Mod3 - Molecular Cell Biology of Disease

Question 1

What kind of mutation can lead to defects in mitochondrial protein targeting, and why?

Answer 1

Point mutation (Arg -> Pro), because Proline is a helix breaker - it is unable to form H bonds to keep the amphiphilic alpha-helix in the right conformation, which disrupts the helix

Question 2

What is the consequence (for the cell) of an Arg -> Pro point mutation in the signal sequence of Pyruvate Dehydrogenase?

Answer 2

Inefficient import -> lower PDH levels in mitochondria -> Pyruvate accumulates -> converted to lactate -> build-up of lactic acid in the blood -> congenital lactic acidosis

Question 3

What kind of mutation (mentioned in lecture) might lead to defects in ER protein targeting, and why?

Answer 3

Point mutation (Arg -> Cys), because this will result in the formation of Disulphide bonds, disrupting interaction with Sec61

Question 4

What is the consequence (for the cell) of an Arg -> Cys point mutation in the signal sequence of Insulin?

Answer 4

Insulin incorrectly localised to cytosol -> forms toxic aggregates causing ß cell death

Question 5

How does the toxin Mycolactone lead to ulceration?

Answer 5

Mycolactone binds Sec61 and inhibits protein translocation into ER -> inhibits production of immune proteins e.g. cytokines and membrane receptors -> cells cannot effectively fight infection and die, leading to ulceration

Question 6

What happens to proteins which are not correctly targeted?

Answer 6

Localise to cytosol (“default”) and are degraded into amino acids by proteolysis

Question 7

What is the proteosome?

Answer 7

A large protease complex which degrades short-lived and misfolded proteins by proteolysis

Question 8

How are proteins “marked” for proteosome degradation (proteolysis)?

Answer 8

Attachment of a protein called ubiquitin - the proteosome recognises the polyubiquitin chain

Question 9

How does general “quality control” of proteins/protein folding work?

Answer 9

Molecular chaperones bind to misfolded proteins and retain them in the ER

Question 10

Briefly describe the normal function of CFTR and the effect cystic fibrosis has on it

Answer 10

CFTR pumps Cl- ions out of epithelial cells, causing water to leave the cell via osmosis, hydrating the mucous on the surface; in CF, this pumping is inhibited, so mucous becomes dehydrated and cilia cannot beat

Question 11

Describe how the dF508 mutation causes cystic fibrosis

Answer 11

CFTR protein cannot fold correctly, so it is retained in the ER and does not reach the plasma membrane

Question 12

What happens to dF508 CFTR protein?

Answer 12

It is moved back into the cytosol and degraded by the proteosome - this is ER-associated degradation (ERAD)

Question 13

Name the two types of potential therapies for CF

Answer 13

1 - gene therapy to express wild-type gene
2 - CORRECTORS -> pharmacological chaperones which enhance CFTR folding and allow it to escape from ER quality control

Question 14

What can happen if misfolded proteins are not effectively degraded?

Answer 14

Accumulation of misfolded proteins in ER lumen can trigger an Unfolded Protein Response (UPR) -> can lead to apoptosis and cell death if homeostasis not restored

Question 15

Three strategies to prevent cell death due to UPR?

Answer 15

1. Proteosome activators
2. Autophagy enhancers
3. Drugs to alter UPR signalling, prevent apoptosis

Question 16

Define CRD

Answer 16

Chylomicron Retention Disease - preChylomicrons (although correctly folded) accumulate in the ER and are unable to reach the Golgi

Question 17

Which “stage” is defective in CRD?

Answer 17

ER EXPORT - the COPII coat responsible for cargo export from the ER fails to assemble correctly and preCM remains in the ER

Question 18

Name the protein (and what “type” of protein) controls formation of COPII vesicles

Answer 18

Sar1-p (a GTPase!)

