Solubility Profiles of Amyloidogenic Molecular Structures

Transcription

1 Solubility Profiles of Amyloidogenic Molecular Structures Key Theories Towards Meaningful Experiments Florin Despa, PhD Department of Pharmacology The University of California, Davis

2 Outline 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) Despa, Fernandez, Scott & Berry, JBP (008) B. hiapp: relationship between the state of aggregation of the protein and the degree of toxicity induced in cells

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4 How does human islet amyloid polypeptide (amylin, hiapp) become toxic? P agg Amyloid Formation Beta Cell dysfunction Hyperinsulinemia & Hyperamylinemia Molecular denaturation induced by molecular crowding Time (min) Despa, Biophys. Chem. (009) Oversecretion of PPinsulin & PPamylin in the ER hiapp: KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY riapp: KCNTATCATQRLANFLVRSSNNLGPVLPPTNVGSNTY Hyperglycemia & Increased Metabolic Demands

5 toxic molecular species 1 nm 10 nm 100 nm 100 nm 1 μm 100 μm ex vivo detection electron microscopy light microscopy non invasive detection NO YES adapted from

6 In Vivo MR Microimaging of Individual Amyloid Plaques in Alzheimer s Transgenic Mice Jack et al., J. Neurosci. 5: , 005

7 Amyloidogenic Proteins are characterized by: an increased number of poorly dehydrated backbone HBs Fernandez, Kardos, Scott, Goto & Berry, PNAS, (003) Fernandez & Berry, PNAS, (003) large surface densities of patches of bulk-like water De Simone et al., PNAS, 005 favor protein association Despa, Fernandez & Berry, PRL, 004 Despa and Berry, Biophys. J, (007); Biophys. J, (008)

8 Pathogenic Proteins Have Poor Desolvations of the Intramolecular HBs Fernandez, Kardos, Scott, Goto & Berry, PNAS, (003) Fernandez & Berry, PNAS, (003)

9 The DEHYDRON is a hydrophilic structural defect Fernandez, Kardos, Scott, Goto & Berry, PNAS, (003) Water in the desolvation region is mostly in contact with hydrophobes.

10 Water Molecular Dipoles Correlated in Pairs Through an Entropic Effect P r ( E) = ( A π ) E 1 4 A e Polarized Water f =1 f = r ( μ f ) α ( f ) A = r μ N EL r, μ N 18 = r dep r 0 r r ( E) μ( E) Q Nd f =3 Despa, Fernandez & Berry, PRL, 004 τ S ( ps) η βe L d correlated water l = 0 1 nm l0 = 0. 75nm Despa, PCCP, 008 experiment Tan et al., JCP (005) R ( nm) Despa, PCCP, R (nm) The fraction of correlated water and its characteristic relaxation time depend on the degree of confinement.

11 r c b b Water structured at the interface between hydrophobes is a source of induction-dispersion effects that favor protein association () () r r g f v ASAr d l r b r b r U c h = μ πε γ ε r ( ) 1 Kcal mol U h ( ) o A r wetting regime Despa and Berry, Biophys. J, (007) Despa and Berry, Biophys. J, (008)

12 Proteins presenting structural defects are characterized by an increased fraction of water with a bulk-like behavior. τ np ~ s (bulk-like water) (structural defects) τ ~ 10 b 1 s (bulk water) τ >10 8 s c (caged water) τ p ~10 9 s (highly structured water) Prion (PrP C ) Solvation Map De Simone et al., PNAS, 005

13 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.

