Amyloid diseases Flashcards

Question

what was discovered about amyloid deposits by xray diffraction and how?

Answer 1

X-ray diffraction can be applied to any repeating structure: – Fibre diffraction can be used to provide information on order in fibres – We need repeating structure (typically need a crystal, so can get diffraction in three dimensions) – order in one dimension can give some information but not so much when we do fibre diffraction experiments, we get information about how the repeating structures are present in the fibre. * This gives rise to unique reflection that report on the periodicity and the relative orientation of the structures with respect to the fibril axis.

Answer 2

Braggs Law

Answer 3

Normally when we do X-ray crystallography, we have 3D crystals so that we have repeating structures in 3-dimensions so that we can obtain diffraction data in all directions. * In the case of fibrils as you will see we do have order, just not 3D order. So when we do fibre diffraction experiments, we get information about how the repeating structures are present in the fibre. * This gives rise to unique reflection that report on the periodicity and the relative orientation of the structures with respect to the fibril axis.

Answer 4

X-ray diffraction relies on the repeating properties of a system to diffract X-rays. Relies on the constructive/destructive interference of waves as they are scatted after interacting with the atom. Intensity at a given location depends on the wavelength (l), the spacing and the angle of the incident beam. essentially to get diffraction spots we need for the diffracted waves to be in phase. This occurs when the separation between the plane is equal to (n times the wavelength of the X_ray)/2 time the sine of the incident angle. * Further away from the centre of the plate the shorter the distance is

Answer 5

* Meridional reflections (top/bottom) indicate a regular spacing of 4.8Å * Equatorial reflection indicate a spacing of between 10-11Å

Answer 6

the separation between strand of a beta sheet is about 4.8A * spacing of fribrils are indicative of the gap between beta strands * the meridional reflections indicate that we have a repeating structure running perpendicular to the fibre long axis.

Answer 7

the equatorial reflections, these are indicative of 10-11 A spacing and the repetitive structure is parallel to the fibril long axis. | packing of beta sheets across the width of the fibre

Answer 8

cross beta structure - beta strands run perpendicular to the fibres axis as well as packing of beta sheets across the width of the fibre the meriodonal reflections 4.8A - spacing is regular because they are fixed by H-bonding between beta strands the equitorial reflections 5-12A are variable depending on the sidechains (primary sequence) packing between sheets ## Footnote they can be formed from proteins, that are frequently highly soluble and structured

Answer 9

* Proteins tend to be hydrophobic (exception PolyQ – htt in huntingtons disease) * Slight preference for β-strands * Minimum size 6 residues

Answer 10

it does not depend on the fold of the original protein (alpha-helica proteins and beta-sheet proteins can both make the transition into the cross-beta structures required for amyloid fibril formation).

Answer 11

the cross-β motif is formed from the lamination of successive β-sheet layers, and it is abundantly observed in the core of insoluble amyloid fibrils associated with protein-misfolding diseases. It is a double β-sheet, with each sheet formed from parallel segments stacked in-register. Sidechains protruding from the two sheets form a dry, tightly self-complementing steric zipper, bonding the sheets

Answer 12

protein misfolding or mis processing

Answer 13

Levinthal's paradox is that finding the native folded state of a protein by a random search among all possible configurations can take an enormously long time. Yet proteins can fold in seconds or less.

Answer 14

* protein folding is a directed process. * When a protein is unfolded it has a high energy, with many degrees of conformational entropy. * Proteins fold by sequentially making favorable interactions, * These are frequently more local (perhaps small regions of secondary structure) – so call compact configurations. * These slowly start to coalesce, forming more favorable interactions and the formation of a so called transition state. * This continues to fold, until we end up with the native protein fold. (wild type structure).

Answer 15

if during the course of folding and unfavorable interaction is made, that leads to a local energy minima where there is insufficient thermal energy to extract it, then the protein will be trapped in a non-native state. In the case of amyloid proteins this indeed thought to be the case, and perturbingly, it appears that the amyloid fibrils formed have perhaps the lowest energy conformation a protein can adopt (think about it we have maximized the number of H-bonds, the sidechains are all interacting).

