Transcript Cu(II)

Chemistry of Alzheimer’s Disease:
Role of amyloid-beta, metal ions, and
reactive oxygen species
Peter Faller
LCC, Toulouse
[email protected]
Metals
8% of Cu
Metal
disorder
Brain
2% w/w of body
Neurodegeneration
Oxygen
Consumes
20% of O2
Oxidative
Stress (ROS)
Neurons in the brain
Neuron (nerve cell)
The nerve impulse. In the resting neuron, the interior of the axon membrane is negatively
charged with respect to the exterior (A). As the action potential passes (B), the polarity is
reversed. Then the outflow of K+ ions quickly restores normal polarity (C).
Synapse and Neurotransmitter
Nerve
impulse
continues
Molecular mechanism of learning
• Donald O. Hebb (1949) (Hebb’s rule):
« When an axon of a cell A is near enough to
excite a cell B and repeatedly or persistently
takes part in firing it, some growth process or
metabolic change takes place in one or both
cells such that A’s efficiency, as one of the cells
firing B, is increased »
Long term potentiation (LTP): a mechanism for establishing
memory
stimulation
(EPSPs excitatory postsynaptic potentials)
Long term
Potentiation
(LTP):
a mechanism
for establishing
memory
FIGURE 53-3 An illustration of a synapse
between the presynaptic and postsynaptic
neurons. The glutamate released from
presynaptic terminals activates both
AMPA and NMDA receptors. While the
AMPA receptor is responsible for basal
synaptic transmission, the NMDA receptor
acts like the volume controller regulating
the efficacy of synaptic transmission.
Synaptic transmission is enhanced if the
NMDA receptor detects the co-activity of
the presynaptic (release and binding of
glutamate) and postsynaptic neuron
(enough depolarization to expel Mg2+
from the channel pore). When such a
coincidence event occurs, the NMDA
receptor is activated, which opens the
channel pore and allows Na+ and Ca2+
to rush in and K+ to rush out. The influx
of Ca2+ then activates biochemical
cascades that eventually strengthen
the synapse. It is believed that some of
these kinases bind directly to the
C-terminus of the NR2B subunit,
allowing efficient signal detection and
amplification .
Metals in the cell
Essential and toxic metal ions
Essentiel
Toxique
(non-essentiel)
physiological effect
positiv
death
deficiency
health
toxic
toxic
death
health
death
negativ
concentration
concentration
Biological system tries to keep the metal content constant (steady state)
General features: Metabolism of essential metals
M
M
Protein to
stock metals
Cell (microrganisms)
ATP
ADP
M
M
M
M
M
ADP
M
ATP
Sensor for Regulation
Active transport
diffusion
Metal Specificity
Different metals have different roles:
-e.g. Alcohol dehydrogenase : Zn(II) enzyme
-Cytochrome c oxydase Cu and heme for oxygen reduction
Make sure that the right metal goes to the right place!
How to Reach Metal Specificity
How to make sure that the right metal ion goes to the right place?
1) thermodynamics:
engeneer the site that it binds « specifically », i.e. prefentially the wanted metal
-> coordination chemistry
2) kinetics
specific transporters/carriers called « metallo-chaperones » bring the right metal
to the right place. Then the metals is well bound so that koff is very low
Thermodynamic versus Kinetic Control of Metal-binding
Thermodynamic control
+ metal
ion
2
1
4
3
3
2
K1
1
Si K2 >> K1,3,4
K2
K3 K4
4
K1
1
2
K2
K3 K4
3
4
Kinetic control
1
3
k1
k3
2
k2
k4
2
1
4
« k1 >> k2,3,4 »
4
3
1
3
2
4
For Cu kon diffusion controlled
k1 = k2 = k3 = k4
1
3
k1
k3
2
k2
k4
4
k1
Mn+
+
Mn+-Prot
Prot
k-1
K1 = k1/k-1
Thermodynamic control:
parameters to optimize;
- size of the site (ion radius Ca2+: 100pm is larger than Mg2+ 72 pm)
- charge (metal-ligand most stable when neutral)
- number of ligands (Ca2+ 6-8 ligands; Cu+ 2-4)
- type of ligands (Pearsons model of hard/soft acid/base)
- geometry (Cu(II) likes square planar, Zn(II) tetrahedral, or pentacoordinated)
Chelate Effect
M
+
M
+
2L
L
L
L
M
L
L
M
L
Association constant: monodentate < bidentate < tridentate etc.
