Disease In Molecular Cell Biology

Question 1

What are diseases associated with defects in protein targeting?

Answer 1

Diseases caused by protein misfolding, defective mitochondrial targeting signal, defective ER signal sequence, inhibition of Sec61 translocator by bacterial toxin

Examples include congenital lactic acidosis and diabetes.

Question 2

What is the consequence of a defective mitochondrial targeting signal?

Answer 2

Inefficient import of pyruvate dehydrogenase in mitochondria, leading to accumulation of pyruvate and lactate and thus a build up of lactic acid in the blood
= resulting in congenital lactic acidosis.

Question 3

What is the role of mitochondrial targeting sequences?

Answer 3

High content of positively charged residues (Arg, R) and a strong tendency to form an amphiphilic α-helix.

Question 4

What happens when proteins fail to be correctly targeted?

Answer 4

Localize incorrectly to cytosol, aggregate, and are degraded by the proteasome or lysosome.

Question 5

What is ER-associated degradation (ERAD)?

Answer 5

The process by which misfolded proteins in the ER are recognised and retained by ER quality control and must be removed

Moved back into the cytosol and degraded by the proteasome

Question 6

What triggers the unfolded protein response (UPR)?

Answer 6

Accumulation of misfolded proteins in the ER lumen.

Question 7

What is cystic fibrosis caused by?

Answer 7

Mutations in a chloride channel called CFTR, leading to misfolding and loss of function.

Question 8

What is the most common mutation associated with cystic fibrosis?

Answer 8

Deletion of phenylalanine at position 508 (dF508).

Question 9

What are potential therapies for cystic fibrosis?

Answer 9

Gene therapy, gene editing, pharmacological chaperones (act as correctors), and combination therapies like Orkambi, Symdeko, Kaftrio.

Question 10

What is the role of chaperones in protein misfolding diseases?

Answer 10

Chaperones bind to misfolded proteins and prevent them from leaving the ER.

Question 11

What is the result of a point mutation in insulin signal sequence?

Answer 11

Diabetes due to inefficient translocation into the ER and formation of toxic aggregates.

Question 12

True or False: Misfolded proteins can cause cell death.

Answer 12

True.

Question 13

Fill in the blank: Proteins marked for proteolysis are tagged with _______.

Answer 13

ubiquitin.

Question 14

What does the term ‘loss-of-function disease’ refer to?

Answer 14

Diseases caused by the degradation of misfolded proteins leading to a lack of functional protein.

Question 15

What is the effect of the unfolded protein response (UPR) when homeostasis cannot be restored?

Answer 15

It can trigger programmed cell death (apoptosis).

Question 16

What is the main consequence of protein misfolding in the ER?

Answer 16

Accumulation of misfolded proteins can lead to diseases such as cystic fibrosis.

Question 17

What are some therapeutic strategies to treat protein misfolding diseases?

Answer 17

Pharmacological chaperones, gene therapy, gene editing, RNAi therapies, drugs that alter protein degradation and UPR signaling.

Question 18

What is the role of the Sec61 translocator?

Answer 18

It facilitates the translocation of proteins into the ER.

Question 19

What can happen if misfolded proteins are not efficiently degraded?

Answer 19

They can build up in the ER and activate the unfolded protein response.

Question 20

Cause of defective mitochondrial targeting

Answer 20

A point mutation in the targeting sequence of a pyruvate dehydrogenase (PDH) complex subunit = Arginine is mutated into proline

Proline is a helix breaker as its side chain is a ring structure so cannot form hydrogen bonds

Question 21

Role of ER targeting

Answer 21

• ER targeting sequences are 8+ hydrophobic amino acids that are often at the N-terminal and sometimes cleaved
• They are recognised by SRP, targeted to the ER membrane and cause Sec61 translocator channel to open = initiates translocation

Question 22

Cause of defective ER targeting

Answer 22

A point mutation where Arg is mutated into Cistinine

Question 23

Consequence of defective ER targeting

Answer 23

• Point mutation in insulin signal sequence causes diabetes
• Does not interact correctly with Sec61 and not efficiently translocated into the ER
• Less insulin made
• Incorrectly localised to the cytosol so is non-functional
• Forms toxic aggregates causing beta cell death —> as cystine forms Disulfide bonds

Question 24

Role of cytosolic proteasome

Answer 24

• A large protease complex which degrades proteins by proteolysis
• Short-lived proteins and misfolded proteins are degraded
• Proteins are marked for proteolysis by the attachment of a protein called ubiquitin
• Polyubiquitin chain is recognised by the proteasome

Question 25

How error prone is protein folding?

