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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 O2ˉ + 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?