Ex: EDTA (hexadentate)
Co-EDTA
Chelate Effect
Example: Complexes of Ni(II):
Ni2+ (aq) + 6 NH3
[Ni(NH3) 6] 2+ (aq)
log K = 8.6
Ni2+ (aq) + 3 en
[Ni(en)3] 2+ (aq)
log K = 18.3
Mainly entropic effect
Ni(NH3) 6] 2+ (aq) + 3 en
[Ni(en)3] 2+ (aq) + 6 NH3
log K = 9.7; ΔG° = -67kJ/mol, ΔH°= -12 kJ/mol; -TΔS° = -55 kJ/mol
CHIMIE FONDAMENTALE, Chottard, Depezay, Leroux
Metal-ions binding and pKa of ligands
2+
MII-OH2
+
MII-OH
+
H+
Metal ion
(2+)
pKa
No metal
Ca
Mn
Cu
Zn
14,0
13,4
11,1
10,7
10,0
« pKa »
+
HN
NH
+ M2+
MII- N
2+
+
NH
H+
No metal
Co
Ni
Cu
Competition between metal ion and proton
(low pH used to remove metal ion from ligand)
6,0
4,6
4,0
3,8
Concept of hard/soft acid/base (HSAB, Pearson)
In biology:
Bases:
Oxygen (hard)
Nitrogen (intermediate)
Sulfur (soft)
Acids
hard: Fe(III), Co(III), Ca(II)
intermediate: Fe(II), Zn(II), Cu(II)
soft: Cu(I), Hg(II)
Hard acids prefer hard bases: more ionic bond
Soft acids prefer soft bases: more covalent bond
Hard Lewis acids: weakly polarizable, small ionic radii, high positive charge, strongly
solvated, empty orbitals in the valence shell and with high energy LUMOs.
Soft Lewis acids highly polarizable, large ionic radii, low positive charge, completely filled
atomic orbitals and with low energy LUMOs.
Hard Lewis bases weakly polarizable, small ionic radii, strongly solvated, highly
electronegative, high energy HOMO
Soft Lewis bases highly polarizable,large ionic radii, intermediate electronegativity, low
energy HOMOs.
Biological Ligands: amino acides (peptide/proteines)
amino acid
side chain
Histidine
H
Méthionine
Cysteine
Selenocysteine
Tyrosine
aspartique acid
glutamique acidH
Backbone : terminal amine pK ~8;, terminal COO- pKa ~4
pKa
Irving-Williams series
Stability constant (log K1) of divalent mtal ions
Problem: even coordination optimized for a specific metal
There is the possibility that other ions binds stronger
Example of a thermodynamic contol: Calcium
Normally Ca2+ concentrations are high extracellularly (~2mM) and unbound Ca2+
is low in the cytosol (~10nM). Ca2+ influx is used for signalling (secondary messanger).
Upon entrance Ca2+ binds to proteins, e.g. calmodulin
Ca2+ Kd: 0.1 µM – 1µM
Ca-binding induces conformational
Change, and opens binding
site for protein (red star)
(Mg2+ Kd: ~1mM (intracellular
free Mg2+ : 0.5 -1 mM))
Ca-binding site
Asn
H2O
Thr
Glu
Asp
Asp
Apo-Calmodulin
Ca-Calmodulin
Example: Metallothionein
Metallothioneins are cysteine rich proteins binding metal ions,
They are thought to be involved in metal metabolism (Zn and Cu)
and in metal detoxification (Cd, Hg)
normally they bind Zn(II) and Cu(I), but under high exposure to other metals,
in particular Cd(II) and Hg(II) they will bind them as well.