Answer 16

In cells, no single molecular species is present at an extremely high concentration, but a significant proportion of the volume is physically occupied by various macromolecules and cell organelles. ## Footnote the presence of other macromolecules influence the activity and folding of the proteins present.

Answer 17

– Reduces configurational entropy – Increase in ΔG (ΔG= ΔH –TΔS) (reduces the entropy of the system) – Shown to increase rate of protein aggregation ## Footnote the presence of other macromolecules influence the activity and folding of the proteins present.

Answer 18

Here the environment is rich in long fibrous molecules such as collagen, glycosaminoglycans and proteoglycans, all of which demand large volumes and limit the space proteins have to fold.

Answer 19

monomer nucleation event (oligomers) protofibril elongation fibril formation

Answer 20

rate using a t assay < - light phase while nucleation is about to occurs then

Answer 21

the degree of hydrogen bonding and the stability of the sturctures

Answer 22

view the process through an electron microscope

Answer 23

There is evidence that each part of the aggregation pathway could be the culpret

Answer 24

Ecoli extracellular matrix used to colonise surfaces and from biofilms now used as a scaffold for colonizing inert surfaces and mediate binding of host proteins

Answer 25

A small protein normally found on the surface of many cells, including lymphocytes, and in small amounts in the blood and urine. An increased amount in the blood or urine may be a sign of certain diseases, including some types of cancer, such as multiple myeloma or lymphoma. It is part of the light chain of MHC class 1 its role in antigen presentation is one of faulty control

Answer 26

Virus taken up by the cells and broken down by proteosome Viral peptides loaded into endoplasmic reticulum, they need to be presented, Antigen are loaded in the heavy chain of MHC1, beta 2 macroglobulin acts as a quality control mechanism – so the binding of the beta microglobulin is a necessary step before the protein is shuttled to the cell surface where the antigens will be presented to the T cells

Answer 27

* Fibres – β2 microglobulin – Truncated β2m (DN6) * Proteoglycans – Heparan sulphate, dermatan sulphate, glycosaminoglycans * Collagen * Serum Amyloid P Component (SAP)

Answer 28

* Seven stranded b sheet fold typical of Ig superfamily * Two sheets composed of residues A, B, E, D and C,F,G * Disulphide bond C25/C80 * Strand D is split (has break in the beta sheet structure)

Answer 29

See fibrillar structures under electron microscopy or congo red birefringence we see the firbirllar like structures and apple green birefringnecne the hallmarks of amyloid fibres If we look at these fibres in vitro (we can create them by lowering the pH to pH2.5-3.5) We find these fibre diffraction pattern that point to a cross beta structure

Answer 30

Twisted structures shown via electron microscopy Two types of packing (morphology) named a and b

Answer 31

two types of reflections meridional (4.8A)(further out so smaller distance correspond to space between beta strands) and equitoral (10-11A)(closer to centre so larger distance so is the packing between beta sheets)

Answer 32

cryo Electron microscopy combined with solid state NMR cryo EM provide global structure at lower resolution solid state NMR provides local structure at high resolution

Answer 33

Fibril formation can be detected by a change in the emission spectrum of ThioflavinT (ThT

Answer 34

when incubated with amyloid fibres it binds to them and the fluorescence of molecule increases – so we can use the fluorescence as a proxy for the amoint of amyloid that is forming We can then measure emission Makes assays quicker and easier – monitor in real time

Answer 35

Thioflavin T assay

Answer 36

Added tht to monomeric beta 2 microglobulin and then added seed Different compounds like heparin, SAP, ApoE, collagen, mixtures of these, human serum, uremic fluid, synovial fluid, DRA tissue (things people are finding in the deposits)

Answer 37

* destabilise protein structure * stabilise structure/ act as a template for aggregation

Answer 38

destabilise protein structure * Cu2+ * Lysolipids * Truncated protein * Advanced glycation end products (AGE) stabilise structure/ act as a template for aggregation * Apolipoproteins * Heparin (GAGs) * Collagen * SAP

Answer 39

destabilise protein structure * Detergents * Acidic condition * Ionic strength * TFE

Answer 40

o Deletion of the six N-terminal residues renders allows b2m to spontaneously form fibrils o In-vivo deposits contain >25% of b2m-ND6 o Not clear if cleavage occurs whilst b2m is in its globular form or when it is assembled into fibrils o Folded structure is 2.5 kcal mol-1 less stable o Has a greater propensity to oligomerise in solution (size exclusion chromatography)

