Solubility Profiles of AmyloidogenicMolecular Structures

Key Theories Towards Meaningful Experiments

Florin Despa, PhD

Department of Pharmacology The University of California, Davis

A. Hydration profiles of amyloid proteins, embryonic amyloid composites and mature amyloid fibrils

• each structural archetype has distinct long-lived water structures;• magnetic resonance (MR) signals of these waters are structure-specific

and differ from the MR signal of normal protein background. (theory & experiment)

B. hIAPP: relationship between the state of aggregation of the protein and the degree of toxicity induced in cells

Despa, Fernandez, Scott & Berry, JBP (2008)

Outline

aggP

Time (min)

Despa, Biophys. Chem. (2009)

hIAPP:KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY

rIAPP:KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY

Hyperinsulinemia &Hyperamylinemia

Molecular denaturationinduced by molecular

crowding

Oversecretion of PPinsulin & PPamylin in

the ER

Hyperglycemia &Increased Metabolic

Demands

Beta Cell dysfunctionAmyloid Formation

How does human islet amyloid polypeptide (amylin, hIAPP) become toxic ?

10 nm 100 nm 100 nm 1 μm 100 μm1 nm

toxic molecular species

electron microscopy light microscopy

adapted from

ex vivo detection

non invasive detection NO YES

Jack et al., J. Neurosci. 25:10041–8, 2005

In Vivo MR Microimaging of Individual Amyloid Plaques in Alzheimer’s Transgenic Mice

Amyloidogenic Proteins are characterized by:

• an increased number of poorly dehydrated backbone HBsFernandez, Kardos, Scott, Goto & Berry, PNAS, (2003)

Fernandez & Berry, PNAS, (2003)

• large surface densities of patches of bulk-like waterDe Simone et al., PNAS, 2005

• favor protein associationDespa, Fernandez & Berry, PRL, 2004Despa and Berry, Biophys. J, (2007); Biophys. J, (2008)

Fernandez, Kardos, Scott, Goto & Berry, PNAS, (2003)

Pathogenic Proteins Have Poor Desolvations of the Intramolecular HBs

Fernandez & Berry, PNAS, (2003)

Fernandez, Kardos, Scott, Goto & Berry, PNAS, (2003)

The DEHYDRON is a hydrophilic structural defect

Water in the desolvation region is mostly in contact with hydrophobes ….

dELβ

dNQ

0

1=f2=f

3=f

20 40 60 80 100

0.05

0.1

0.15

0.2

0.25Polarized Water

Despa, Fernandez & Berry, PRL, 2004

( ) ( )2

2

43

2

1 AE

eA

EP−

=π

r

( ) ( ) 22

0

2

18, μαμ rr LE

NNffA =

( ) ( ) 22EEPEdrrrrr μμ ∫=

Water Molecular Dipoles Correlated in Pairs Through an Entropic Effect

( )psSτ

2 4 6 8 10 12 14( )nmR

10

20

30

40nml 75.00 =

nml 10 =50

60

experimentTan et al., JCP (2005)

Despa, PCCP, 2008

The fraction of correlated water and its characteristic relaxation time depend on the degree of confinement.

2

0.2

0.4

0.6

0.8

4 6 8 10 12 14

η

)(nmR

correlated water

Despa, PCCP, 2008

cr2b

2b

Water structured at the interface between hydrophobes is a source of induction-dispersion effects that favor protein association

( )

( ) 32

0

22

3

612

0

142

2

rrg

fvrASAd

l

rb

rbrU

c

h

⎟⎟⎠

⎞⎜⎜⎝

⎛⎟⎠⎞

⎜⎝⎛

−⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎠⎞

⎜⎝⎛−⎟

⎠⎞

⎜⎝⎛=

μπε

γ

ε

r

( )1−molKcalUh

( )oAr“wetting” regime

Despa and Berry, Biophys. J, (2007)

Despa and Berry, Biophys. J, (2008)

sc810−>τ (caged water)

sp910~ −τ (highly structured water)

sb1210~ −τ (bulk water)

snp1010~ −τ (bulk-like water)

(structural defects)

Prion (PrPC) Solvation MapDe Simone et al., PNAS, 2005

Proteins presenting structural defects are characterized by an increased fraction of water with a bulk-like behavior.