Cysteines contain a thiol group, i.e. RSH. Metals bind to the thiolate
(R-S-, deprotonated thiol)
R-SH + Mn+
[R-S-M] (n-1)+ + H+
General affinity of metal ions for thiolates (and metallothioneins):
Zn(II) < Cd(II) <Cu(I) < Hg(II)
Example:
Snail has 2 metallothioneins: HpCdMT and HpCuMT
Apparent Kd
HpCdMT
HpCuMT
Cu
1 pM
0.1 fM
Zn
30 pM
20 fM
Although HpCdMT binds Cu stronger than Zn,
HpCdMT binds Zn in the cell!
Because it depends also on the concentration of metal ions available
Estimated fee [Zn] : ~10 pM
Estimated free [Cu]: ~ 1 fM
Kd = [free M+] [unbound HpMT]
---------------------------------[M+-HpMT]
HpCdMT
[M+-CdHpMT]
---------------- =
[unbound HpCdMT]
free M+
1fM
---------- = ------- = 0.001
Kd
1pM
Example:
Although HpCdMT binds
Cu stronger than Zn, due to the
Avilability in a cell it will
Bind Zb (red triangle)
Question for training:
You have two chelators A and B
In line with Irving-Williams:
Kd of A for Cu(II) 1µM
Kd of A for Zn(II) 10µM
Kd of B for Cu(II) 10µM
Kd of B for Zn)(II 20µM
Define Kd (dissociation constant) and Ka (association constant)
Tell which chelator binds which metal when you do the following mixtures
1) 1mM A, 1mM B and 1mM Cu(II)
2) 1mM A, 1mM B and 1mM Zn(II)
3) 1mM A, 1mM Cu(II) and 1mM Zn(II)
4) 1mM B, 1mM Cu(II) and 1mM Zn(II)
5) 1mM A, 1mM B, 1mM Cu(II) and 1mM Zn(II)
Zinc in a classic cell: thermodynamic control?
Zinc(II) is buffered by proteins, small molecules (amino acids etc)
Zn(II) proteins and enzymes take Zn(II) up from « free » Zn(II)
Question for training:
The concentration of Zn(II) in mamalian cells is controled
by the transcription factor MTF1.
In simple way: MTF1 is a Zn-sensor, i.e. if Zn is bound to MTF1, this means
there is too much « free » Zn in the cell.
What is a transcription factor?
The dissociation constant of Zn to MTF-1 has not been exactly determined,
but was estimated to be 30 pM (Berg and coworkers, Biochemsitry 2004, p5437)
Define dissociation constant
Assuming when half or more of the MTF-1 in a cell is bound to Zn(II),
MTF-1 initiates the transcription of the protein metallothionein to bind
the excess Zn(II).
What is the « free » Zn-concentration at which this happens? Calculate.
Make a general conclusion about the concentration of a « free » metal
and the affinity of its sensor
Thermodynamic versus Kinetic Control of Metal-binding
Thermodynamic control
+ metal
ion
2
1
4
3
3
2
K1
1
Si K2 >> K1,3,4
K2
K3 K4
4
K1
1
2
K2
K3 K4
3
4
Kinetic control
1
3
k1
k3
2
k2
k4
2
1
4
« k1 >> k2,3,4 »
4
3
1
3
2
4
For Cu kon diffusion controlled
k1 = k2 = k3 = k4
1
3
k1
k3
2
k2
k4
4
k1
Mn+
+
Mn+-Prot
Prot
k-1
K1 = k1/k-1
Kinetics: Rate exchange of ligands
Reedijk Platinum Metals Rev., 2008, 52, (1), 2–11
Copper trafficking pathways in eukaryotes (kinetic control)
O'Halloran T V , Culotta V C J. Biol. Chem. 2000;275:2505725060
©2000 by American Society for Biochemistry and Molecular Biology
Cu(I) trafficking is under kinetic control
Kd of Cu(I)-proteins (in cell) 10-15 to 10-18 M
With Kd = koff/kon and assumed kon diffusion controlled
(fastest possible) koff 106 – 109 s-1, i.e. 11 days to 350 years
Proposed pathway for copper transfer from ATX1 to CCC2.
O'Halloran T V , Culotta V C J. Biol. Chem. 2000;275:2505725060
©2000 by American Society for Biochemistry and Molecular Biology
Copper in a classic cell
Banci et al. Nature 2010
Question for training:
You want to be able to add a very strong and specific chelator for Zn(II)
and Cu(I) into a cell,
1) How would you design a very strong (as strong or stronger than
proteins in the cell) and « specific » chelator for Zn(II) and Cu(I).