Answer 25

Up to a third of proteins fold incorrectly

Question 26

Quality control in the ER

Answer 26

- Misfolded proteins are potentially harmful - These are retained in the ER - Chaperone proteins bind to the misfolded protein and stop them leaving the ER - Mutant protein doesn’t reach site of function & typically gets degraded - Lack of functional proteins can cause disease

Question 27

Role of CFTR in cystic fibrosis

Answer 27

- CFTR is expressed in epithelial cells in many organs - It pumps Cl- ions out of cells, water follows by osmosis, keeps mucus on surface of epithelial cells hydrated and cilia can beat to remove bacteria and debris - In CF, mucus becomes dehydrated and cilia cannot function

Question 28

What mutations prevent CFTR folding?

Answer 28

- Most common is the deletion of phenylalanine at position 508 (dF508) - 90% of patients have at least one copy of dF508 mutant gene - dF508 CFTR cannot fold correctly = non functional & can’t leave ER - Retained by ER quality control system - Does not reach plasma membrane - Loss of functional CFTR

Question 29

Disadvantages of correctors & combination therapies for CF

Answer 29

Very effective but very expensive Ethical questions = is it worth the cost?

Question 30

What is proteotoxicity?

Answer 30

Gain of function Misfolded protein has toxic properties = activates UPR or other toxic functions

Question 31

Treatment of diseases caused by accumulation of misfolded proteins

Answer 31

1. Reduce synthesis of mutant protein —> gene editing or RNA interference 2. Stimulate degradation of mutant protein —> proteasome enhancers (IU1) or autophagy enhancers (carbamazepine) 3. Drugs to alter UPR signalling —> prevent activation of programmed cell death or increase protein ER folding & degradation capacity

Question 32

What is the molecular mechanism of COPII-coated vesicle formation?

Answer 32

Involves the Sar1 GTPase, which controls vesicle formation and cargo transport ## Footnote Mutations in Sar1 can impair vesicle assembly.

Question 33

What genetic mutation is linked to Chylomicron Retention Disease (CRD)?

Answer 33

Mutations in the Sar1b gene ## Footnote These mutations affect the export of prechylomicrons from the ER.

Question 34

What is the consequence of Sar1b mutations in CRD?

Answer 34

Sar1b promotes export of preCM 20 mutations linked to CRD are identified in Sar1b gene - results in no Sar1b being made - GTP binding site is defective = won’t be activated Loss of Sar1b function prevents preCM export from the ER

Question 35

What are the symptoms associated with Chylomicron Retention Disease?

Answer 35

* Accumulation of preChylomicrons in the ER * Lipid droplets in cytoplasm of intestinal cells * Impaired absorption of fats, cholesterol and fat-soluble vitamins * Slow growth and weight gain * Effects on GI and nervous system

Question 36

What treatments are available for Chylomicron Retention Disease?

Answer 36

A low-fat diet to minimize accumulation of intracellular preChylomicrons ## Footnote Dietary management is crucial for symptom control.

Question 37

What is Familial Hypercholesterolemia (FH)?

Answer 37

An autosomal-dominant disease that leads to high cholesterol levels and increased risk of coronary heart disease ## Footnote Incidence is approximately 1:200 to 1:500.

Question 38

What are the classes of LDL receptor mutations in FH?

Answer 38

* Class 1: No receptor synthesis * Class 2: Impaired receptor formation * Class 3: Abnormal LDL binding * Class 4: Disrupted internalization * Class 5: Receptors rapidly degraded * Class 6: Mis-targeting to the membrane ## Footnote Each class has distinct effects on receptor function.

Question 39

What is the effect of Class 2 mutations in LDL receptors?

Answer 39

They prevent proper folding of the receptor so the receptor is retained by ER quality control & is degraded Approximately 50% of FH patients have this mutation.

Question 40

What is the role of LDLRAP1 in LDL receptor function?

Answer 40

It is an adaptor protein required for endocytic uptake of the LDL receptor ## Footnote Mutations in LDLRAP1 can cause autosomal recessive hypercholesterolemia.

Question 41

What are the treatment options for Familial Hypercholesterolemia?

Answer 41

**Inhibit cholesterol synthesis** - Stimulate LDL receptor expression - Effective for heterozygotes who have one copy of wild type (normal) gene - Increased uptake of LDL - STATINS = inhibit HMG-CoA reductase **Inhibit dietary cholesterol absorption** - EZETIMIBE = acts in the intestine

Question 42

What are lysosomal storage diseases?