14 Aqueous Environment Containing Protein Background + Test Isomers η 0.69 C test isomer native protein η A protein background N0 1mM V 1μm 3 B + ΔN ( 0. mm + ΔN ) Dimers Compact Aggregates negative control η 0.69 Floppy Aggregates

15 Partition of Water in the Protein System f ( A) test isomer (increased number of surface defects) native protein f surface density of defects ( f A ) > f ( A) ( A) V water = N ( Vs + Vc ) + ΔN ( Vs + Vc ) + V r (surface water) V s V ( 1 f ) V p V s f np V s τ τ p np ~10 ~ 10 9 s 10 s (highly structured water) (bulk-like water) (structural defects) (caged water) V c ( ρ ρ ) = Vm VW = Vm max τ >10 8 s c (caged water) Despa, Fernandez, Scott & Berry, JBP (008)

16 Water Fractions protein background + test isomers scaling theory (Reiss, 1965) VS 0. 11V m c p np r = ( ρ ρ)( 1 η) 1 η = η 1 η Nf + ΔNf = 0.11 η N + ΔN = 1 max c η p ( A) fn + f ΔN N + ΔN np ( A) (caged water) (highly structured water) (bulk-like water) (bulk water) f ( A) f ( A) m (soluble oligomers) Despa, Fernandez, Scott & Berry, JBP (008)

17 Histograms describing the partition of constrained water in a system of protein background plus test isomers water fractions highly structured water caged water less structured water (bulk-like) τ >10 8 s c τ p ~10 9 s residence time τ np ~ s ( ) f A f f ( A) ( A) = f = 0.3 = 0.5 = 0.1 (control)

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

19 1. at equilibrium, most of the surface defects are buried inside the fibril:. packing density of fibrils is high so they exclude surrounding water (Petkova et al., Science, 005) f ( A) f ( ASA) c = ( ρ ρ )( 1 η) max η (caged water) Compact Aggregates ( ASA) np = 0.11 f 1 η N + ΔN η N + ΔN / 3 (bulk-like water) ( ASA) p = 0.11 ( 1 f ) 1 η N + ΔN η N + ΔN / 3 (highly structured water) ( ASA) r = 1 ( ASA) c ( ASA) np ( ASA) p (bulk water) Despa, Fernandez, Scott & Berry, JBP (008)

20 1. most of the surface defects are buried inside the aggregate;. packing density of fibrils in a plaque is sufficiently low so that, plaques contain additional caged water molecules (Nelson et al., Nature, 005) Floppy Aggregates ( agg ) c = ( ρ ρ )( 1 η) max η ΔN N + ΔN 1 η η ( ρ ρ ) a (caged water) ( agg ) np ( agg ) p ( agg ) r = 0.11 f = 0.11 = 1 1 η N + ΔN η N + ΔN ( 1 f ) ( agg ) c ( agg ) np / 3 1 η N + ΔN η N + ΔN / 3 ( agg ) p (bulk-like water) (highly structured water) (bulk water) caged water Despa, Fernandez, Scott & Berry, JBP (008)

21 Histograms describing the partition of constrained water in a system of protein background plus test isomers water fractions highly structured water caged water less structured water (bulk-like) τ >10 8 s c τ p ~10 9 s τ np ~ s residence time control protein background + test isomers dimers of test isomers compact protein aggregates Despa, Fernandez, Scott & Berry, JBP (008) floppy protein aggregates

22 = j j j j k T k TE T TR S S, 1, max exp exp 1 = j j j T T, 1 ( ) ( ) ( ) ( ) = =, 1, j L j j L j j j j L j j L j j C T C T ω τ τ ω τ τ τ ω τ τ ω τ τ Magnetic Relaxation Response

23 A Control Protein Background D Protein Background + Compact Aggregates B Protein Background + Test Isomers E Protein Background + Floppy Aggregates C Protein Background + Dimers Less Structured Water MR Intensity Signal (grayscale) S S max C D B ( c) ( b) T > T > T ( b) T > T T ( control) ( control) ( control) A (control) ( a) T < T ( control) 9.5 Highly Structured Water E TE (ms) AD samples can display both bright and dark spots on MR images; Bright spots are likely to indicate oligomers and protofibrils.