Make a propostion.
2) What could be the difference between a such strong chelator
for Zn(II) and Cu(I) in terms of the abilility to bind Zn(II) and Cu(I)
In the cell? Will the Zn and Cu-chelator be equal efficient?
Metals in the brain
Zinc
Copper
Becker et al.
Anal Chem.
2005 77:3208-16
Zn-Pools
Different Zn-pools:
A) tightly coordinated (thermodynamicly and kineticly)
more or less existing in all cells
e.g. catalytic site of enzymes (peptidase),
structural site of proteins (super-oxide dismutase)
Zn-fingers
only accessible to very strong chelators (and long incubaiton)
B) labile Zn-pool
The “extra” Zn in the Zn-containing neurons (absent in other neurons and cells)
not so tightly bound
accessible to complexation of chelators
How to Measure Zn in the Zn-Containing Neurons ?
Different Zn-pools:
- tightly coordinated (e.g. catalytic site of enzymes, structural site of proteins)
not accessible
- labile Zn-pool (The “extra Zn in the Zn-containig neurons)
not tightly bound accessible
Can be measured by
fluorophores “specific” to Zn
Examples for Fluorescent Detector of Zn
There are many more known:
Jiang & Guo
Coord. Chem. Rev. 2004, 248, 205-29
Examples for Fluorescent Detector of Zn
A) 2-Me-TSQ
B) Ratiometric Zn-sensor: FluoZin-3
Jiang & Guo
Coord. Chem. Rev. 2004, 248, 205-29
How to Bring a Chelator in a Cell?
Example zinquin:
Zinc homeostasis in neurons
(Colvin et al., 2003, Eur. J. Pharmacol.)
Roles of synaptic zinc
• Modulation of glutamic
responses
• Modulation of GABA
responses
• Antagonism on Ca2+, K+ and
Na+ conductances
• Probable role in disease-
associated neurodegeneration
(e.g. Alzheimer’s disease)
(Colvin et al., 2003, Eur. J. Pharmacol.)
Zinc-Release in the Synaptic Cleft upon Stimulation
Qian & Noebels, J. Physiol. 2005
Fluorescence increase of Zn-sensor
without ZnT-3
with ZnT-3
Extracellular Zn-chelator
Ca-EDTA
Intracellular Zn chelator
DEDTC diethyldithiocarbamate
Training
before
chelator
Addition
Training
after
chelator
addition
Question for training:
You study a process x in Zn-rich neurons, in which you
suspect that the labile Zn-concentration is changing
either extra or intracellularly.
Design an experiment, which allows you to conclude where (intra
or extracellualrly ) the labile Zn concentration is changing
What about Cu in the Brain?
much less known than for zinc, but evidence accumulates that copper
Can be released into synaptic cleft (like zinc)
Cu(I)
Cu(I) specific fluorescence based sensor for
biological applications developed (Fahrni et al. ; Cheng et al. etc)
Spatial resolved X-ray absorption
Cu(II):
Problem: Cu(II) normally quenches fluorescence,
thus difficult to design fluorescent Cu-chelators
And difficult to measure a labile Cu(II) pool
(if it exists)
Porphyrin-Fl is quenched: indicates Cu(II)? Release upon stimulation
Synaptic Copper and Zinc
M-Aβ
agrégats
ZnT3
Zn
MT-3
Zn
Aβ
Cu
CuATP7a
APP
Aβ
Presynapse
MT-3
MT-3
postsynapse
Indicate that up to 15µM Cu can be released into synptic cleft
Metals and Oxygen
Respiratory Chain: Metals and Oxygen
Why NADH does not react fast with oxygen?
Dioxygen: triplet ground state (two unpaired electrons)
Organic molecules: mostly paired electrons
Halliwell & Gutteridge put it on page 24 [11]:
“(The triplet ground state of O2)…imposes a restriction on electron
transfer which tends to make O2 accept its electron one at the time, and
contributes to explaining why O2 reacts sluggishly with many nonradicals.