Answer 41

In-vivo deposits contain >25% of b2m-ND6

Answer 42

Dilute unfolded b2m into buffer and monitor Trp flourescence (we see flourescence getting buried as it folds) * Folding is a 2 step process (one fast step and one slow) and this can be mapped on a graph. Dilute the folded b2m in guanidium cloride and monitor Trp flourescence (* Dissolution single step reaction) plot on a graph

Answer 43

typical of cis/trans isomerisation of Pro proline32 is responsible (changing Pr32 to GLy removes the slow step)

Answer 44

Rate of fibril formation is much higher when proline32 is locked into trans state The molecular switches that help protect aggregation include conserved proline (Pro) residues, which not only modulate the rates of folding but also affect both oligomerization and amyloid fibril formation. A growing body of evidence suggests that certain Pro residues increase the fibrillation propensity due to the process of cis/trans isomerization of the peptide bond preceding Pro. **Stabilization of P32 in the trans configuration leads to the disappearance of the β-bulge in the D-strand**

Answer 45

chaperone machinery and two degradation systems, these inherent protective mechanisms include phosphorylation, acetylation, and the inherent properties of the amino acid sequence.

Answer 46

n terminal deletion 6 residues

Answer 47

* Used liquid state NMR methods to determine the structure of a folding intermediate of β2-microglobulin * Confirmed that the folding intermediate had similar/identical spectral properties to a N-terminal deletion with known propensity to form fibrils * Identified areas of instability in the N-terminal deletion that contribute to ability to form fibrils * Demonstrate that small populations of the N-terminal deletion are able to destabilize native protein and induce fibril formation

Answer 48

o Presence of Cu2+ enhances rate of fibrillization Cu2+ stabilizes Pro32 amide bond in trans configuration * Crystal structure of b2m reveals Cu2+ ligated to His31 * Copper acts as Lewis Acid interacting with lone pair electrons of nitrogen of Pro32 * Destabilizes amide bond permitting cistrans conversion

Answer 49

the bulge prevents the formation of hydrogen bonds that allow dimer formation (without the bulge, the D strand can now form additonal H bonds, so the long continuous d strand can form the bond to allow dimer formation, enhance the rate)

Answer 50

– “A chronic or persistent disorder of the mental processes caused by brain disease or injury and marked by memory disorders, personality changes, and impaired reasoning”

Answer 51

* In 2001, 24 million people had dementia * By 2040 it is expected to rise to 80 million due to improvements in life expectancy (aging population)

Answer 52

* Alzheimer’s is the most common form of dementia (50-60% of all cases)

Answer 53

* First described in 1906 by Alois Alzheimer * Published a paper entitled ''About a peculiar disease of the cerebral cortex”

Answer 54

* Describes a 51 year old female patient Auguste, D. with progressive senile dementure * Symptoms: – progressive memory loss, focal symptoms, delusions, hallucinations – He describes the presence of plaques and tangles present in the brain post-mortem.

Answer 55

* Two types of deposits characterized by their different appearance under the microscope. – Neuritic plaques: amyloid beta - extracellular – Neurofibrillary tangles: hyperphosphorylated tau - intracellular * Found in – Medial temporal lobe – Cortical areas

Answer 56

plaques are rich in amyloid-beta peptide and serum amyloid P component

Answer 57

tangles are formed from hyperphosphorylated tau, and serum amyloid-P component.

Answer 58

serum amyloid P

Answer 59

**serial and parallel: ** Serial suggest elevated levels of amyloid beta peptide leads to an increase in tau phosphorylation and so increase in synaptic loss and cell loss. Parallel says that there are increased levels of amyloid beta and increased levels of tau then you have synaptic loss and cell loss

Answer 60

Tau is characterized as a: * Microtubule associated protein * 50-64kDa little secondary structure when purified * Role in tubulin assembly, inducing microtubule bundling and promoting neurite outgrowth. * In AD it becomes hyperphosphorylated, and starts to fibrilise via small oligomeric intermediates * Ultimately it form paired helical filaments, with normal amyloid morphology

Answer 61

1) EM images show the classical fibrillar type structures. (amyloid fibre) 2) The fibre diffraction studies reveal a typical cross-beta structure. 3) Solid-state NMR data (H/D exchange) reveal the location of the beta-strands.

