Apoptosis

Question 1

Define Necrosis.

Answer 1

Unregulated cell death associated with trauma, cellular disruption + an inflammatory response

Question 2

Define Apoptosis.

Answer 2

Regulated cell death; controlled disassembly of cellular contents without disruption– no inflammatory response

Question 3

Describe the process of necrosis.

Answer 3

Plasma membrane becomes more permeable: the cell swells + membrane ruptures
Proteases are released leading to dissolution + autodigestion of the cell
There is localised inflammation

Question 4

What are the two phases of apoptosis? Describe them.

Answer 4

Latent phase: Death pathways are activated, but cells appear morphologically the same
Execution phase:
Loss of microvilli + intercellular junctions
Cell shrinkage
Loss of plasma membrane asymmetry
Chromatin + nuclear condensation
DNA fragmentation
Formation of membrane blebs
Fragmentation into membrane enclosed apoptotic bodies (digested by macrophages)

Question 5

What is an important feature of apoptosis that distinguishes it from necrosis?

Answer 5

Plasma membrane remains intact so there is no inflammation

Question 6

What DNA modification is seen during apoptosis?

Answer 6

Fragmentation of DNA ladders (seen in agar gel)

Formation of more “ends”, which are labelled by adding an extra fluorescently-labelled bases in a TUNEL assay

Question 7

What other types of cell death are there other than necrosis and apoptosis?

Answer 7

Cell death is GRADED:
Apoptosis-like cell death (display phagocytic recognition molecules before membrane lysis)
Necrosis-like cell death (features of apoptosis before lysis, like an aborted apoptosis)

Question 8

What are caspases?

Answer 8

Cysteine-dependent aspartate-directed proteases
Executioners of apoptosis
Activated by cleavage (Proteolysis)

Question 9

Which caspases are effector caspases?

Answer 9

3, 6 + 7

Question 10

Which caspases are initiator caspases?

Answer 10

2, 9, 8 + 10

Question 11

Describe the structure of effector caspases.

Answer 11

Single chain polypeptides consisting of a small (p10)+ large (p20) subunit
Subunits are released by proteolytic cleavage

Question 12

Describe the structure of initiator caspases.

Answer 12

They have the same 2 subunits found in effector caspases but they also have a targeting subunit (for homotypic protein-protein interactions)

Question 13

What are the two types of targeting subunit that initiator caspases can have?

Answer 13

CARD: caspase recruitment domain (Caspase 2 + 9)
DED: death effector domain (Caspase 8 + 10)

Question 14

How are active caspases formed?

Answer 14

Cleavage of inactive procaspases is followed by the folding of 2 large + 2 small chains to form an active L2S2 heterotetramer

Question 15

What are the two mechanisms of apoptosis

Answer 15

Death by design (receptor-mediated, extrinsic)

Death by default (mitochondrial (intrinsic))

Question 16

Describe the structure of death receptors.

Answer 16

Cysteine-rich extracellular domain
Transmembrane domain
Intracellular tail with a death domain (DD)

Question 17

What are the two important adaptor proteins in the death by design pathway and how are they different?

Answer 17

FADD: positive regulator that promotes cell death (DED + DD)
FLIP: negative regulator (DED + DED)

Question 18

Describe signalling of apoptosis through Fas.

Answer 18

Fas ligand (on CTLs) binds to Fas receptor
Fas receptors undergo trimerisation, which brings the 3 DDs together
Trimerised DDs recruit FADD, which binds via its own DD
FADD recruits + oligomerises procaspase 8 through the DED of procaspase 8
Binding of procaspase 8 to FADD forms DISC (death-induced signalling complex)
DISC formation results in cross-activation of procaspase 8
Active caspase 8 is released, which then activates effector caspases

Question 19

Describe the importance of oligomerisation in the death by design pathway

Answer 19

Some initiator caspases have intrinsic low catalytic activity
Oligomerisation brings them close enough together to allow transcleavage
Thus, at least 2 procaspases are required to form an active caspase

Question 20

Describe how FLIP acts as an inhibitor of apoptosis

Answer 20

FLIP has 2 DED domains but no proteolytic activity so can compete with procaspase 8 to bind to the DED domains of FADD
It can incorporate into receptor-procaspase complexes + interfere with transcleavage

Question 21

As an overview, describe death by default.

Answer 21

Cellular stress causes change in mitochondrial membrane potential
Mitochondrion releases cytochrome C + other apoptotic inducing factors
This stimulates formation of the heptameric apoptosome complex

Question 22

What does the apoptosome consist of?

Answer 22

APAF-1 (apoptotic activating factor 1)
Cytochrome C
ATP
Procaspase 9

Question 23

Describe the domains found within APAF-1.

Answer 23

CARD domain
ATPase domain
WD-40 repeats (for protein-protein interactions)

Question 24

Explain fully, how death by default leads to caspase activation.

