquid makeups, many other skin preparations.
Alternatives: coconut oil; see alternatives for Animal Oils and Fats.
STEARIC ACID: Tallow (see). Stearamide. Stearate. Quaternium 27. Stearin. Fat from
cows, sheep, etc. (could be dogs and cats from shelters). Most often refers to a fatty
substance taken from the stomachs of pigs. Can be harsh, irritating. Used in cosmetics,
soaps, lubricants, candles, hairsprays, conditioners, deodorants, creams. Alternatives:
can be found in many vegetable fats, e.g., coconut.
STEROID: Sterol. From various animal glands or from plant tissues. Steroids include
sterols. Sterols are alcohols from animals or plants (e.g., cholesterol). Used in hormone
preparations. In creams, lotions, hair conditioners, fragrances, etc. Alternatives: plant
tissues, synthetics.
STEARYL ALCOHOL: Stenol. A mixture of solid alcohols; can be prepared from sperm
whale oil. In medicines, creams, rinses, shampoos, etc. (Federal regulations currently
prohibit the use of ingredients derived from marine mammals.) Alternatives: plant
tissues, synthetics.
Figure 5.12 The structure of a phospholipid
5.3 Phospholipid structure
Figure 5-27a
Figure 5-28
Cholesterol, polar steroid
with acyl chain
Membrane Cont’d
Ganglioside
Fluidity depends on temperature and composition
Temp: phase transition
Composition: acyl chain length & saturation, cholesterol
Short or kinks=>fewer van der Waals interactions
Cholesterol has opposing effects & is tightly regulated:
•Polar head group restricts phospholipid head group movement-> decreases fluidity
•Planar steroid separates phospholipid acyl tails->increases fluidity
Fluorescence polarization and intensity were obtained by a simultaneous measurement of 11 I/II and 11, where II 1 and II are the
fluorescence intensit,ies detected through a polarizer oriented
parallel and perpendicular to the direct,ion of polarization of the
excitation beam. The lil/Il and the 11 values relate to the degree of fluorescence polarization, P, to the fluorescence anisotropy,
r, and to the total fluorescence intensity, F, by the following
equations:
- P= 4, 1, w.L - 1 z?4, + 1, Ill/I, + 1
I,, - I, I,,/I, - 1 r=-----=I,, + 21, I,,/I, + 2
0)
F = I,, + 21, = Il(Z,,/I, f 2)