Module 3 - Molecular cell biology of disease

Question 1

Mitochondrial targetting

Answer 1

A high content of positively charged residues with a strong tendency to form an amphipathic α-helix

Question 2

Defective mitochondrial targetting: what is it caused by and what is the effect?

Answer 2

A point break mutation from an arginine amino acid into a proline which acts as a helix breaker

In a normal MTS, essentially 100% reaches the matrix, but in a mutated MTS, ~25% reach the matrix
This causes less pyruvate hydrogenase to reach the matric which greatly increases blood lactic acid concentration - congenital lactic acidosis

Question 3

Defective ER targetting

Question 4

Defective ER targetting: what is it caused by and what is the effect?

Answer 4

Point mutation of arginine into a cysteine

ERTS does not correctly interact with Sec61 and not be efficiently translocated into the ER and instead mostly be released into the cytosol. This causes toxic insulin build-up in the cytosol, killing beta cells

Question 5

What happens to incorrectly targetted proteins?

Answer 5

Build-up in the cytosol and may be broken down by lysosomes and proteasomes

Question 6

Cystolic proteasomes: what are they, what do they do, and what is the process behind them?

Answer 6

Large protease complex which degrades proteins by proteolysis

They degrade Short-lived proteins and misfolded proteins

• Proteins marked for proteolysis by attachment of a protein called ubiquitin
• Polyubiquitin chain recognised by the proteasome
• Ubiquitin recycled
• Protein degraded into amino acids

Question 7

What quality control is there in the ER?

Answer 7

Misfolded proteins (as they are harmful if left around) are recognised by chaperone proteins and prevented from leaving the ER

Question 8

Cystic fibrosis: what is it, how fatal is it, and what is it caused by?

Answer 8

A recessive genetic disorder that affects 1 in 3000 live births

Often fatal by the age of 40

Caused by mutations in a chloride channel called CFTR (cystic fibrosis transmembrane conductance regulator)

Question 9

Cystic fibrosis mutation: what happens when there is no mutation and what happens when there is a mutation?

Answer 9

CFTR is expressed in epithelial cells in many organs (lung, liver, pancreas) and it pumps Cl⁻ ions out of cells, causing water to follow by osmosis which keeps mucus on the surface of epithelial cells hydrated

Cilia can beat to remove bacteria and debris

In cystic fibrosis, mucus becomes dehydrated and cilia cannot function

Question 10

Common mutations of cystic fibrosis: the most popular form

Answer 10

The most common mutation is the deletion of phenylalanine at position 508 (dF508)

~90% of patients have at least one copy of the dF508 mutant gene

dF508 CFTR cannot fold correctly, so is retained by ER quality control system and does not reach the plasma membrane

Question 11

What happens to misfolded CFTR proteins

Answer 11

Misfolded proteins that are recognised and retained by ER quality control must be removed

They are moved back into the cytosol and degraded by the proteasome

ER-associated degradation: ERAD

Question 12

Potential treatments for CFTR mutations: trafficking/folding

Answer 12

The df508 mutation still functions as a Cl⁻ but just can’t make it to the plasma membrane.

Pharmacological chaperones can be used as ‘correctors’ (lumacaftor, tezacaftor) to allow CFTR to fold correctly and reach the membrane

Question 13

Potential treatments for CFTR mutations: synthesis

Answer 13

Genetic approaches (gene editing etc)

Question 14

Potential treatments for CFTR mutations: function

Answer 14

Potentiators (increasing channel opening frequency)

Question 15

Accumulation of misfolded proteins: what does it do?

Answer 15

Triggerds the unfolding protein response

UPR - a response that attempts to restore homeostasis by increasing chaperone concentration and inhibiting protein synthesis

Question 16

What happens if the UPR fails and what are the potential treatments for accumulated proteins?

Answer 16

Apoptosis may occur

• Reduce synthesis of the mutant protein (Gene editing, RNA interference)
• Stimulate degradation of the mutant protein (Proteasome activators (IU1), Autophagy enhancers (carbamazepine))
• Drugs to alter UPR signalling (Prevent activation of programmed cell death, Increase protein ER folding and degradation capacity )

Question 17

Chylomicron biosynthesis: what is the process behind it?

Answer 17

Lipids and fatty acids enter the intestines where a Prechylomicron is formed in the ER and it is then sent to the Golgi in a transport vesicle to mature before being secreted as a mature Chylomicron

Question 18

Chylomicron retention disease (CRD): what is it and what causes it to occur?