Answer 42

- Disorders caused by defects in acid hydrolases and transporters - Leads to progressive accumulation of uncleaved/untransported substrates Examples include Niemann-Pick type C —> 95% of cases = caused by mutations in membrane protein NPC1 Gaucher disease —> caused by mutations in the lysosomal acid-B-glucosidase

Question 43

What is the molecular basis of Gaucher disease?

Answer 43

Caused by mutations that cause misfolding of lysosomal acid β-glucosidase = retention in the ER

Question 44

What are the potential treatments for Gaucher disease?

Answer 44

- Enzyme replacement therapy —> inject synthetic enzymes that are taken up from outside the cell by endocytosis, M6P receptors in the plasma membrane and reach the lysosome via endocytosis - Substrate reduction therapy —> reduce amount of glucosylceramide in lysosomes through dieting or inhibitory enzymes (so don’t need as much lysosomal acid β-glucosidase), Miglustat inhibits glucosylceramide synthesis - Pharmacological chaperones —> drugs that promote correct folding can increase amount of enzyme that escapes ER quality control and reaches the lysosome

Question 45

Fill in the blank: The Sar1 GTPase is required for the formation of _______ coated vesicles.

Answer 45

COPII

Question 46

True or False: Sar1a and Sar1b are identical in amino acid sequence.

Answer 46

False

Question 47

What is the primary function of COPII coated vesicles?

Answer 47

To mediate transport from the ER to the Golgi ## Footnote This is crucial for the proper secretion of proteins.

Question 48

Biosynthesis of chylomicrons

Answer 48

- Occurs in ER & Golgi - Carry lipids from the intestine around the body in capillaries - Pre-chylomicrons are assembled in the ER from triglycerides and large proteins - Packaged into transport vesicles (PCVT) for delivery to the Golgi - Mature into chylomicrons in the Golgi - Released by exocytosis and enter the capillaries

Question 49

What happens in chylomicron retention disease (CRD)?

Answer 49

**NOT AN ER FOLDING DISEASE** - pre-chylomicrons accumulate in the ER and are unable to reach the Golgi - Apoproteins that initiate assembly of pre-chylomicrons are properly folded & preCM are correctly folded - BUT the COPII coat responsible for cargo export from the ER fails to assemble correctly - This leaves the newly assembled preCM trapped inside the ER and unable to progress to the cis Golgi

Question 50

Role of Sar1 GTPase

Answer 50

Controls formation of COPII vesicles - SAR1p in the cytosol is in the GDP-bound form = off - A regulatory protein on the ER membrane activates SAR1p so it can form COPII coated vesicles = on - SAR1-GTP initiates assembly of COPII coat proteins (Sec23/24) on the ER membrane

Question 51

What are the 2 Sar1 genes in humans?

Answer 51

2 different isoforms of Sar1 - Sar1a - Sar1b (mutation in this cause CRD) These are 90% identical in amino acid sequence BUT encoded by different genes on different chromosomes

Question 52

Why does loss of Sar1b selectively disrupt preCM export?

Answer 52

- Sar1a is still active and functions to mediate vesicular transport of most cargo (smaller vesicles) - Sar1b required for transport of specific cargo including preCM - PreCM are large so require large transport vesicles

Question 53

What causes FH?

Answer 53

Defects in cholesterol uptake Cholesterol accumulates in the blood = atherosclerosis

Question 54

Effect of class 4 mutations on LDL receptors

Answer 54

Disrupt LDL receptor endocytosis - Mutations in the cytoplasmic tail of the LDL receptor prevents binding of an adaptor protein (at the plasma membrane) that recruits the LDL receptor to clathrin coated vesicles - LDL binds to the receptor as normal but the complex is not internalised - LDL receptor associated protein 1 (LDLRAP1) is an adaptor protein required for endocytic uptake of the LDL receptor - Mutations in the gene for LDLRAP1 leads to its loss - Causing ARH (autosomal recessive hypercholesterolaemia) with symptoms similar to FH

Question 55

Effect of class 3 mutations on LDL receptors

Answer 55

Disrupt LDL binding to the receptor

Question 56

What is Gaucher disease?

Answer 56

A lysosomal storage disease Absence of acid β-glucosidase causes the accumulation of glucosylceramide and other glycolipids in the lysosome Cleavage of glucosylceramide to produce glucose and ceramide requires lysosomal acid β-glucosidase

Question 57

How can bacterial toxin inhibit ER translocation?

Answer 57

- A toxin called Mycolactone causes ulcers - Myoalactone binds Sec61 translocator and inhibits protein translocation - Inhibits production of immune proteins (e.g. cytokines & membrane receptors) - Cannot effectively fight infection - Causes cell death resulting in ulceration