Theoretically, the complex organic compounds of the human body
should immediately combust in the O2 of the air but the spin restriction
and other factors slow this down, fortunately!”
Reductant (NADH)
Reductant
e-
e-
O2
Metal ion (Cu(II))
slow electron transfer
e-
O2
Fast electron transfer
Reactive Oxygen Species (ROS)
Strong link between redox metal and oxygen
Redox metals can be «good» or «bad»
So called reducing agents (ascorbate, glutathione etc) can
be prooxidants
Depends on the Coordination
Conclusion
O2 metabolism
Oxidative Stress
(HO●, O2 ●-…)
Redox
metals
(Cu, Fe, Mn..)
Tight link between redox metals and ox stress
Redox metals (e.g.Copper, iron) are ideal to abolish or produce radicals
Coordination of the metal ion defines reactivity
Reactive Oxygen Species (ROS)
Free or loosely bound redox-metals(e.g. Cu)
+ eˉ
O2
O2ˉ
+ eˉ + 2H+
SOD (Cu,Zn)
H2O2
+ eˉ
HO ˉ + HO 
+ eˉ + 2H+
CytC Oxidase (Cu,Fe)
Strong link between redox metal and oxygen
Redox metals can be «good» or «bad»
Depends on the Coordination
2H2O
Redox Metals and Reducing Agents
Reducing agents of organic molecule type (VitC, VitE, glutathion etc)
Antioxidants like to give an electron
e.g. VitC:
°R +
VitC ->
H-R + °VitC
But another possibility:
PROoxidant: R +
VitC ->
°R + °VitC (VitC as prooxidant)
Proxidant activity of VitC can be catalyzed by redox metals (often loosly bound)
Production of HO° by different Cu-Complexes
Under aerobic conditions
and with ascobate
Coordination chemistry of Cu determines the amount of HO°
Metal metabolism has to be tighly controlled
Guilloreau et al. ChemBioChem, (2007)
Alzheimer’s Disease
And the Amyloid Cascade Hypothesis
Alzheimer’s Disease: Morphological Hallmarks
Neuronal death
Amyloid plaques
Neurofibrillary tangles
Important Factors in Alzheimer’s Disease (AD)
- Aggregation of the peptide amyloid-beta (Aβ)
- Hyperphosphorylation of the protein tau (neurofibrillary tangles)
- Genetic factors (mutations) increasing the risk of AD
- Diminution of acetylcholine concentration in the brain
- Role of metal ions
- Role of membranes
- Oxidative stress (production dreactive oxygen species (ROS) like
OH°, H2O2, O2-, NO°)
- lipide peroxydation
- protein oxydation
- DNA/RNA adducts
- etc
Drugs on the market:
Approved by FDA:
Inhibitors of Acetylcholinerase
Donepezil (Aricept ™)
ENA-713 (Exelon ™)
Galantamine (Reminyl ™)
Tacrine (Cognex ™)
NMDA- receptor antagonist.
Memantine (Namenda ™)
Excessive activation of N-methyl-D-aspartate (NMDA) receptors may underlie
the degeneration of cholinergic cells. Memantine is a fast, voltage-dependent
NMDA- receptor antagonist. It blocks the NMDA receptor in the presence
of sustained release of low glutamate concentrations and thus attenuate
NMDA receptor function.
Not approved by FDA, but medication sold over the counter
Alpha-tocopherol (Vitamin E)
Melatonin ???
Source: Alzheimer research forum
http://www.alzforum.org/new/
Amyloid- in Alzheimer’s Disease
α-secretase
β- and γsectretase
soluble A
APP
not toxic
Healthy
neuron
healthy brain
aggregates
Amyloid
plaques
toxic
(ROS)
Degenerated
neuron
Alzheimer brain
Amyloid-beta (Aβ) peptides
Native Aβ peptides
Form Aβ42 (42 amino acids)
D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-G-A-I-I-G-L-M-V-G-G-V-V-I-A
hydrophylic
hydrophobic
Form Aβ40 (40 amino acids)
D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-G-A-I-I-G-L-M-V-G-G-V-V
Metal binding site
Aggregation of Amyloid-beta (Aβ)
Aβ monomer
Structure in water:
random coil
micelle environment:
Alpha-helical
(Zagorski et al.