Association of Proteins to Form Oligomers and Fibrils Rescales the Long-lived Water Structures

Oligomers

Fibrils

The change in the distribution of long-lived water structures provides the MRI signal.

mMN 10 ≅

NΔ+

NΔ+

protein background

31 mV μ≅

)2.0( mM≅

69.0≅η

74.00 ≅η

69.0≅η

B

C

A

test isomer

native protein

negative control

Aqueous Environment Containing Protein Background + Test Isomers

Dimers

Compact Aggregates

Floppy Aggregates

rA

cA

scswater VVVNVVNV ++Δ++= )()( )()(

Partition of Water in the Protein System

test isomer(increased number of surface defects)

native proteinff A >)(

)( Af

surface density of defects

f

sp VfV )1( −≅

snp VfV ≅sV(surface water)

( )ρρ −=−= maxmWmc VVVV(caged water)

sp910~ −τ (highly structured water)

snp1010~ −τ (bulk-like water)

(structural defects)

sc810−>τ (caged water)

Despa, Fernandez, Scott & Berry, JBP (2008)

Water Fractionsprotein background + test isomers

( )( )

nppcr

A

np

A

p

c

XXXXNN

NfNfX

NNNffNX

X

−−−=Δ+Δ+−

=

⎟⎟⎠

⎞⎜⎜⎝

⎛Δ+Δ+

−−

=

−−=

1

111.0

1111.0

1

)(

)(

max

ηη

ηη

ηηρρ

scaling theory (Reiss, 1965)

(caged water)

(highly structured water)

(bulk-like water)

(bulk water)

Despa, Fernandez, Scott & Berry, JBP (2008)

mf A)()( Af (soluble oligomers)

mS VV 11.0≅

sc810−>τ sp

910~ −τ snp1010~ −τ

water fractions

residence time

( ) 1.0== ff A

( ) 3.0=Af( ) 5.0=Af

caged water

highly structured water

less structured water(bulk-like)

(control)

Histograms describing the partition of constrained water in a system of protein background plus test isomers

Change of the hydration profile following the formation of fibrils and plaques.

(caged water)

(highly structured water)

(bulk-like water)

(bulk water)

Compact Aggregates

( )( )

( ))()()()(

3/2)(

3/2)(

max)(

1

1111.0

111.0

1

ASAp

ASAnp

ASAc

ASAr

ASAp

ASAnp

ASAc

XXXXNN

NNfX

NNNNfX

X

−−−=

Δ+Δ+−

−=

Δ+Δ+−

=

−−=

ηη

ηη

ηηρρ

1. at equilibrium, most of the surface defects are buried inside the fibril:2. packing density of fibrils is high so they exclude surrounding water

(Petkova et al., Science, 2005)

Despa, Fernandez, Scott & Berry, JBP (2008)

ff A →)(

(caged water)

(highly structured water)

(bulk-like water)

(bulk water)

Floppy Aggregates

caged water

( )( ) ( )

( ))()()()(

3/2)(

3/2)(

max)(

1

1111.0

111.0

111.01

aggp

aggnp

aggc

aggr

aggp

aggnp

aagg

c

XXXXNN

NNfX

NNNNfX

NNNX

−−−=

Δ+Δ+−

−=

Δ+Δ+−

=

−−

Δ+Δ

+−−

=

ηη

ηη

ρρηη

ηηρρ

1. most of the surface defects are buried inside the aggregate;2. packing density of fibrils in a plaque is sufficiently low so that, plaques

contain additional caged water molecules (Nelson et al., Nature, 2005)

Despa, Fernandez, Scott & Berry, JBP (2008)

sc810−>τ sp

910~ −τ snp1010~ −τ

water fractions

residence time

caged water

highly structured water

controlprotein background + test isomersdimers of test isomers compact protein aggregates

floppy protein aggregates

less structured water(bulk-like)