Answer 62

Amyloid beta peptide (Aβ) is produced through the proteolytic processing of a transmembrane protein, amyloid precursor protein (APP), by β- and γ-secretases. Aβ accumulation in the brain is proposed to be an early toxic event in the pathogenesis of Alzheimer's disease, physiolpogical function is unknown

Answer 63

* The deposits are composed of different types of amyloid beta that vary in length from about 39 to 42 (can find 37) – in contrast to other types of amyloid proteins

Answer 64

Poor Fidelity of cleavage event of the C terminus causes the variation, a reflection of the environment where it occurs ## Footnote ultimately the variation depending of where it is cleaved at the C-terminus

Answer 65

– 1-42  most amyloidogenic (spontaneously forms amyloid fibres, no seed needed) – 1-40 – 1-39

Answer 66

In solution the peptide is largely unstructured. When expressed as part of a phage display library we see that the C-terminus adopts a strand-turn-strand type structure, with the N-terminus largely unstructured. * Structure of monomeric Aβ: – Beta strand structure C-terminus – N-term (in solution unstructured)

Answer 67

* Strong miridional reflections with a spacing of 4.7Å – Spacing between β-strand * Weaker equatorial reflections with a spacing of 10Å – Spacing between β-sheets * Classical cross β-structure

Answer 68

The HSQC (Heteronuclear Single Quantum Coherence) In the case of many of the H/D NMR exchange studies a commonly used experiment is the HSQC. The HSQC is widely used in protein NMR as it correlates the resonance frequency of two adjacent atoms – typically the amide proton and amide nitrogen. (can be carbon) This means we will have a spectrum which contains a peak for each amino acid (not Pro). As the position of the spectrum depends on the local structure – these can provide a finger print of the protein

Answer 69

NMR uses microwaves to probe the local structure of the molecule. Relies on the interaction of the nuclear spin with an external magnetic field resulting in the presence of two or more energy levels. As with any form of spectroscopy, we can look at the absorption of electromagnetic radiation (in this case a radiowave) to report on the energy separation.

Answer 70

a chemical reaction in which a covalently bonded hydrogen atom is replaced by a deuterium atom, or vice versa an indirect way to probe the dynamics and conformational properties of biomolecules in NMR and mass spectrometry, providing information about the solvent accessibility of labile proton A proton and a deuterium are two isotopes of hydrogen We detect them on entirely different regions of the spectrum. If we replace a proton with a deuterium the signal disappears from the spectrum

Answer 71

1) Take a fully protonated fibril. 2) Dissolve in D2O (deuterium oxide – water with the protons replaced with deuterons)(all protons accessible to solvent are exchanged, the ones of the edges of the structure) 3) Leave it for a while (to let the H and D exchange – this need optimizing) 4) After a period of time the sample is flash frozen and freeze dried (lyophilise). This removes the water whilst retaining its frozen state. 5) The sample is then resuspended in deuterated DMSO (an aprotic solvent) 6) We can then run NMR and MS analysis on the protein to see which sites have undergone exchange.

Answer 72

DMSO has two important properties: amyloid proteins are soluble in it (so you have a small molecule which can tumble rapidly that you do liquid state NMR on), it is an Aprotic solvent (it cannot facilitate the transfer of protons) - which means we cant cant exchange of protons and deuterium meaning that any protons and deuterium that were switched in the earlier step are going to stay in the same place ## Footnote a proton or deuteron that was in a particular position remains in that position.

Answer 73

Take fully protonated proton and do HSQC experiment on it – we get a spectrum and one dot for every NH pair (indicating the proton). Then if we do the same experiment after H/D exchange and resuspension in DMSO then we see all the protons on the surface (blue) they exchange with deutriums and disappear form the spectrum. So the dots that remain are the protons which are integrated in the secondary structure In amyloid fibrils these are typically the ones contributing to the cross beta structure – so now we know which residues in you polypeptide are actually forming the cross beta structure

Answer 74

this technique makes use of the fact that hydrogen and deuterium (two of the isotopes of hydrogen – resonate a different frequencies in the NMR spectrum). So if a proton exchanges with a deuteron it will essentially disappear from the proton spectrum.