JACS 126; 1992 (2004))
15’
?
1h
?
Craik et al. Biochem. 1998, 11064
Beta-sheet
Riek et al. PNAS 2005
24h
Nielsen Methods Enzym.
(1999) 309: 491
Metals in Alzheimer’s Disease
Role of Metals in the Aggregation of
the peptide -amyloid
Amyloid- (A)
mM concentrations
of Cu, Zn, Fe
Evidence for a Role of Metals in Amyloid-β Aggregation
Some examples:
- mM concentrations of metals in the ßA-plaques
- metal homeostasis affected in AD
- Zn and Cu enhance the aggregation of ß-A in vitro
Cl
- metal chelator clioquinol
(5-chloro-7-iodo-8-hydroxyquinoline)
5
reduce plaques in mice model
8
1
I
N
clinical trials in phase II
clioquinol
OH
-mice with knocked out Zn-transporter (ZnT3): less plaques because less
Zn in the synapse
A
Zn
Zn Zn
Zn
Zn Zn
Zn
A plaques
Zn
Metals are involved in Alzheimer’s disease
In healthy conditions: redox metal metabolism is very well regulated
Concentration, compartimentation, transport, excretion etc
(by transport proteins, sequestering proteins, chaperons etc.)
Deregulation of metal metabolism in Alzheimer’s disease
Oxidative stress
Questions:
- Is this deregulation of metals an early event (important) or late event (less
important) ?
- What type of deregulation occurs?
- Can we fix that with metal chelators ? (Some Cu(II) chelators entered clinical
phase II studies)
Metals and Amyloid- in Alzheimer’s Disease
No ROS
+
APP
Zn
Cu
not
toxic
Healthy
neuron
toxic
(ROS
with Cu)
Degenerated
neuron
healthy brain Alzheimer brain
Cu and Zn binding supposed only to occur in Alzheimer’s
Cu promotes neurodegeneration of Aβ, Zn rather protects
Metals and Amyloid- in Alzheimer’s Disease
1
+
APP
No ROS
Zn
Cu
not
toxic
Healthy
neuron
toxic
(ROS
with Cu)
Degenerated
neuron
healthy brain Alzheimer brain
Cu and Zn binding supposed only to occur in Alzheimer’s
Cu promotes neurodegeneration of Aβ, Zn rather protects
Metals and Amyloid- in Alzheimer’s Disease
No ROS
+
APP
2
Zn
Cu
not
toxic
Healthy
neuron
toxic
(ROS
with Cu)
Degenerated
neuron
healthy brain Alzheimer brain
Cu and Zn binding supposed only to occur in Alzheimer’s
Cu promotes neurodegeneration of Aβ, Zn rather protects
Metals and Amyloid- in Alzheimer’s Disease
No ROS
+
APP
Zn
Cu
not
toxic
Healthy
neuron
toxic
(ROS
with Cu)
3
Degenerated
neuron
healthy brain Alzheimer brain
Cu and Zn binding supposed only to occur in Alzheimer’s
Cu promotes neurodegeneration of Aβ, Zn rather protects
Metals and Amyloid- in Alzheimer’s Disease
No ROS
+
APP
not
toxic
Healthy
neuron
Zn
Cu
4
Chelator
(native, therapeutic)
toxic
(ROS
with Cu)
Degenerated
neuron
healthy brain Alzheimer brain
Cu and Zn binding supposed only to occur in Alzheimer’s
Cu promotes neurodegeneration of Aβ, Zn rather protects
Dynamics of Metal-Amyloid-β
1. Intramolecular
2. Intermolecular
NMR study of Cu(II) interaction with A
NMR study of Cu(II) interaction with Aβ : 13C data
pH 6.5
I
Hureau, C.; Coppel, Y. et al. Angew. Chem. Int. Ed. 2009, 48 (50), 9522-9525.