Answer 18

When there is a buildup of Prechylomicrons in the ER

Everything is folded correctly (so not a folding disease) but the ER export is defective - COPII fails to assemble correctly

Question 19

What controls COPII assembly?

Answer 19

Sar1 GTPase controls the formation of COPII vesicles

Sar1p in the cytosol is in the ‘OFF’ form when bound with GDP but Sar1-GEF causes GDP to dissociate and nearby GTP to bind, activating the Sar1p

Sar1p then binds with Sec23 which binds with Sec24 to form the coat for the vesicle

Question 20

What are the two forms of the Sar1 protein and which causes CRD

Answer 20

Sar1a and Sar1b - there are 90% identical but are just encoded by different genes

Sar1b can have 20 known gene mutations which may cause the GTP binding site to be ineffective or the protein to not be made

Question 21

Why can the ER have normal cargo transport despite having Sar1b mutated?

Answer 21

Sar1a functions properly and so, since Sar1b focuses on only specific types of cargo, only a few cargos, including chylomicrons, are affected by the mutation

Question 22

What are the symptoms and treatment of CRD?

Answer 22

Symptoms - Impaired fat, cholesterol and soluble vitamin absorptions which lead to slow growth, weight gain, and gastrointestinal/nervous system effects

Treatment - low-fat diet to minimise Prechylomicron concentration in the ER

Question 23

Familial hypercholesterolemia: what is it and what can it cause?

Answer 23

An autosomal-dominant disease

The leading cause of coronary heart disease

Question 24

What causes familial hypercholesterolemia?

Answer 24

Accumulation of cholesterol in the blood due to defects in cholesterol uptake (caused by LDL receptor defects)

Question 25

The 6 classes of LDL receptor mutations

Answer 25

Class 1 - Receptor not synthesised Class 2 - Absent from membrane (due to misfolding or withholding in the ER) Class 3 - Normal synthesis but abnormal binding Class 4 - Clustering into pits (LDL binds but does not enter the cell) Class 5 - Receptors not recycled after endocytic complex occurs and readily degraded Class 6 - Receptors fail to target the membrane

Question 26

How many FH sufferers are affected by the class 2 mutation?

Answer 26

~50%

Question 27

What is LDLRAP1, what does it do, and

Answer 27

LDL-receptor assistor protein - an adaptor protein required for the endocytic uptake of the LDL receptor Mutations in the gene for LDLRAP1 lead to its loss, autosomal recessive hypercholesterolemia (ARH) occurs with symptoms similar to FH

Question 28

What is LDLRAP1, what does it do, and

Answer 28

LDL-receptor assistor protein - an adaptor protein required for the endocytic uptake of the LDL receptor Mutations in the gene for LDLRAP1 lead to its loss, and autosomal recessive hypercholesterolemia (ARH) occurs with symptoms similar to FH

Question 29

Treatments for FH

Answer 29

Inhibit cholesterol synthesis Inhibit dietary cholesterol absorption

Question 30

Lysosomal storage diseases: how many are there, what do they do, and what are the main types?

Answer 30

Over 50 known types Causes progressive accumulation of uncleaved/untransported substrates * Niemann-pick type C (95%) - caused by NPC1 mutation * Gaucher disease - caused by lysosomal acid β-glucosidase

Question 31

Niemann-pick type C: what occurs with this, what type of disease is it, and what is the effect of the disease?

Answer 31

NPC1 should transport cholesterol (that came from LDLs) from lysosomes into cytosol Misfolding disease Inefficient cholesterol transport leads to the cell producing more of its own cholesterol which disrupts the production of other lipids

Question 32

Gaucher disease: what occurs with this and what is the effect of the disease?

Answer 32

The absence of acid β-glucosidase in the lysosome causes the accumulation of glucosylceramide and other glycolipids A range of symptoms

Question 33

What are the treatments for Gaucher disease?

Answer 33

* Enzyme replacement therapy * Substrate reduction therapy: * Pharmacological chaperones

Question 34

Enzyme replacement therapy: how does it work?

Answer 34

Inject synthetic enzymes which are added with M6P to bind to the M6P receptor and function in the lysosome

Question 35

Substrate reduction therapy: how does it work?

Answer 35

Reduce the amount of glycosylceramide in lysosomes – don’t need so much lysosomal acid β-glucosidase (Miglustat inhibits glucosylceramide synthesis and is used to treat Gaucher disease)

Question 36

Pharmacological chaperones: how does it work?

Answer 36

Drugs that promote correct folding can increase the amount of enzyme that escapes ER quality control and reaches the lysosome