Question 19

Name the ER membrane regulatory protein which activates Sar1-p

Answer 19

Sar1-GEF

Question 20

Describe how Sar1-GEF activates Sar1-p

Answer 20

It converts Sar1-p from GDP-bound (inactive) to GTP-bound (active) form, causing a conformational change that allows the alpha-helix to bind to the membrane

Question 21

Describe the genetics underlying the Sar1 protein and the mutations that affect it and cause CRD

Answer 21

Humans have two Sar1 genes - Sar1a and Sar1b - which are isoforms of each other. Mutations in SAR1B(!) cause CRD, as this gene promotes export of preCMs

Question 22

How do Sar1b mutations cause CRD?

Answer 22

They either cause NO Sar1p to be made, or the GTP binding site to be defective - loss of Sar1b function prevents preCM export from the ER

Question 23

Why do Sar1b defects selectively affect preCMs but not other cargo?

Answer 23

Sar1a is still active and functions to mediate export of most other cargo - Sar1b is required for transport of specific cargo, including preCMs, as they are large and require large vesicles

Question 24

What are the cellular and physiological symptoms of CRD?

Answer 24

Cell: accumulation of preCMs in ER; appearance of lipid droplets in the cytoplasm of intestinal cells
Physio: Impaired fat/cholesterol/lipid-soluble vitamin absorption; slow growth; weight gain; effects on GI and nervous systems

Question 25

What is the main treatment for CRD?

Answer 25

Low-fat diet to reduce accumulation of intracellular preCMs

Question 26

Summarise (few words) causes and symptoms of Familial Hypercholesterolemia

Answer 26

Caused by defects in cholesterol uptake into cells; cholesterol accumulates in blood -> atherosclerosis

Question 27

Describe Class 2, 3 and 4 mutations of LDL receptor proteins (RPs)

Answer 27

Class 2: FOLDING (RP formation is absent or impaired; misfolded and retained in ER by quality control)
Class 3: BINDING (normal RP synthesis but abnormal LDL binding)
Class 4: ENDOCYTOSIS (mutations in RP cytoplasmic tail prevents binding of adaptor protein that would recruit RP to clathrin-coated vesicles -> complex not internalised)

Question 28

What mutation can cause Autosomal RECESSIVE Hypercholesterolemia?

Answer 28

Mutation in the LDLRAP1 gene (LDL Receptor Associated Protein) - an adaptor required for endocytic uptake of RP after binding

Question 29

Name the two types of treatment for Familial Hypercholesterolemia

Answer 29

1 - Inhibit Cholesterol Synthesis with statins to stimulate LDL receptor expression (effective for heterozygotes as it upregulates the WT gene)
2 - Inhibit Dietary Cholesterol Absorption (e.g. Ezetimibe works in intestine)

Question 30

What is NPC1 (full name and function)?

Answer 30

Niemann-Pick Type C 1 - a lysosomal membrane protein which transports cholesterol released from LDLs into the cytosol

Question 31

What is the impact of the most common mutation affecting NPC1?

Answer 31

It causes it to misfold and be retained in the ER (this is an ER misfolding disease)

Question 32

What is the most common cause of Gaucher disease?

Answer 32

A mutation which causes misfolding of Lysosomal acid ß-glucosidase causing retention in ER

Question 33

What is the impact of Gaucher disease?

Answer 33

Accumulation of Glucosylceramide and other glycolipids in the lysosomes causing a range of symptoms

Question 34

Name the 3 types of potential treatments for Gaucher disease

Answer 34

1 - Enzyme Replacement Therapy
2 - Substrate Reduction Therapy
3 - Pharmacological Chaperones

Question 35

Describe how Enzyme Replacement Therapy can treat Gaucher disease

Answer 35

Inject synthetic Lysosomal Acid ß-Glucosidase (modified with M6P), which is taken up from outside the cell by M6P receptors

Question 36

Name the drug used in Substrate Reduction Therapy and how this treats Gaucher disease

Answer 36

Miglustat - inhibits Glucosylceramide synthesis - less Lysosomal Acid ß-Glucosidase required

Question 37

What protein is found in the brain plaques associated with Alzheimer’s?

Answer 37

Beta-Amyloid

Question 38

NOTE - FLASHCARDS NEARLY DONE BUT THING ABT ALZHEIMERS AND BETA AMYLOID