Answer 75

This factor is basically the ratio of the peak intensity for the H/D exchanged sample divided by that for the fully protonated sample. When no exchange takes place, we see high levels of protection, when exchange occurs rapidly we see low levels of protection. High protection factor means they are integrated in the structure, low means on the edges

Answer 76

The first 15 residue show a relatively low protection factor which means they are accessible to the solvent and exchange occurs. Residues from 15 to 23 and again 28-35 we have high protection factors.(shows integration into the structural motif). This arise from the fact the we have a strand/turn/strand structure. The N-terminal domain has low protection factors indicative of an unstructured region. Similar that look region 23-28, shows high rates of exchange as the N-H are not in H-bonds. ## Footnote High protection factor means they are integrated in the structure, low means on the edges

Answer 77

Use magic angle spinning where we mechanically introduce the tumbling that you would normally see in solution to get a high resolution spectrograph – to get these plots In these 2D experiments above what we see are carbon-13 resonances in the NMR spectrum. Along the diagonal in the spectra we have essentially a carbon-13 spectrum of the fibril. Off diagonal peaks correspond to carbon atoms which are in close proximity to one another If we can assign each of the resonances (peaks) to a particular atom, then we can use this information to start to produce a structure of AB fibrils. We can start to measure distances between aa – to build a picture of what they look like One the basis of this we can start to produce a model ## Footnote Here we see a largely disordered N-terminus, with a strand-turn-strand structure and the C-termius.

Answer 78

No Apart from the fact that Tycko knew that he could generate multiple conformations (diff students were ending up with doff structures), there was also a lot of medical data that suggested that the polymorph formed is important – depending on which source you saw different levels of toxicity - some lead to rapid cell death and others dont

Answer 79

1) Structurally distinct fibrils exhibit difference levels of toxicity. 2) Propagation of Aβ in transgenic mice depends on source of exogenous Aβ. (When administered to mice the source of the AB fibrils could give rise to very different rates of spread.) 3) Binding of imaging agents varies significantly between in-vivo and in-vitro fibrils – suggesting that a molecular level there could be differences between patients

Answer 80

Extract amyloid fibres from the postmortem brain tissue of two patients (one F72 diagnose with lewby body dementia, with neuritib Abeta plque, tangles and mild strophy of frontal and pareital lobes, the other F80 severe AD, gross atrophy of brain, neurons with graulovacuolar degeneration, plaques and tangles) and use them to seed recombinant protein to generate new amyloid fibres with the expectation that their structure would mirror that of the initial seeds, whilst at the same time increasing the amount of fibril present and introducing NMR isotopes. After growing the fibrils, he used EM compared those from the patient with those from the in-vitro growth, see that they have the same morphology. then used solid state nmr (homonuclear and heteronuclear) to provide a molecular fingerprint (local molecular structure) of each of the fibril types - in order to compare and contrast

Answer 81

1) Patients can have structurally distinct species – implications for drug development (eg blocker of fibril growth/Ab therapy) and diagnosis (imaging agents) 2) Does it have implications for disease progression? – a big question.

Answer 82

AB is derived from the processing of amyloid precursor protein (APP) – a type I integral membrane protein. APP is cleaved by 3 proteases, alpha, beta and gamma. Cleavage by beta and gamma results in the formation of AB – with variation in the C-terminus due to the variability in the cleavage by the gamma secretase. (Gamma cleaves the APP half way through the lipids bilayer) Cleavage by the alpha secretase prohibits the formation of AB as the cleavage site is part way through the AB.

Answer 83

we have 3D crystals so that we have repeating structures in 3-dimensions so that we can obtain diffraction data in all directions

Answer 84

it gives rise to unique reflection that report on the periodicity and the relative orientation of the structures with respect to the fibril axis.

Answer 85

a unit of length equal to 10−10 m (one ten-billionth of a meter) or 0.1 nm.

Answer 86

X-ray diffraction relies on the repeating properties of a system to diffract X-rays. Relies on the constructive/destructive interference of waves as they are scatted after interacting with the atom.

Amyloid diseases Flashcards

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