pH 8.7
II
NMR and EPR study of CuII-Amyloid-
13C-NMR
(and 2D 13C-1H experiments) in solution,
EPR (pulsed and ENDOR) on specifically isotopically labeled Aβ1-16
Major form at pH 7.4 (pure at pH 6.5)
at pH 9)
Minor form at pH 7.4 (pure
Very dynamic, equilibrium between different coordination modes
Hureau et al. Angew. Chem. 2009
Murine Amyloid-beta (Aβ) peptides
Comparison mouse/rat and human Aβ
Mouse:
-No Aβ aggregation in brain
-Less toxic to cells
-Less aggregation in vitro (+/- Cu(II))
-Cu(II) binds differently to human and mouse
human
D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-G-A-I-I-G-L-M-V-G-G-V-V-I-A
mouse
D-A-E-F-G-H-D-S-G-F-E-V-R-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-G-A-I-I-G-L-M-V-G-G-V-V-I-A
Difference in Cu(II)-binding of mouse and human?
Murine Amyloid-beta (Aβ) peptides
Comparison mouse/rat and human Aβ
Mouse:
-No Aβ aggregation in brain
-Less toxic to cells
-Less aggregation in vitro (+/- Cu(II))
-Cu(II) binds differently to human and mouse
human
D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-G-A-I-I-G-L-M-V-G-G-V-V-I-A
mouse
D-A-E-F-G-H-D-S-G-F-E-V-R-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-G-A-I-I-G-L-M-V-G-G-V-V-I-A
Which replacement of amino acid(s) is responsibme for the different Cu(II)binding?
-> Replacement of Arg to Gly in human Aβ at position 5 induces mouse like Cu(II)
binding (spectroscopic techniques: CD and EPR) -> sufficient
13C
Nuclear Magnetic Resonance (NMR) of Aβ and Cu(II)-Aβ
Human Aβ
Mouse Aβ
Cu(II)
added
No Cu(II)
Cu(II)-coordination is different for human and mouse Aβ
Model of Cu(II)-binding to human and mouse Aβ ?
Human
pKa 7.7
Mouse
pKa 6.2
Eury, et al.
Angew. Chem.
2011
Predominant forms of Cu(II)-binding to human and mouse Aβ at phys. pH?
Human
pKa 7.7
Mouse
pKa 6.2
Eury, et al.
Angew. Chem.
2011
Comparison of human and mouse Cu(II)-Aβ:
What is the consequence of the different Cu(II) coordination?
1) Different affinity: Cu(II): mouse Aβ 3 x stronger than human Aβ
2) Redox activity: mouse Cu(II)-Aβ: lower redox activity
-> generates less ROS
Eury, et al.
Angew. Chem.
2011
Transgenic mice as Alzheimer’s model:
Transgenic mice
Express human and mouse Aβ:
Cu(II) preferentially bound to mouse
Less aggregation, less ROS production
Humans
Express only human Aβ:
Cu(II) bound to human
More aggregation, more ROS production
Amyloid plaques in AD model mice bind less metals than human (Leskovjan et al. 2009)
Limitation of transgenic mouse as AD model?
Eury, et al.
Angew. Chem.
2011
Copper, Zinc and Abeta in Alzheimer’s
M-Aβ
agrégats
Metals
dysfunction
ZnT3
Zn
MT-3
Zn
Aβ
Cu
CuATP7a
APP
Aβ
Presynapse
MT-3
MT-3
postsynapse
Source: wikipedia
- Deregulation of metal ions modulate Abeta toxicity
-Could affect LTP (memory) and lead to neuronal death
-Still not clear who triggers whom (Abeta and metals)
QUESTION:
• One can find from time to time publications, in which the authors try
to identify the metal that is bound to a certain protein under
physiological conditions. The reason that they do not know the
identity of the metal is that they started from the protein gene and
gene analysis proposed a metalloprotein (e.g. by the identification of
a metal-binding motive in the sequence). Then they overexpress
the protein in a bacterium, purify it and measure the dissociation
constant of the complexes of Cu(II), Zn(II), Fe(III), Co(II), Mn(II),
Ca(II), K(I),Na(I),Mg(II) with that apo-protein (apo: demetallated
protein). Then they conclude that the metal ion that has the highest
affinity is the physiological bound one.
• What do you think about this strategy?
• Can you propose an alternative method to confirm the identity of the
metal in the metalloprotein?