Amyloid diseases Flashcards
what was the first documented cases of an amyloidogenic disease?
who described it and when?
First documented cases: Theophili Boneti pub. 1679, a text book of the anatomy of diseased “organs and parts” two cases:
1. Described a young man with an abscess in the liver and a large spleen filled with white stones.
2. Young woman “whose spleen was so hard it could hardly be cut with a knife. Incision of the spleen produced a sound like that of the cutting of spongy timbers”
who first coined the term amyloid?
by the German physician scientist Rudolph Virchow in 1854
* Identified that deposits stained positive with iodine (test for starch) went white
* Derived name from Latin, Latin amylum and the Greek amylon
what did Friedreich and Kekule demonstrate about the deposits in 1859?
Friedreich and Kekule demonstrate that the deposits contained a high nitrogen content suggesting the deposits were proteinacious
what was discovered about amyloid deposits in the later part of 19th to the start of the 20th century?
polarized light microscopy studies of deposits and staining with Congo-red indicated that these were not only amorphous aggregates but ordered structures.
what was adopted as the first criteria/definition of amyloid?
is still the defacto standard in hospitals
- Birefringence when stained with Congo-red
- With advent of azo-dyes – such as Congo-Red, can stain specifically and see that these structures were not amorphous aggregates.
- Bifrifigrance is optical property only occurs with highly ordered structures
what are the five main types of amyloidogenic diseases?
- systemic
- hereditary
- central nervous system
- ocular
- localised
what fibril protein causes cutaneous amyloidosis?
keratin
what fibril protein causes pituitary amyloidosis?
prolactin
what fibril protein causes familial corneal amyloidosis?
lactoferrin
what fibril protein causes creutzfeld-jakob disease?
prion protein
what fibril proteins can cause familial systemic amyloidosis?
fibrinogen alpha chain
apolipoprotein A1
apolipoprotein A2
lysozyme
what are the classes of familial amyloidogenic disease?
- primary
- secondary
- familial genetic
- other localised types
what is primary familial amyloidosis?
Deposition of amyloid throughout body. Disease manifests when enough amyloid has built up to cause organ dysfunction – commonly heart, kidneys, nervous system and gastrointestinal tract. Mostly deposits of antibody light chains.
what is secondary familial amyloidogenic disease?
“secondary” to a chronic infection or inflammatory disease e.g. rheumatoid arthritis, familial Mediterranean fever, osteomyelitis… Mostly deposits made of amyloid A protein, deposition patterns vary.
what techniques can we use to identify amyloid deposits?
in the lab
- congo red dye - binds specifically to amyloid fibrils.
under polarised ight when bound it has birefringence
also
use UV/visible spectra to look at binding through red shift in spectra of CR and an increase in intensity at 541nm - other dyes increase fluorescence bases dye ThT
how is amyloidosis diagnosed clincally
Identification of amyloid deposits in vivo: 123I-SAP scintigraphy
how is 123SAP scintigraphy performed to analyse amyloid deposits
Take SAP from patient and radiolabel it
SAP labelled with radioactive iodine (see your 1st year Chemistry notes).
Normally patient SAP metabolised and the iodine eliminated from the body
When amyloid deposits present SAP binds, retained within the body.
Can use whole body scintigraphy to localise the deposits
what are the major components of amyloidogenic deposits?
- Fibres (proteinaceous): One type of protein (depending on disease)
- Proteoglycans: Heparan sulphate, dermatan sulphate, glycosaminoglycans
- Collagen
- Serum Amyloid P Component (SAP)
what are the conventional techniques we use to determine the structure of proteins?
X ray Diffraction
solution state Nuclear Magnetic Resonance (NMR)
why is x ray diffraction/crystallography difficult to do with amyloid fibres?
need a (regular 3D) crystalised sample for this, amyloid fibrils do not readily form such species (don’t form nice crystals) which is an issue so cant create nice diffraction patterns.
(diffraction typically needs crystals however can also be used on other materials)
why cant liquid state NMR to determine the structure of amyloid fibrils
because amyloid fibrils are insoluble, which makes it difficult to use solution state NMR for structure determination
what alternative methods can be used to determine the structure of amyloid fibrils?
(6)
– Electron microscopy over fibril morphology
– Atomic Force Microscopy over fibril morphology
– Circular Dichroism secondary structure
– Fluorescence fibril assembly
– Fibre diffraction repeat structures in fibre
– Solid-state NMR local structure/overall folds
electron microscopy of amyloidogenic deposits can identify what features?
- We can get an idea of the overall morphology of the fibrils e.g. number of fibres, packing etc.
- Even fibrils composed of the same protein, when studied by EM can appear different (eg twisted, non-twisted ribbons etc).
what is one disadvantage of electron microscopy of amyloidogneic deposits analysis?
it doesn’t give the high-resolution structural information that we need if we are to understand the molecular structure of the fibrils.
what was discovered about amyloid deposits by xray diffraction and how?
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.
what physics underpins xray diffraction?
Braggs Law
why can we use xray diffraction but not xray crystallography?
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.
describe the science behind xray diffraction
not needed for exam
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
what are the two major measurements that come form xray diffraction of amyloid fibrils?
- Meridional reflections (top/bottom) indicate a regular spacing of 4.8Å
- Equatorial reflection indicate a spacing of between 10-11Å
how do we know amyloidogenic proteins posses beta sheets?
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.
how do we know amyloidogenic proteins have a cross beta structure?
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
what is the common structure of amyloidogenic fibres?
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
they can be formed from proteins, that are frequently highly soluble and structured
what are the few key properties that have been identified that favor amyloid formation?
- Proteins tend to be hydrophobic (exception PolyQ – htt in huntingtons disease)
- Slight preference for β-strands
- Minimum size 6 residues
although there is no evidence for a common sequence between amyloidogenic proteins - what is feature is similar about the structure despite this?
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).
what is a cross beta structure?
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
why do fibrils form?
protein misfolding or mis processing
what is levinthals paradox?
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.
how does protein folding occur?
- 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).
describe in terms of protein folding the route to amyloid fibrils
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).
what is molecular crowding?
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.
the presence of other macromolecules influence the activity and folding of the proteins present.
what effect does molecular crowding have on protein folding?
– Reduces configurational entropy
– Increase in ΔG (ΔG= ΔH –TΔS) (reduces the entropy of the system)
– Shown to increase rate of protein aggregation
the presence of other macromolecules influence the activity and folding of the proteins present.
many of the amylodogenic proteina re found in the extracellular environment - what feature of this encourages protein aggregation/ fibriliation?
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.
what are the four forms/steps that amyloidogenic proteins go through to form fibrils?
monomer
nucleation event (oligomers)
protofibril elongation
fibril formation
how do you monitor the rate of fibrilisation?
rate using a t assay < - light phase while nucleation is about to occurs then
why can amyloidogenic structure be thought of as a super stable state?
with lower energy than the native state
the degree of hydrogen bonding and the stability of the sturctures
