Amyloidosis

Question 1

What is amyloid?

Answer 1

Abnormal extracellular protein deposit composed of amyloid fibrils, Glycosaminoglycans and serum Amyloid P Component.

It is similar to what is found on the cell surface of many bacteria

Question 2

Describe amyloid structure

Answer 2

Different globular precursor proteins can form fibrils which are rigid, non-branching and insoluble

~50-100Å diameter

Pathognomonic cross-β core structure

Question 3

Why is in vivo amyloid clearance so low?

Answer 3

Because cells like macrophages ‘ignore’ it

Question 4

What are the components of amyloid fibrils?

Answer 4

Fibrilar proteins

Heparan/dermatan sulphate

Serum amyloid P component (SAP)

Question 5

What are the two general classes of amyloidosis?

Answer 5

Local (one organ) and Systemic (many organs)

Question 6

Describe systemic amyloidosis (prevalence and severity)

Answer 6

Rare (incidence 0.4/100,000)

Usually fatal ~ 1 per 1,000-1,500 deaths

Question 7

Why is amyloidosis often diagnosed late?

Answer 7

It is so rare that it is not well known to doctors

Question 8

How is the current state of amyloidosis treatment?

Answer 8

Treatment is very challenging and it is still an important unmet medical need.

Major recent advances and better outcomes in specialist centres

Question 9

What is the most common type of amyloidosis?

Answer 9

AL Amyloidosis

Question 10

Inheritance of amyloidosis

Answer 10

Can be hereditary or it can be acquired

Question 11

Give examples of acquired and hereditary amyloidoses

Answer 11

AL is acquired. It is associated with myelomas which make many immunoglobulin light chains and fragments. Can eventually lead to beta-sheet and then amyloid fibril formation.

Familial amyloid polineuropathy or cardiopathy is hereditary and is related to transthyretin variants (mutation) and fragments.

Question 12

With amyloid fibrils there is a large heterogeneity in what?

Answer 12

Concentration of protein and protein type

Question 13

What is the most common genetic amyloidosis?

Answer 13

Familial amyloid polyneuropathy or cardiopathy

Question 14

How is the location of the disease determined?

Answer 14

By the location of the amyloid deposits

Question 15

Why is it hard to figure out which organs will be affected by AL amyloidosis?

Answer 15

Because light chains which form the amyloid fibrils have high heterogeneity and thus can target different organs

Question 16

What dictates survival in amyloidosis?

Answer 16

The protein precursor

Question 17

What is an important determinant of amyloidosis prognosis?

Answer 17

Type of organ involved in deposition is important in prognosis

Cardiac involvement is worse

Question 18

Describe amyloid structure at a molecular level

Answer 18

Structure is considered a cross-beta structure because the angles are consistent with beta sheet

Make multiple hydrogen bonds among the aggregates.

At the end you have the most stable form of the protein found in nature.

In addition to the one amyloidogenic protein which generate the structure, other proteins provide additional stability to the structure

Question 19

What is the structure of the serum amyloid P component?

Answer 19

Pentamer

Question 20

How do the pathogenic variants of the precursour proteins differ from the wild type form?

Answer 20

They are partially denatured

Question 21

What would happen if the precursor protein was fully denatured?

Answer 21

It would not form amyloid

Question 22

What do all the amyloid precursors have in common?

Answer 22

All lose thermodynamic stability

Question 23

Is amyloidosis a gain of function or loss of function disease, explain?

Answer 23

Gain-of-function

Normally proteins with decreased stability would be detected and broken down

Instead, these are not and go on to form amyloid.

Question 24

What is the genotype of most patients?

Answer 24

Heterozygous = dominant

Question 25

When is amyloidosis onset and why is it at this stage in life?

Answer 25

Middle age

Partially folded proteins will be broken down

At some point there is a high enough concentration for nucleation and after this elongation occurs.

Question 26

What is nucleation?

Answer 26

It is a term for aggregate formation

Question 27

How does amyloidosis progress?

Answer 27

Lag phase –> nucleation–> elongation.

After nucleation you can have a strong acceleration in 2-3 years

(You use different drugs pre- and post-nucleation)

Question 28

What are synonyms for nuclei?

Answer 28

Seeds and template

Question 29

What can reduce the lag phase of amyloid fibril formation and what does this suggest?

Answer 29

When seeds are added, the lag phase decreases suggesting that seeds have a key role in amyloid fibril formation

Question 30

Why do the monomers in the amyloid fibril not have the same structure as the precursor?

Answer 30

The fibril can be made up of variant and WT protein

The variant protein first forms the fibril

The variant for then converts the WT protein so that it can elongate

Question 31

When can wild type monomers be found in the fibrils?

Answer 31

If there is not too much destabilization of the variant compared to the wild type

Question 32

Give an exmaple of how amyloidosis was studied

Answer 32

The structure of the precursor (beta2-microglobulin) was found–> mutation behind it was found–> fibril formation was modeled with biomechanical conditions found in the human body –> showed that forces in the body provided the energy to change from native state to partially folded one

Question 33

What can hydrophobic/hydrophilic interface and turbulent fluid flow do?

Answer 33

They can generate forces in the range of 20-50 pN sufficient to cause protein unfolding

Question 34

What is the mechanism of amyloid toxicity?

Answer 34

Oligomeric state is considered by many as the source of toxicity

Oligomers can associate with membranes and make holes messing with the separation between the intracellular and extracellular space - can activate apoptotic pathways

In vivo though we don’t have disease without amyloid

Question 35

What is an oligomer?

Answer 35

An intermediate between precursor and fibres

Question 36

What is the relevance of amyloid in in vivo toxicity of amyloidosis?

Answer 36

Histology shows the tissue is completely remodelled

Amyloid can greatly affect glucose diffusion, oxygen gradient etc

Once fibrils are formed, molecular crowding significantly increases: COLLOIDAL STABILITY IS REDUCED and OLIGOMERISATION IS FAVOURED

There is still a gap in the investigation of toxicity in vivo and in vitro models

Question 37

Protein cleavage with and without mechanical forces was studied in a family with a transthyretin mutation. What was found?

Answer 37

Showed that shear stress/mechanical forces can act as a cofactor to protein cleavage (a mechanoenzymatic mechanism)