Amyloids

Question 1

What are amyloid deposits made of (simple)

Answer 1

Amyloids
Fibrils

Question 2

When were amyloid deposits first identified? How can they be identified?

Answer 2

1854 the term was first used by Rudolph virchow
identified through staining positive with iodine (starch test)

Question 3

Although first identified with the starch test, what was the first criteria/definition of amyloid?

Answer 3

Birefringence when stained with Congo-red

Question 4

What are the different types of amyloidogenic diseases? Give examples for each (and include the fibril protein (FP) involved)

Answer 4

Systemic – amyloid deposition throughout the body
e.g.
FP = b2 microglobulin - Dialysis related amyloidosis (DRA)
FP = Immunoglobulin heavy chain - systemic amyloidosis

Hereditary – genetic – gene mutations in particular genes
e.g.
FP= Fibrinogen alpha chain, apolipoprotein AI/II, Lysoszyme - all are Familial systemic amyloidosis

CNS – amyloid deposition in the CNS – or linked to CNS
e.g. FP = Beta protein precursor - Alzheimers disease

Ocular - eye
e.g. FP = lactoferrin - familial corneal amyloidosis

Localized - one part of body
e.g. FP = Calcitonin - Medullary thyroid carcinoma

Question 5

What are the different classes of amyloidogenic diseases?

Answer 5

Primary:
- Deposition of amyloid throughout body
- organ dysfunction – commonly heart, kidneys, nervous system and gastrointestinal tract
-Mostly deposits of antibody light chains.

Secondary:
“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

Familial:
-Found in a small number of families of nearly every ethnic origin
-Transthyretin most common, deposition patterns vary (some forms of Alzheimers as well)

Other types:
Localised amyloid, renal dialysis amyloid -DRA (β2M), Alzheimers disease (both tangles and plaques), prion diseases, diabetes…

Question 6

Explain how/why amyloid fibrils can be observed using cogo red and birefringence

Answer 6

Congo red has delocalised aromatic sequences

If you add to protein solution – if it’s a disordered aggregate - see nothing

When fibrils are forming (when bound to ordered amyloid fibrils) – under a polarising light - see Birefringence - resulting in an apple green colour

Question 7

How is the congo red/birefringence amyloid detection technique advanced to allow for measurement of formation. of amyloid fibrils?

Answer 7

Can also use UV/Visible spectra to look at binding through red shift in spectra of CR and an increase in intensity at 541 nm

routinely used in the laboratory to measure the formation of amyloid fibrils

(plot wavelength (nm) against absorbance (AU))

Question 8

Name another dye that can be used instead of congo red and why it may be used

Answer 8

more convenient
Fluorescent based dyes such as ThT

Question 9

How do we detemine location of amyloid deposits in the body (in-vivo)?

Answer 9

123I-SAP scintigraphy

Radiate with iodine 123 (I-SAP)

See where radioactovity is localised within patient

SAP is a protein which is ubiquitously present in amyloid deposits and is thought to bind to them and help stabilize the fibrils

Question 10

Describe the composition of amyloidogenic deposits

Answer 10

Fibres :One protein - linked to a particular type of disease

Proteoglycans : Heparan sulphate, dermatan sulphate, glycosaminoglycans

Collagen

Serum Amyloid P Component (SAP)

Question 11

What component defines the nature of the amyloid deposit?

Answer 11

the fibres (protein) e.g. b2m

Question 12

Explain why classic techniques for determining protein structure don’t work for determining fibril structure

Answer 12

X-ray diffraction:

Needs a crystalised structure

Amyloids form fibres – fibres don’t form crystals

NMR :

NMR requires a small molecules that solubilize rapidly in solution to give a well resolved spectra - These large deposits don’t tumble so spectra challenging.

NMR (liquid state) requires small moelcules that tumble rapidly in solution to give well resolved spectra. These large deposits don’t tumble so spectra challenging

Question 13

What alternative methods can be used to determine fibre protein structure?

Answer 13

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

Question 14

Explain fibril morphology and structure of protofilaments

Answer 14

Fibril morphology:
- Long thin fibres
- Composed of a number of protofilaments
- Typically helical

Protofilaments:
- Vary in width depending on proteins
- Repeats along length

Question 15

Explain how EM can be used to determine overall fibril morphology, and its challenges

Answer 15

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).

doesn’t give the high-resolution structural information that we need if we are to understand the molecular structure of the fibrils, important if we want to develop small molecules that prevent fibril growth

Question 16

What technique is used to determine order in fibres?

Answer 16

X-ray diffraction:

Protein structures solved by the analysis of 3D crystals

X-ray diffraction can be applied to any repeating structure:

Fibre diffraction can be used to provide information on order in fibres

Question 17

Why dont we use X-ray crystallography to determine fibril structure

Answer 17

Fibrils do have order but not 3D order

Question 18

Explain the distinctive pattern we see in amyloid fibrils when X-ray diffraction is done

Answer 18

Meridional reflections (top and bottom) indicate a regular spacing of 4.8Å

Equatorial reflection (left/right) indicate a spacing of between 10-11Å (more variable)