Histograms describing the partition of constrained water in a system of protein background plus test isomers

Despa, Fernandez, Scott & Berry, JBP (2008)

∑ ⎟⎟⎠

⎞⎜⎜⎝

⎛−

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎟⎠

⎞⎜⎜⎝

⎛−−=

j jjjk T

TEkTTRXSS

,2,1max expexp1

∑=j j

j

TX

T ,22

1

( ) ( )

( ) ( ) ⎟⎟⎠

⎞⎜⎜⎝

⎛

++

++=

⎟⎟⎠

⎞⎜⎜⎝

⎛

++

+=

22,2

22,1

212

15

32

1

214

11

jL

j

jL

jj

j

jL

j

jL

j

j

CT

CT

τωτ

τωτ

τ

τωτ

τωτ

Magnetic Relaxation Response

40 50 60 70

9.35

9.45

9.25

A (control)

BC

Less Structured Water

Highly Structured Water

D

E

TE (ms)

maxSSMR Intensity Signal

(grayscale)

BC

Protein Background + Test Isomers

Control Protein BackgroundA DE

Protein Background + Dimers

Protein Background + Compact Aggregates

Protein Background + Floppy Aggregates

• AD samples can display both bright and dark spots on MR images;• Bright spots are likely to indicate oligomers and protofibrils.

)(2

)(2

)(2

controlbc TTT >>

)(2

)(2

controlb TT >

)(2

controlT

)(2

)(2

controla TT <

hIAPP in serum

5 µM 50 µM 100 µM

Aβ1-40 in serum

5 µM 50 µM 100 µM

Objectives:• To detect the MR response of water following structural modifications of the amyloidogenic assemblies;• To test the hypothesis that the formation of oligomers and/or fibrils leads to an increase of the T2 values, shifting the MR signal towards values corresponding to bulk-like water.

1 2 3 1 2 3

serum+water

1 2 3

Water Proton NMR Spectroscopy of Amyloidogenic Structures

Dr. J. Walton Dr. S. Anderson

NMR Setup

ParaVision 3.0.2 to collect data, Nonlinear least square (NNLS) fit to analyze data

Data Analysis

200 250 300 350 40090

92

94

96

98

100

hIAPPAbeta

T2 (ms)

% o

f sam

ple

Water Proton NMR Spectroscopy of Amyloidogenic Structures

200

250

300

350

400hIAPPAbeta

m c1 c2

T 2 (m

s)

5 µM 50 µM 100 µM

92

94

96

98

100hIAPPAbeta

m c1 c2

% o

f sam

ple

5 µM 50 µM 100 µM

0.4, 0.441.2, 5.792.6, 97.48, 96.3 (95.46)

350, 370, 370363.33

6.3, 0.48, 1.61

0.71, 7.2, 1Abeta(c2)

93.5, 96.8, 95.9 (95.4)

310, 310, 330316.66

0.5, 0.47, 0.77

9.1, 5.7, 5.15.5, 1.27, 1.74

0.85, 2.4, 1.4Abeta(c1)

94.5, 95.97, 96.8 (95.76)

210, 210, 230216.66

0.26, 0.697.2, 5.14.3, 1.88, 2.01

1.1, 1.9, 3.6Abeta(m)

96.9, 98.27, 98.1 (97.76)

310, 310, 3102.2, 0.42, 0.51

2.1, 7.2, 7.2control(2)

96, 97.99, 97.7 (97.23)

310, 310, 310310

3, 0.58, 0.681.8, 8.1, 6.8hIAPP(c2)

0.33, 0.585.4, 6.495, 97.5, 97 (96.5)

230, 250, 250 (243.33)

4.1, 1.25, 1.81.1, 2.4, 1.1control (1)

94.4, 97.38, 97.4 (96.39)

250, 250, 260253.33

0.28.63.6, 1.11, 0.87

1.4, 5.1, 6.4hIAPP(c1)

