Amyloid Disease

Question 1

what is required in vivo for protein folding

Answer 1

enzymes, chaperones and energy input

Question 2

what are the possible states of equilibrium between folded, unfolded, misfiled and aggregated

Answer 2

folded-unfoled, misfolded-unfolded, aggregated-unfolded

Question 3

what does the energy landscape of protein folding look like

Answer 3

rough containing troughs

Question 4

what sort of proteins sometimes aggregate from an unfolded conformation to form amyloid fibres

Answer 4

intrinsically disordered proteins which are proteins that are functional without adopting a tertiary structure (e.g. transcription factors)

Question 5

what are the 2 categories of protein misfolding disease

Answer 5

1) they are identified by proteasome leading to degradation and loss of function
2) they are allowed to aggregate leading to loss of function but also gain of toxic function

Question 6

what happens to misfolded proteins in the ER

Answer 6

they are tagged for degradation and go down the ERAD pathway

Question 7

what happens to misfolded proteins in the cytosol

Answer 7

they are ubiquitinated and degraded

Question 8

give 2 examples of diseases caused by errors in the degradation system

Answer 8

Tay-Sachs and Gauchers

Question 9

how many diseases are associated with amyloid

Answer 9

> 50

Question 10

what is the structure of amyloid fibrils

Answer 10

they are long thin and unbranched. generic cross beta structure on protofilaments, b strands are organised perpendicular to the finer. many protofilaments form a fibril which is always around 10nm in diameter

Question 11

give an example of amyloid-like fibres which are good

Answer 11

melanin used to store secretory hormones in vesicles

Question 12

where to amyloid fibres localise in transthyretin amyloidosis

Answer 12

the heart

Question 13

where do amyloid fibres localise in systemic amyloidosis

Answer 13

liver

Question 14

what are the common features of all amyloid fibres

Answer 14

x ray diffraction pattern always gives a 4.8a repeat due to inter strand repeat and 10.7a repeat between cross linking strands
red birefringence
all grow by nucleated growth- monomers form oligomers which then undergo conformational change to form nucleus. at this point many monomers can add quickly- highly structured and ordered

Question 15

in what ways are amyloid fibres highly polymorphic

Answer 15

can have different numbers of protofilaments, form in different ways

Question 16

what are the main mutations which occur in lysozyme and why are each of these amyloidogenic

Answer 16

I56T and D67H because they cause misfiling and aggregation

Question 17

what is the difference between WT lysosyme and mutant lysosyme

Answer 17

variants are less stable and more aggregation prone. in HX ESI MS variant entire b domain concurrently exchange hydrogen atoms en masse

Question 18

why are camelid antibodies used to prevent lysozyme aggregation rather than human

Answer 18

they bind tightly and specifically like human antibodies but only contain the heavy chain so can be recumbently reproduced more easily

Question 19

why is it possible to use antibodies to treat amyloid diseases despite them being unable to enter cells

Answer 19

most amyloid diseases are extracellular

Question 20

how does camelid antibody stabilise mutant lysozyme

Answer 20

it binds the native state and pulls the ab towards the stabilised native state. it also clamps the a and b domains together stopping non-cooperative dissociation and exchange of beta sheet domain which leads to the amyloid precursor

Question 21

what sort of mutation (gain or loss of function) is tranthyretin amyloidosis

Answer 21

gain of toxic function

Question 22

where can transthyretin amyloidosis deposit

Answer 22

heart or neurones

Question 23

what is the disease caused by transthyretin (TTR) amyloidosis deposits in the heart

Answer 23

senile systemic amyloidosis

Question 24

what is the disease caused by transthyretin amyloidosis deposits in the

Answer 24

familial amyloid polyneuropathy

Question 25

what is the structure of TTR

Answer 25

127aa, 55kDa homotetramer

Question 26

what is the function of TTR

Answer 26

it carries retinol binding protein

Question 27

what molecule is TTr able to bind, despite this function not being used in humans

Answer 27

thyroxin

Question 28

which mutations in TTR give rise to amyloidosis

Answer 28

over 50 known- basically any mutation in 127aa protein

Question 29

describe the TTR aggregation cascade

Answer 29

monomers dissociate along the 2-fold axis breaking into 2 dimers and then monomers from the tetramer leading to misfiling and aggregation to form fibrils

Question 30

how does the dissociation path of TTR open up possibilities for therapeutics

Answer 30

a ligand which can stabalise native tetramer would prevent further steps of the aggregation cascade- potentially utilising the thyroxin binding site

Question 31

how was the T199M mutation found to counter V30M FAP causing mutation

Answer 31

a family with V30M mutation but no FAP had genome sequenced and found T199M mutation on other allele. this creates a higher transition state barrier and so slows aggregation

Question 32

how was it shown that stabilising the tetramer you could also increase the barrier to the transition state

Answer 32

TTR was tirade with different concentrations of thyroxin as well as 2 other small molecules which all inhibited aggregation

Question 33

what are the properties required for a TTR amyloid drug

Answer 33

binding tightly to TTR as negative cooperativity means a large excess of ligand is required to bind both sites therefore it’s better to have a ligand which binding 1 site is sufficient so you can work with a lower conc and so lower side effects
it needs to bind with high selectivity
it mustn’t interfere with thyroid hormone receptor

Question 34

describe the screening process to discover potential small molecule drugs for TTR amyloidosis

Answer 34

formed small molecules by varying group x, group y and group z and selected for the best of each and put those together

Question 35

whats the name of the drug now in the clinic to treat TTR amyloidosis

Answer 35

tafamidis