24 Water Proton NMR Spectroscopy of Amyloidogenic Structures 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 T values, shifting the MR signal towards values corresponding to bulk-like water. hiapp in serum Aβ 1-40 in serum serum+water µm 50 µm 100 µm 5 µm 50 µm 100 µm

25 NMR Setup Dr. J. Walton Dr. S. Anderson

26 Data Analysis ParaVision 3.0. to collect data, Nonlinear least square (NNLS) fit to analyze data

27 Water Proton NMR Spectroscopy of Amyloidogenic Structures T (ms) hiapp Abeta 5 m µm 50 c1 µm 100 cµm % of sample hiapp Abeta % of sample hiapp Abeta T (ms) 9 m c1 c 5 µm 50 µm 100 µm

28 Water Proton NMR Spectroscopy of Amyloidogenic Structures T (A) (ms) % of sample T (B) (ms) % of sample T (C) (ms) % of sample hiapp(m) 1.5, 7., , 1.11, , 0, , 97.38, 97.7 (96.33) control 0.95, 1.5, , 1.1, , 8.1, , 0.77, , 00, , 96.58, 96.8 (95.59) hiapp(c1) 1.4, 5.1, , 1.11, , 50, , 97.38, 97.4 (96.39) control (1) 1.1,.4, , 1.5, , 50, 50 (43.33) 95, 97.5, 97 (96.5) 5.4, , 0.58 hiapp(c) 1.8, 8.1, 6.8 3, 0.58, , 310, , 97.99, 97.7 (97.3) control().1, 7., 7.., 0.4, , 310, , 98.7, 98.1 (97.76) Abeta(m) 1.1, 1.9, , 1.88,.01 7., , , 10, , 95.97, 96.8 (95.76) Abeta(c1) 0.85,.4, , 1.7, , 5.7, , 0.47, , 310, , 96.8, 95.9 (95.4) Abeta(c) 0.71, 7., 1 6.3, 0.48, , 370, , 97.48, 96.3 (95.46) 1., , 0.44

29 The formation of oligomers and/or fibrils leads to an increase of the T value T (ms) Control hiapp Abeta m c1 c 5 µm 50 µm 100 µm % of sample Control hiapp Abeta m c1 c 5 µm 50 µm 100 µm hiapp in serum Aβ 1-40 in serum serum+water µm 50 µm100 µm 5 µm 50 µm 100 µm

30 How does the amyloid toxicity manifest at the cellular level?

31 Amylin-Induced Toxicity in Cardiac Myocytes cardiac myocyte Objective: To assess the functional alteration of myocytes induced by hiapp. Method: Monitor the intracellular Ca signal.

32 3Na Na Na K Sarcolemma ATP NC NHE PLM ATP Ca RyR Ca Ca H 3Na I Ca SR PLB ATP Ca T-Tubule Ca Ca Na- Ca 3Na Ca AP (E m ) Myofil [Ca] i Contraction Na H Cyt H Ca Ca Na Mito

33 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±0 nm; - the fluorescence ratio F340/F380 was calculated after background subtraction.

34 Ca-mediated Cardiomyocyte Contraction AP (E m ) [Ca] i Contraction

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36 Intensity of Magnetic Response of Water Concluding Remarks S bulk-like water fibrils soluble amyloidogenic proteins Amyloid proteins and embryonic amyloid composites can be differentiated based on their hydration profiles and characteristic MR signals of the surrounding water. 0.5 normal protein background structured water large, floppy aggregates TE (ms)

37 Clinical Implication: MRI Contrast Mechanism for Detection of AD In Vivo Magnetic Resonance Microimaging of Individual Amyloid Plaques in Alzheimer s Transgenic Mice Jack et al., J. Neurosci. 5: , 005

38 Intensity of Magnetic Response of Water Concluding Remarks S bulk-like water fibrils soluble amyloidogenic proteins normal protein background structured water large, floppy aggregates TE (ms) Amyloid proteins and embryonic amyloid composites 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. Oligomers are the most toxic species. Fibril growth at the membrane is equally toxic. T 13ms T 53ms 5 µm 50 µm

39 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)