93.4, 96.58, 96.8 (95.59)

200, 200, 2000.23, 0.77, 1.03

7.7, 8.1, 6.85.4, 1.21, 0.82

0.95, 1.5, 1.4control

93.9, 97.38, 97.7 (96.33)

200, 220, 220213.33

4.1, 1.11, 0.77

1.5, 7.2, 9.1hIAPP(m)

% of sampleT2(C) (ms)% of sampleT2

(B) (ms)% of sampleT2(A) (ms)

Water Proton NMR Spectroscopy of Amyloidogenic Structures

The formation of oligomers and/or fibrils leads to an increase of the T2 value

200

250

300

350

400ControlhIAPPAbeta

T 2 (m

s)

m c1 c290

92

94

96

98

100

ControlhIAPPAbeta

m c1 c2

% o

f sam

ple

5 µM 50 µM 100 µM 5 µM 50 µM 100 µM

hIAPP in serum

5 µM 50 µM100 µM

Aβ1-40 in serum

5 µM 50 µM 100 µM

1 2 3 1 2 3

serum+water

1 2 3

How does the amyloid toxicity manifest at the cellular level ?

cardiac myocyte

Amylin-Induced Toxicity in Cardiac Myocytes

Objective: To assess the functional alteration of myocytes induced by hIAPP.

Method: Monitor the intracellular Ca signal .

Sarcolemma

ICa

CaCaCa

Ca

3Na

MyofilCa

SRR

yRCa

3Na

Ca

T-Tu

bule

Na-CaX

NCXATP

PLB ATP

Na

Mito

3Na

2K

ATP

PLM

Na

NHE

H

AP(Em)

[Ca]i

Contraction

Ca

CaNa

H

2NaHCytCyt

Amylin-Induced Toxicity in Cardiac Myocytes

Experimental Protocol:- myocytes were plated on laminin-coated coverslips; - loaded with Fura-AM (10 μmol/L, for 45 min);- Fura was alternately excited at 340 and 380 nm (F340 and F380) using an Optoscan monochromator (Cairn Research, Faversham, UK);- fluorescence was collected at 510±20 nm;- the fluorescence ratio F340/F380 was calculated after background subtraction.

AP(Em)

[Ca]i

Contraction

Ca-mediated Cardiomyocyte Contraction

40 50 60 70

0.50

0.75

0.25normal protein background

bulk-like water

structured water

TE (ms)

S

Intensity of Magnetic Response of Water

fibrils

soluble amyloidogenic proteins

large, floppy aggregates

Concluding Remarks

• Amyloid proteins and embryonic amyloidcomposites can be differentiated based on their hydration profiles and characteristic MR signals of the surrounding water.

MRI Contrast Mechanism for Detection of AD

Jack et al., J. Neurosci. 25:10041–8, 2005

In Vivo Magnetic Resonance Microimaging of Individual Amyloid Plaques in Alzheimer’s Transgenic Mice

Clinical Implication:

40 50 60 70

0.50

0.75

0.25normal protein background

bulk-like water

structured water

TE (ms)

S

Intensity of Magnetic Response of Water

fibrils

soluble amyloidogenic proteins

large, floppy aggregates

Concluding Remarks

• Amyloid proteins and embryonic amyloidcomposites can be differentiated based on their hydration profiles and characteristic MR signals of the surrounding water.

• Toxicity induced by soluble amyloid composites manifests at the cellular level in a time-dependent manner: progressive damage of the membranes.

5 µM

msT 2132 ≅

50 µM

msT 2532 ≅

• Oligomers are the most toxic species. Fibril growth at the membrane is equally toxic….

Acknowledgements

• R. Stephen Berry (Chicago)• Ariel Fernandez (Rice) • L. Ridgway Scott (Chicago)• Christopher Rhodes (Chicago) • Donald Steiner (Chicago)• Ulrich Hansmann (Juelich)

• Sanda Despa (Davis)• Jeff Walton (Davis)• Steve Anderson (Davis)