Answer 24

Cytochrome C released from mitochondria binds to WD-40 repeats of APAF-1 + leading to formation of a heptamer structure (apoptosome)
This requires ATP
It has 7 central CARD domains, which can interact with CARD domains of procaspase 9
7 procaspase 9’s bind via their CARD domains to the apoptosome
Their close contact allows them to cross-cleave each other to generate active caspase 9, which initiates a caspase cascade, resulting in apoptosis

Question 25

What pro-apoptotic protein links the death by default and death by design pathways? Explain how it works.

Answer 25

``` Bid Caspase 8 (generated by death by design pathway) cleaves Bid, which travels to the mitochondrion + promotes release of cytochrome C, thus triggering the mitochondrial death pathway ```

Question 26

What may determine whether a cell undergoes apoptosis or necrosis?

Answer 26

Apoptosis requires energy (ATP) whereas necrosis does not

Question 27

What is an important family of proteins that act as intrinsic modulators of apoptosis?

Answer 27

Bcl-2 family

Question 28

There are three main groups of Bcl-2 proteins. What is common to all three groups?

Answer 28

BH3 domain: a dimerisation motif, which allows members of the family to form dimers with each other

Question 29

What are the anti-apoptotic Bcl-2 proteins and where are they found?

Answer 29

Bcl-2 Bcl-xL Found localised on the mitochondrial membrane

Question 30

What are the pro-apoptotic Bcl-2 proteins and where are they found?

Answer 30

``` Bid Bad Bax Bak Found in the cytoplasm + in the mitochondrial membrane ```

Question 31

Other than Ras signalling, what other pathway does growth factor binding to growth factor receptors activate?

Answer 31

PI3-kinase

Question 32

What type of molecule is PI3-K?

Answer 32

Lipid kinase

Question 33

What are the main subunits of PI3-K?

Answer 33

``` Adaptor domain (binds to tyrosine phosphorylated receptor) Kinase domain ```

Question 34

What is the main action of PI3-K?

Answer 34

PI3-K converts PIP2 to PIP3

Question 35

What effect does conversion of PIP2 to PIP3 have that leads to inhibition of apoptosis?

Answer 35

PIP3 is recognised by the adaptor subunit of Protein Kinase B (PKB/Akt) This allows PKB to move to the cell membrane where it becomes activated Has mitogenic + anti-apoptotic signal

Question 36

Describe the arrangement of the anti-apoptotic and pro-apoptotic proteins when growth factor signalling and the PI3-K pathway is active.

Answer 36

PKB/Akt is activated meaning Bad is phosphorylated + inactivated (held in an inactive heterodimer with 14-3-3 protein) On the mitochondrial membrane, Bak + Bax are held in inactive heterodimers with Bcl-xL + Bcl-2

Question 37

Describe how loss of growth factor signalling can lead to apoptosis.

Answer 37

Loss of activation of the PI3K pathway so less PIP3, so less activation of PKB/Akt Bad gets dephosphorylated + dissociated from its inactive heterodimer Bad moves to the mitochondrial membrane + binds to the anti-apoptotic proteins (Bcl-2 + Bcl-xL) via its BH3 domain This displaces Bax + Bak from their inactive heterodimers Bax + Bak form a pore in the mitochondrial membrane allowing release of cytochrome C from the mitochondrion– this leads to apoptosis

Question 38

Name two extrinsic regulators of apoptosis and describe their actions.

Answer 38

PTEN (Lipid phosphatase): Counteracts the activation of PKB Reduces cell survival + promotes apoptosis IAPs (Inhibitor of Apoptosis proteins): Binds to procaspases + prevents their activation Can bind to activate caspases + inhibit their activity

Question 39

Are the following tumour suppressor genes or oncogenes? a. Bcl-2 b. PTEN c. PKB/Akt

Answer 39

Bcl-2 Oncogene: increased activation would mean reduced likelihood of apoptosis (cancers are anti-apoptotic) PTEN Tumour suppressor gene: inactivation will mean reduced likelihood of apoptosis PKB/Akt Oncogene

Question 40

What cells would require programmed cell death?

Answer 40

``` Harmful cells (damaged DNA/ Infected) Developmentally defective cells (B cells expressing antibodies against self) Excess cells (sculpting of digits + organs) Obsolete cells (mammary epithelial at end of lactation) ```

Question 41

What are caspase cascades? What do they result in?

Answer 41

Initiator caspases trigger apoptosis by cleaving + activating effector caspases, which carry out the apoptotic programme Cascades generate amplification of signal, divergent responses + points for regulation

Question 42

What actions can caspases have?

Answer 42

Cleave + inactivate proteins/ complexes e.g. nuclear lamins for nuclear breakdown Activate enzymes e.g. protein kinases by direct cleavage or cleavage of inhibitory molecules

Question 43

Summarise the effects of PKB/Akt in promoting cell survival.

Answer 43

Phosphorylates + inactivates Bad + caspase 9 Inactivates FOXO transcription factors (which promote expression of apoptosis promoting genes) Stimulates ribosome production + protein synthesis

Question 44

List the cytoprotective anti-apoptotic pathways

Answer 44

Bcl-2, Bcl-xL (intrinsic pathway) FLIP, IAPs (extrinsic pathway) Growth factor pathways via PI3'-K + PKB/Akt